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2021 Virtual World Medical Innovation Forum, Mass General Brigham, Gene and Cell Therapy, VIRTUAL May 19–21, 2021

The 2021 Virtual World Medical Innovation Forum will focus on the growing impact of gene and cell therapy. Senior healthcare leaders from all over look to shape and debate the area of gene and cell therapy. Our shared belief: no matter the magnitude of change, responsible healthcare is centered on a shared commitment to collaborative innovation–industry, academia, and practitioners working together to improve patients’ lives.

About the World Medical Innovation Forum

Mass General Brigham is pleased to present the World Medical Innovation Forum (WMIF) virtual event Wednesday, May 19 – Friday, May 21. This interactive web event features expert discussions of gene and cell therapy (GCT) and its potential to change the future of medicine through its disease-treating and potentially curative properties. The agenda features 150+ executive speakers from the healthcare industry, venture, startups, life sciences manufacturing, consumer health and the front lines of care, including many Harvard Medical School-affiliated researchers and clinicians. The annual in-person Forum will resume live in Boston in 2022. The World Medical Innovation Forum is presented by Mass General Brigham Innovation, the global business development unit supporting the research requirements of 7,200 Harvard Medical School faculty and research hospitals including Massachusetts General, Brigham and Women’s, Massachusetts Eye and Ear, Spaulding Rehab and McLean Hospital. Follow us on Twitter: twitter.com/@MGBInnovation

Accelerating the Future of Medicine with Gene and Cell Therapy What Comes Next

https://worldmedicalinnovation.org/agenda/

Virtual | May 19–21, 2021

#WMIF2021

@MGBInnovation

Leaders in Pharmaceutical Business Intelligence (LPBI) Group

will cover the event in Real Time

Aviva Lev-Ari, PhD, RN

Founder LPBI 1.0 & LPBI 2.0

member_60221522 copy

will be in virtual attendance producing the e-Proceedings

and the Tweet Collection of this Global event expecting +15,000 attendees

@pharma_BI

@AVIVA1950

LPBI’s Eighteen Books in Medicine

https://lnkd.in/ekWGNqA

Among them, books on Gene and Cell Therapy include the following:

Topics for May 19 – 21 include:

Impact on Patient Care – Therapeutic and Potentially Curative GCT Developments

GCT Delivery, Manufacturing – What’s Next

GCT Platform Development

Oncolytic Viruses – Cancer applications, start-ups

Regenerative Medicine/Stem Cells

Future of CAR-T

M&A Shaping GCT’s Future

Market Priorities

Venture Investing in GCT

China’s GCT Juggernaut

Disease and Patient Focus: Benign blood disorders, diabetes, neurodegenerative diseases

Click here for the current WMIF agenda  

Plus:

Fireside Chats: 1:1 interviews with industry CEOs/C-Suite leaders including Novartis Gene Therapies, ThermoFisher, Bayer AG, FDA

First Look: 18 briefings on emerging GCT research from Mass General Brigham scientists

Virtual Poster Session: 40 research posters and presenters on potential GCT discoveries from Mass General Brigham

Announcement of the Disruptive Dozen, 12 GCT technologies likely to break through in the next few years

AGENDA

Wednesday, May 19, 2021

8:00 AM – 8:10 AM

Opening Remarks

Welcome and the vision for Gene and Cell Therapy and why it is a top Mass General Brigham priority. Introducer: Scott Sperling

  • Co-President, Thomas H. Lee Partners
  • Chairman of the Board of Directors, PHS

Presenter: Anne Klibanski, MD

  • CEO, Mass General Brigham

3,000 people joined 5/19 morning

30 sessions: Lab to Clinic,  academia, industry, investment community

May 22,23,24, 2022 – in Boston, in-person 2022 WMIF on CGT 8:10 AM – 8:30 AM

The Grand Challenge of Widespread GCT Patient Benefits

Co-Chairs identify the key themes of the Forum –  set the stage for top GCT opportunities, challenges, and where the field might take medicine in the future. Moderator: Susan Hockfield, PhD

  • President Emerita and Professor of Neuroscience, MIT

GCT – poised to deliver therapies

Inflection point as Panel will present

Doctors and Patients – Promise for some patients 

Barriers for Cell & Gene

Access for patients to therapies like CGT Speakers: Nino Chiocca, MD, PhD

  • Neurosurgeon-in-Chief and Chairman, Neurosurgery, BWH
  • Harvey W. Cushing Professor of Neurosurgery, HMS

Oncolytic virus triple threat: Toxic, immunological, combine with anti cancer therapies

Polygenic therapy – multiple genes involved, plug-play, Susan Slaugenhaupt, PhD

  • Scientific Director and Elizabeth G. Riley and Daniel E. Smith Jr., Endowed Chair, Mass General Research Institute
  • Professor, Neurology, HMS

Ravi Thadhani, MD

  • CAO, Mass General Brigham
  • Professor, Medicine and Faculty Dean, HMS

Role of academia special to spear head the Polygenic therapy – multiple genes involved, plug-play, 

Access critical, relations with IndustryLuk Vandenberghe, PhD

  • Grousbeck Family Chair, Gene Therapy, MEE
  • Associate Professor, Ophthalmology, HMS

Pharmacology Gene-Drug, Interface academic centers and industry

many CGT drugs emerged in Academic center 8:35 AM – 8:50 AM FIRESIDE

Gene and Cell Therapy 2.0 – What’s Next as We Realize their Potential for Patients

Dave Lennon, PhD

  • President, Novartis Gene Therapies

Hope that CGT emerging, how the therapies work, neuro, muscular, ocular, genetic diseases of liver and of heart revolution for the industry 900 IND application 25 approvals Economic driver Skilled works, VC disease. Modality one time intervention, long duration of impart, reimbursement, ecosystem to be built around CGT

FDA works by indications and risks involved, Standards and expectations for streamlining manufacturing, understanding of process and products 

payments over time payers and Innovators relations Moderator: Julian Harris, MD

  • Partner, Deerfield

Promise of CGT realized, what part?

FDA role and interaction in CGT

Manufacturing aspects which is critical Speaker: Dave Lennon, PhD

  • President, Novartis Gene Therapies

Hope that CGT emerging, how the therapies work, neuro, muscular, ocular, genetic diseases of liver and of heart revolution for the industry 900 IND application 25 approvals Economic driver Skilled works, VC disease. Modality one time intervention, long duration of impart, reimbursement, ecosystem to be built around CGT

FDA works by indications and risks involved, Standards and expectations for streamlining manufacturing, understanding of process and products 

payments over time payers and Innovators relations

  • Q&A 8:55 AM – 9:10 AM  

8:55 AM – 9:20 AM

The Patient and GCT

GCT development for rare diseases is driven by patient and patient-advocate communities. Understanding their needs and perspectives enables biomarker research, the development of value-driving clinical trial endpoints and successful clinical trials. Industry works with patient communities that help identify unmet needs and collaborate with researchers to conduct disease natural history studies that inform the development of biomarkers and trial endpoints. This panel includes patients who have received cutting-edge GCT therapy as well as caregivers and patient advocates. Moderator: Patricia Musolino, MD, PhD

  • Co-Director Pediatric Stroke and Cerebrovascular Program, MGH
  • Assistant Professor of Neurology, HMS

What is the Power of One – the impact that a patient can have on their own destiny by participating in Clinical Trials Contacting other participants in same trial can be beneficial Speakers: Jack Hogan

  • Patient, MEE

Jeanette Hogan

  • Parent of Patient, MEE

Jim Holland

  • CEO, Backcountry.com

Parkinson patient Constraints by regulatory on participation in clinical trial advance stage is approved participation Patients to determine the level of risk they wish to take Information dissemination is critical Barbara Lavery

  • Chief Program Officer, ACGT Foundation

Advocacy agency beginning of work Global Genes educational content and out reach to access the information 

Patient has the knowledge of the symptoms and recording all input needed for diagnosis by multiple clinicians Early application for CGTDan Tesler

  • Clinical Trial Patient, BWH/DFCC

Experimental Drug clinical trial patient participation in clinical trial is very important to advance the state of scienceSarah Beth Thomas, RN

  • Professional Development Manager, BWH

Outcome is unknown, hope for good, support with resources all advocacy groups, 

  • Q&A 9:25 AM – 9:40 AM  

9:25 AM – 9:45 AM FIRESIDE

GCT Regulatory Framework | Why Different?

  Moderator: Vicki Sato, PhD

  • Chairman of the Board, Vir Biotechnology

Diversity of approaches

Process at FDA generalize from 1st entry to rules more generalizable  Speaker: Peter Marks, MD, PhD

  • Director, Center for Biologics Evaluation and Research, FDA

Last Spring it became clear that something will work a vaccine by June 2020 belief that enough candidates the challenge manufacture enough and scaling up FDA did not predicted the efficacy of mRNA vaccine vs other approaches expected to work

Recover Work load for the pandemic will wean & clear, Gene Therapies IND application remained flat in the face of the pandemic Rare diseases urgency remains Consensus with industry advisory to get input gene therapy Guidance  T-Cell therapy vs Regulation best thinking CGT evolve speedily flexible gained by Guidance

Immune modulators, Immunotherapy Genome editing can make use of viral vectors future technologies nanoparticles and liposome encapsulation 

  • Q&A 9:50 AM – 10:05 AM  

9:50 AM – 10:15 AM

Building a GCT Platform for Mainstream Success

This panel of GCT executives, innovators and investors explore how to best shape a successful GCT strategy. Among the questions to be addressed:

  • How are GCT approaches set around defining and building a platform?
  • Is AAV the leading modality and what are the remaining challenges?
  • What are the alternatives?
  • Is it just a matter of matching modalities to the right indications?

Moderator: Jean-François Formela, MD

  • Partner, Atlas Venture

Established core components of the Platform Speakers: Katherine High, MD

  • President, Therapeutics, AskBio

Three drugs approved in Europe in the Gene therapy space

Regulatory Infrastructure exists for CGT drug approval – as new class of therapeutics

Participants investigators, regulators, patients i. e., MDM 

Hemophilia in male most challenging

Human are natural hosts for AV safety signals Dave Lennon, PhD

  • President, Novartis Gene Therapies

big pharma has portfolios of therapeutics not one drug across Tx areas: cell, gene iodine therapy 

collective learning infrastructure features manufacturing at scale early in development Acquisitions strategy for growth # applications for scaling Rick Modi

  • CEO, Affinia Therapeutics

Copy, paste EDIT from product A to B novel vectors leverage knowledge varient of vector, coder optimization choice of indication is critical exploration on larger populations Speed to R&D and Speed to better gene construct get to clinic with better design vs ASAP 

Data sharing clinical experience with vectors strategies patients selection, vector selection, mitigation, patient type specific Louise Rodino-Klapac, PhD

  • EVP, Chief Scientific Officer, Sarepta Therapeutics

AAV based platform 15 years in development same disease indication vs more than one indication stereotype, analytics as hurdle 1st was 10 years 2nd was 3 years

Safety to clinic vs speed to clinic, difference of vectors to trust

  • Q&A 10:20 AM – 10:35 AM  

10:20 AM – 10:45 AM

AAV Success Studies | Retinal Dystrophy | Spinal Muscular Atrophy

Recent AAV gene therapy product approvals have catalyzed the field. This new class of therapies has shown the potential to bring transformative benefit to patients. With dozens of AAV treatments in clinical studies, all eyes are on the field to gauge its disruptive impact.

The panel assesses the largest challenges of the first two products, the lessons learned for the broader CGT field, and the extent to which they serve as a precedent to broaden the AAV modality.

  • Is AAV gene therapy restricted to genetically defined disorders, or will it be able to address common diseases in the near term?
  • Lessons learned from these first-in-class approvals.
  • Challenges to broaden this modality to similar indications.
  • Reflections on safety signals in the clinical studies?

Moderator: Joan Miller, MD

  • Chief, Ophthalmology, MEE
  • Cogan Professor & Chair of Ophthalmology, HMS

Retina specialist, Luxturna success FMA condition cell therapy as solution

Lessons learned

Safety Speakers: Ken Mills

  • CEO, RegenXBio

Tissue types additional administrations, tech and science, address additional diseases, more science for photoreceptors a different tissue type underlying pathology novelties in last 10 years 

Cell therapy vs transplant therapy no immunosuppressionEric Pierce, MD, PhD

  • Director, Ocular Genomics Institute, MEE
  • Professor of Ophthalmology, HMS

Laxterna success to be replicated platform, paradigms measurement visual improved

More science is needed to continue develop vectors reduce toxicity,

AAV can deliver different cargos reduce adverse events improve vectorsRon Philip

  • Chief Operating Officer, Spark Therapeutics

The first retinal gene therapy, voretigene neparvovec-rzyl (Luxturna, Spark Therapeutics), was approved by the FDA in 2017.Meredith Schultz, MD

  • Executive Medical Director, Lead TME, Novartis Gene Therapies

Impact of cell therapy beyond muscular dystrophy, translational medicine, each indication, each disease, each group of patients build platform unlock the promise

Monitoring for Safety signals real world evidence remote markers, home visits, clinical trial made safer, better communication of information

  • Q&A 10:50 AM – 11:05 AM  

10:45 AM – 10:55 AM

Break

  10:55 AM – 11:05 AM FIRST LOOK

Control of AAV pharmacology by Rational Capsid Design

Luk Vandenberghe, PhD

  • Grousbeck Family Chair, Gene Therapy, MEE
  • Associate Professor, Ophthalmology, HMS

AAV a complex driver in Pharmacology durable, vector of choice, administer in vitro, gene editing tissue specificity, pharmacokinetics side effects and adverse events manufacturability site variation diversify portfolios,

Pathway for rational AAV rational design, curated smart variant libraries, AAV  sequence screen multiparametric , data enable liver (de-) targeting unlock therapeutics areas: cochlea 

  • Q&A 11:05 AM – 11:25 AM  

11:05 AM – 11:15 AM FIRST LOOK

Enhanced gene delivery and immunoevasion of AAV vectors without capsid modification

Casey Maguire, PhD

  • Associate Professor of Neurology, MGH & HMS

Virus Biology: Enveloped (e) or not 

enveloped for gene therapy eAAV platform technology: tissue targets and Indications commercialization of eAAV 

  • Q&A 11:15 AM – 11:35 AM  

11:20 AM – 11:45 AM HOT TOPICS

AAV Delivery

This panel will address the advances in the area of AAV gene therapy delivery looking out the next five years. Questions that loom large are: How can biodistribution of AAV be improved? What solutions are in the wings to address immunogenicity of AAV? Will patients be able to receive systemic redosing of AAV-based gene therapies in the future? What technical advances are there for payload size? Will the cost of manufacturing ever become affordable for ultra-rare conditions? Will non-viral delivery completely supplant viral delivery within the next five years?What are the safety concerns and how will they be addressed? Moderators: Xandra Breakefield, PhD

  • Geneticist, MGH, MGH
  • Professor, Neurology, HMS

Florian Eichler, MD

  • Director, Center for Rare Neurological Diseases, MGH
  • Associate Professor, Neurology, HMS

Speakers: Jennifer Farmer

  • CEO, Friedreich’s Ataxia Research Alliance

Ataxia requires therapy targeting multiple organ with one therapy, brain, spinal cord, heart several IND, clinical trials in 2022Mathew Pletcher, PhD

  • SVP, Head of Gene Therapy Research and Technical Operations, Astellas

Work with diseases poorly understood, collaborations needs example of existing: DMD is a great example explain dystrophin share placedo data 

Continue to explore large animal guinea pig not the mice, not primates (ethical issues) for understanding immunogenicity and immune response Manny Simons, PhD

  • CEO, Akouos

AAV Therapy for the fluid of the inner ear, CGT for the ear vector accessible to surgeons translational work on the inner ear for gene therapy right animal model 

Biology across species nerve ending in the cochlea

engineer out of the caspid, lowest dose possible, get desired effect by vector use, 2022 new milestones

  • Q&A 11:50 AM – 12:05 PM  

11:50 AM – 12:15 PM

M&A | Shaping GCT Innovation

The GCT M&A market is booming – many large pharmas have made at least one significant acquisition. How should we view the current GCT M&A market? What is its impact of the current M&A market on technology development? Are these M&A trends new are just another cycle? Has pharma strategy shifted and, if so, what does it mean for GCT companies? What does it mean for patients? What are the long-term prospects – can valuations hold up? Moderator: Adam Koppel, MD, PhD

  • Managing Director, Bain Capital Life Sciences

What acquirers are looking for??

What is the next generation vs what is real where is the industry going? Speakers:

Debby Baron,

  • Worldwide Business Development, Pfizer 

CGT is an important area Pfizer is active looking for innovators, advancing forward programs of innovation with the experience Pfizer has internally 

Scalability and manufacturing  regulatory conversations, clinical programs safety in parallel to planning getting drug to patients

Kenneth Custer, PhD

  • Vice President, Business Development and Lilly New Ventures, Eli Lilly and Company

Marianne De Backer, PhD

Head of Strategy, Business Development & Licensing, and Member of the Executive Committee, Bayer

Absolute Leadership in Gene editing, gene therapy, via acquisition and strategic alliance 

Operating model of the acquired company discussed , company continue independence

Sean Nolan

  • Board Chairman, Encoded Therapeutics & Affinia

Executive Chairman, Jaguar Gene Therapy & Istari Oncology

As acquiree multiple M&A: How the acquirer looks at integration and cultures of the two companies 

Traditional integration vs jump start by external acquisition 

AAV – epilepsy, next generation of vectors 

  • Q&A 12:20 PM – 12:35 PM  

12:15 PM – 12:25 PM FIRST LOOK

Gene Therapies for Neurological Disorders: Insights from Motor Neuron Disorders

Merit Cudkowicz, MD

  • Chief of Neurology, MGH

ALS – Man 1in 300, Women 1 in 400, next decade increase 7% 

10% ALS is heredity 160 pharma in ALS space, diagnosis is late 1/3 of people are not diagnosed, active community for clinical trials Challenges: disease heterogeneity cases of 10 years late in diagnosis. Clinical Trials for ALS in Gene Therapy targeting ASO1 protein therapies FUS gene struck youngsters 

Q&A

  • 12:25 PM – 12:45 PM  

12:25 PM – 12:35 PM FIRST LOOK

Gene Therapy for Neurologic Diseases

Patricia Musolino, MD, PhD

  • Co-Director Pediatric Stroke and Cerebrovascular Program, MGH
  • Assistant Professor of Neurology, HMS

Cerebral Vascular disease – ACTA2 179H gene smooth muscle cell proliferation disorder

no surgery or drug exist –

Cell therapy for ACTA2 Vasculopathy  in the brain and control the BP and stroke – smooth muscle intima proliferation. Viral vector deliver aiming to change platform to non-viral delivery rare disease , gene editing, other mutations of ACTA2 gene target other pathway for atherosclerosis 

  • Q&A 12:35 PM – 12:55 PM  

12:35 PM – 1:15 PM

Lunch

  1:15 PM – 1:40 PM

Oncolytic Viruses in Cancer | Curing Melanoma and Beyond

Oncolytic viruses represent a powerful new technology, but so far an FDA-approved oncolytic (Imlygic) has only occurred in one area – melanoma and that what is in 2015. This panel involves some of the protagonists of this early success story.  They will explore why and how Imlygic became approved and its path to commercialization.  Yet, no other cancer indications exist for Imlygic, unlike the expansion of FDA-approved indication for immune checkpoint inhibitors to multiple cancers.  Why? Is there a limitation to what and which cancers can target?  Is the mode of administration a problem?

No other oncolytic virus therapy has been approved since 2015. Where will the next success story come from and why?  Will these therapies only be beneficial for skin cancers or other easily accessible cancers based on intratumoral delivery?

The panel will examine whether the preclinical models that have been developed for other cancer treatment modalities will be useful for oncolytic viruses.  It will also assess the extent pre-clinical development challenges have slowed the development of OVs. Moderator: Nino Chiocca, MD, PhD

  • Neurosurgeon-in-Chief and Chairman, Neurosurgery, BWH
  • Harvey W. Cushing Professor of Neurosurgery, HMS

Challenges of manufacturing at Amgen what are they? Speakers: Robert Coffin, PhD

  • Chief Research & Development Officer, Replimune

2002 in UK promise in oncolytic therapy GNCSF

Phase III melanoma 2015 M&A with Amgen

oncolytic therapy remains non effecting on immune response 

data is key for commercialization 

do not belief in systemic therapy achieve maximum immune response possible from a tumor by localized injection Roger Perlmutter, MD, PhD

  • Chairman, Merck & Co.

response rates systemic therapy like PD1, Keytruda, OPTIVA well tolerated combination of Oncolytic with systemic 

GMP critical for manufacturing David Reese, MD

  • Executive Vice President, Research and Development, Amgen

Inter lesion injection of agent vs systemic therapeutics 

cold tumors immune resistant render them immune susceptible 

Oncolytic virus is a Mono therapy

addressing the unknown Ann Silk, MD

  • Physician, Dana Farber-Brigham and Women’s Cancer Center
  • Assistant Professor of Medicine, HMS

Which person gets oncolytics virus if patient has immune suppression due to other indications

Safety of oncolytic virus greater than Systemic treatment

series biopsies for injected and non injected tissue and compare Suspect of hot tumor and cold tumors likely to have sme response to agent unknown all potential 

  • Q&A 1:45 PM – 2:00 PM  

1:45 PM – 2:10 PM

Market Interest in Oncolytic Viruses | Calibrating

There are currently two oncolytic virus products on the market, one in the USA and one in China.  As of late 2020, there were 86 clinical trials 60 of which were in phase I with just 2 in Phase III the rest in Phase I/II or Phase II.   Although global sales of OVs are still in the ramp-up phase, some projections forecast OVs will be a $700 million market by 2026. This panel will address some of the major questions in this area:

What regulatory challenges will keep OVs from realizing their potential? Despite the promise of OVs for treating cancer only one has been approved in the US. Why has this been the case? Reasons such have viral tropism, viral species selection and delivery challenges have all been cited. However, these are also true of other modalities. Why then have oncolytic virus approaches not advanced faster and what are the primary challenges to be overcome?

  • Will these need to be combined with other agents to realize their full efficacy and how will that impact the market?
  • Why are these companies pursuing OVs while several others are taking a pass?

Moderators: Martine Lamfers, PhD

  • Visiting Scientist, BWH

Challenged in development of strategies 

Demonstrate efficacyRobert Martuza, MD

  • Consultant in Neurosurgery, MGH
  • William and Elizabeth Sweet Distinguished Professor of Neurosurgery, HMS

Modulation mechanism Speakers: Anlong Li, MD, PhD

  • Clinical Director, Oncology Clinical Development, Merck Research Laboratories

IV delivery preferred – delivery alternative are less aggereable Jeffrey Infante, MD

  • Early development Oncolytic viruses, Oncology, Janssen Research & Development

oncologic virus if it will generate systemic effects the adoption will accelerate

What areas are the best efficacious 

Direct effect with intra-tumor single injection with right payload 

Platform approach  Prime with 1 and Boost with 2 – not yet experimented with 

Do not have the data at trial design for stratification of patients 

Turn off strategy not existing yetLoic Vincent, PhD

  • Head of Oncology Drug Discovery Unit, Takeda

R&D in collaboration with Academic

Vaccine platform to explore different payload

IV administration may not bring sufficient concentration to the tumor is administer  in the blood stream

Classification of Patients by prospective response type id UNKNOWN yet, population of patients require stratification

  • Q&A 2:15 PM – 2:30 PM  

2:10 PM – 2:20 PM FIRST LOOK

Oncolytic viruses: turning pathogens into anticancer agents

Nino Chiocca, MD, PhD

  • Neurosurgeon-in-Chief and Chairman, Neurosurgery, BWH
  • Harvey W. Cushing Professor of Neurosurgery, HMS

Oncolytic therapy DID NOT WORK Pancreatic Cancer and Glioblastoma 

Intra- tumoral heterogeniety hinders success 

Solution: Oncolytic VIRUSES – Immunological “coldness”

GADD-34 20,000 GBM 40,000 pancreatic cancer

  • Q&A 2:25 PM – 2:40 PM  

2:20 PM – 2:45 PM

Entrepreneurial Growth | Oncolytic Virus

In 2020 there were a total of 60 phase I trials for Oncolytic Viruses. There are now dozens of companies pursuing some aspect of OV technology. This panel will address:

  •  How are small companies equipped to address the challenges of developing OV therapies better than large pharma or biotech?
  • Will the success of COVID vaccines based on Adenovirus help the regulatory environment for small companies developing OV products in Europe and the USA?
  • Is there a place for non-viral delivery and other immunotherapy companies to engage in the OV space?  Would they bring any real advantages?

Moderator: Reid Huber, PhD

  • Partner, Third Rock Ventures

Critical milestones to observe Speakers: Caroline Breitbach, PhD

  • VP, R&D Programs and Strategy, Turnstone Biologics

Trying Intra-tumor delivery and IV infusion delivery oncolytic vaccine pushing dose 

translation biomarkers program 

transformation tumor microenvironment Brett Ewald, PhD

  • SVP, Development & Corporate Strategy, DNAtrix

Studies gets larger, kicking off Phase III multiple tumors Paul Hallenbeck, PhD

  • President and Chief Scientific Officer, Seneca Therapeutics

Translation: Stephen Russell, MD, PhD

  • CEO, Vyriad

Systemic delivery Oncolytic Virus IV delivery woman in remission

Collaboration with Regeneron

Data collection: Imageable reporter secretable reporter, gene expression

Field is intense systemic oncolytic delivery is exciting in mice and in human, response rates are encouraging combination immune stimulant, check inhibitors 

  • Q&A 2:50 PM – 3:05 PM  

2:45 PM – 3:00 PM

Break

  3:00 PM – 3:25 PM

CAR-T | Lessons Learned | What’s Next

Few areas of potential cancer therapy have had the attention and excitement of CAR-T. This panel of leading executives, developers, and clinician-scientists will explore the current state of CAR-T and its future prospects. Among the questions to be addressed are:

  • Is CAR-T still an industry priority – i.e. are new investments being made by large companies? Are new companies being financed? What are the trends?
  • What have we learned from first-generation products, what can we expect from CAR-T going forward in novel targets, combinations, armored CAR’s and allogeneic treatment adoption?
  • Early trials showed remarkable overall survival and progression-free survival. What has been observed regarding how enduring these responses are?
  • Most of the approvals to date have targeted CD19, and most recently BCMA. What are the most common forms of relapses that have been observed?
  • Is there a consensus about what comes after these CD19 and BCMA trials as to additional targets in liquid tumors? How have dual-targeted approaches fared?
  • Moderator:
  • Marcela Maus, MD, PhD
    • Director, Cellular Immunotherapy Program, Cancer Center, MGH
    • Associate Professor, Medicine, HMSIs CAR-T Industry priority
  • Speakers:
  • Head of R&D, Atara BioTherapeutics
  • Phyno-type of the cells for hematologic cancers 
  • solid tumor 
  • inventory of Therapeutics for treating patients in the future 
  • Progressive MS program
  • EBBT platform B-Cells and T-Cells
    • Stefan Hendriks
      • Gobal Head, Cell & Gene, Novartis
      • yes, CGT is a strategy in the present and future
      • Journey started years ago 
      • Confirmation the effectiveness of CAR-T therapies, 1 year response prolonged to 5 years 26 months
      • Patient not responding – a lot to learn
      • Patient after 8 months of chemo can be helped by CAR-T
    • Christi Shaw
      • CEO, Kite
      • CAR-T is priority 120 companies in the space
      • Manufacturing consistency 
      • Patients respond with better quality of life
      • Blood cancer – more work to be done

Q&A

  • 3:30 PM – 3:45 PM  

3:30 PM – 3:55 PM HOT TOPICS

CAR-T | Solid Tumors Success | When?

The potential application of CAR-T in solid tumors will be a game-changer if it occurs. The panel explores the prospects of solid tumor success and what the barriers have been. Questions include:

  •  How would industry and investor strategy for CAR-T and solid tumors be characterized? Has it changed in the last couple of years?
  •  Does the lack of tumor antigen specificity in solid tumors mean that lessons from liquid tumor CAR-T constructs will not translate well and we have to start over?
  •  Whether due to antigen heterogeneity, a hostile tumor micro-environment, or other factors are some specific solid tumors more attractive opportunities than others for CAR-T therapy development?
  •  Given the many challenges that CAR-T faces in solid tumors, does the use of combination therapies from the start, for example, to mitigate TME effects, offer a more compelling opportunity.

Moderator: Oladapo Yeku, MD, PhD

  • Clinical Assistant in Medicine, MGH

window of opportunities studies  Speakers: Jennifer Brogdon

  • Executive Director, Head of Cell Therapy Research, Exploratory Immuno-Oncology, NIBR

2017 CAR-T first approval

M&A and research collaborations

TCR tumor specific antigens avoid tissue toxicity Knut Niss, PhD

  • CTO, Mustang Bio

tumor hot start in 12 month clinical trial solid tumors , theraties not ready yet. Combination therapy will be an experimental treatment long journey checkpoint inhibitors to be used in combination maintenance Lipid tumor Barbra Sasu, PhD

  • CSO, Allogene

T cell response at prostate cancer 

tumor specific 

cytokine tumor specific signals move from solid to metastatic cell type for easier infiltration

Where we might go: safety autologous and allogeneic Jay Short, PhD

  • Chairman, CEO, Cofounder, BioAlta, Inc.

Tumor type is not enough for development of therapeutics other organs are involved in the periphery

difficult to penetrate solid tumors biologics activated in the tumor only, positive changes surrounding all charges, water molecules inside the tissue acidic environment target the cells inside the tumor and not outside 

Combination staggered key is try combination

  • Q&A 4:00 PM – 4:15 PM  

4:00 PM – 4:25 PM

GCT Manufacturing | Vector Production | Autologous and Allogeneic | Stem Cells | Supply Chain | Scalability & Management

The modes of GCT manufacturing have the potential of fundamentally reordering long-established roles and pathways. While complexity goes up the distance from discovery to deployment shrinks. With the likelihood of a total market for cell therapies to be over $48 billion by 2027,  groups of products are emerging.  Stem cell therapies are projected to be $28 billion by 2027 and non-stem cell therapies such as CAR-T are projected be $20 billion by 2027. The manufacturing challenges for these two large buckets are very different. Within the CAR-T realm there are diverging trends of autologous and allogeneic therapies and the demands on manufacturing infrastructure are very different. Questions for the panelists are:

  • Help us all understand the different manufacturing challenges for cell therapies. What are the trade-offs among storage cost, batch size, line changes in terms of production cost and what is the current state of scaling naïve and stem cell therapy treatment vs engineered cell therapies?
  • For cell and gene therapy what is the cost of Quality Assurance/Quality Control vs. production and how do you think this will trend over time based on your perspective on learning curves today?
  • Will point of care production become a reality? How will that change product development strategy for pharma and venture investors? What would be the regulatory implications for such products?
  • How close are allogeneic CAR-T cell therapies? If successful what are the market implications of allogenic CAR-T? What are the cost implications and rewards for developing allogeneic cell therapy treatments?

Moderator: Michael Paglia

  • VP, ElevateBio

Speakers:

  • Dannielle Appelhans
    • SVP TechOps and Chief Technical Officer, Novartis Gene Therapies
  • Thomas Page, PhD
    • VP, Engineering and Asset Development, FUJIFILM Diosynth Biotechnologies
  • Rahul Singhvi, ScD
    • CEO and Co-Founder, National Resilience, Inc.
  • Thomas VanCott, PhD
    • Global Head of Product Development, Gene & Cell Therapy, Catalent
    • 2/3 autologous 1/3 allogeneic  CAR-T high doses and high populations scale up is not done today quality maintain required the timing logistics issues centralized vs decentralized  allogeneic are health donors innovations in cell types in use improvements in manufacturing

Ropa Pike, Director,  Enterprise Science & Partnerships, Thermo Fisher Scientific 

Centralized biopharma industry is moving  to decentralized models site specific license 

  • Q&A 4:30 PM – 4:45 PM  

4:30 PM – 4:40 PM FIRST LOOK

CAR-T

Marcela Maus, MD, PhD

  • Director, Cellular Immunotherapy Program, Cancer Center, MGH
  • Assistant Professor, Medicine, HMS 

Fit-to-purpose CAR-T cells: 3 lead programs

Tr-fill 

CAR-T induce response myeloma and multiple myeloma GBM

27 patents on CAR-T

+400 patients treaded 40 Clinical Trials 

  • Q&A 4:40 PM – 5:00 PM  

4:40 PM – 4:50 PM FIRST LOOK

Repurposed Tumor Cells as Killers and Immunomodulators for Cancer Therapy

Khalid Shah, PhD

  • Vice Chair, Neurosurgery Research, BWH
  • Director, Center for Stem Cell Therapeutics and Imaging, HMS

Solid tumors are the hardest to treat because: immunosuppressive, hypoxic, Acidic Use of autologous tumor cells self homing ThTC self targeting therapeutic cells Therapeutic tumor cells efficacy pre-clinical models GBM 95% metastesis ThTC translation to patient settings

  • Q&A 4:50 PM – 5:10 PM  

4:50 PM – 5:00 PM FIRST LOOK

Other Cell Therapies for Cancer

David Scadden, MD

  • Director, Center for Regenerative Medicine; Co-Director, Harvard Stem Cell Institute, Director, Hematologic Malignancies & Experimental Hematology, MGH
  • Jordan Professor of Medicine, HMS

T-cell are made in bone marrow create cryogel  can be an off-the-shelf product repertoire on T Receptor CCL19+ mesenchymal cells mimic Tymus cells –

inter-tymic injection. Non human primate validation

Q&A

 

5:00 PM – 5:20 PM   5:00 PM – 5:20 PM FIRESIDE

Fireside with Mikael Dolsten, MD, PhD

  Introducer: Jonathan Kraft Moderator: Daniel Haber, MD, PhD

  • Chair, Cancer Center, MGH
  • Isselbacher Professor of Oncology, HMS

Vaccine Status Mikael Dolsten, MD, PhD

  • Chief Scientific Officer and President, Worldwide Research, Development and Medical, Pfizer

Deliver vaccine around the Globe, Israel, US, Europe.

3BIL vaccine in 2022 for all Global vaccination 

Bio Ntech in Germany

Experience with Biologics immuneoncology & allogeneic antibody cells – new field for drug discovery 

mRNA curative effort and cancer vaccine 

Access to drugs developed by Pfizer to underdeveloped countries 

  • Q&A 5:25 PM – 5:40 AM  

5:20 PM – 5:30 PM

Closing Remarks

Thursday, May 20, 2021

8:00 AM – 8:25 AM

GCT | The China Juggernaut

China embraced gene and cell therapies early. The first China gene therapy clinical trial was in 1991. China approved the world’s first gene therapy product in 2003—Gendicine—an oncolytic adenovirus for the treatment of advanced head and neck cancer.  Driven by broad national strategy, China has become a hotbed of GCT development, ranking second in the world with more than 1,000 clinical trials either conducted or underway and thousands of related patents.  It has a booming GCT biotech sector, led by more than 45 local companies with growing IND pipelines.

In late 1990, a T cell-based immunotherapy, cytokine-induced killer (CIK) therapy became a popular modality in the clinic in China for tumor treatment.  In early 2010, Chinese researchers started to carry out domestic CAR T trials inspired by several important reports suggested the great antitumor function of CAR T cells. Now, China became the country with the most registered CAR T trials, CAR T therapy is flourishing in China.

The Chinese GCT ecosystem has increasingly rich local innovation and growing complement of development and investment partnerships – and also many subtleties.

This panel, consisting of leaders from the China GCT corporate, investor, research and entrepreneurial communities, will consider strategic questions on the growth of the gene and cell therapy industry in China, areas of greatest strength, evolving regulatory framework, early successes and products expected to reach the US and world market. Moderator: Min Wu, PhD

  • Managing Director, Fosun Health Fund

What are the area of CGT in China, regulatory similar to the US Speakers: Alvin Luk, PhD

  • CEO, Neuropath Therapeutics

Monogenic rare disease with clear genomic target

Increase of 30% in patient enrollment 

Regulatory reform approval is 60 days no delayPin Wang, PhD

  • CSO, Jiangsu Simcere Pharmaceutical Co., Ltd.

Similar starting point in CGT as the rest of the World unlike a later starting point in other biologicalRichard Wang, PhD

  • CEO, Fosun Kite Biotechnology Co., Ltd

Possibilities to be creative and capitalize the new technologies for innovating drug

Support of the ecosystem by funding new companie allowing the industry to be developed in China

Autologous in patients differences cost challengeTian Xu, PhD

  • Vice President, Westlake University

ICH committee and Chinese FDA -r regulation similar to the US

Difference is the population recruitment, in China patients are active participants in skin disease 

Active in development of transposome 

Development of non-viral methods, CRISPR still in D and transposome

In China price of drugs regulatory are sensitive Shunfei Yan, PhD

  • Investment Manager, InnoStar Capital

Indication driven: Hymophilia, 

Allogogenic efficiency therapies

Licensing opportunities 

  • Q&A 8:30 AM – 8:45 AM  

8:30 AM – 8:55 AM

Impact of mRNA Vaccines | Global Success Lessons

The COVID vaccine race has propelled mRNA to the forefront of biomedicine. Long considered as a compelling modality for therapeutic gene transfer, the technology may have found its most impactful application as a vaccine platform. Given the transformative industrialization, the massive human experience, and the fast development that has taken place in this industry, where is the horizon? Does the success of the vaccine application, benefit or limit its use as a therapeutic for CGT?

  • How will the COVID success impact the rest of the industry both in therapeutic and prophylactic vaccines and broader mRNA lessons?
  • How will the COVID success impact the rest of the industry both on therapeutic and prophylactic vaccines and broader mRNA lessons?
  • Beyond from speed of development, what aspects make mRNA so well suited as a vaccine platform?
  • Will cost-of-goods be reduced as the industry matures?
  • How does mRNA technology seek to compete with AAV and other gene therapy approaches?

Moderator: Lindsey Baden, MD

  • Director, Clinical Research, Division of Infectious Diseases, BWH
  • Associate Professor, HMS

In vivo delivery process regulatory cooperation new opportunities for same platform for new indication Speakers:

Many years of mRNA pivoting for new diseases, DARPA, nucleic Acids global deployment of a manufacturing unit on site where the need arise Elan Musk funds new directions at Moderna

How many mRNA can be put in one vaccine: Dose and tolerance to achieve efficacy 

45 days for Personalized cancer vaccine one per patient

1.6 Billion doses produced rare disease monogenic correct mRNA like CF multiple mutation infection disease and oncology applications

Platform allowing to swap cargo reusing same nanoparticles address disease beyond Big Pharma options for biotech

WHat strain of Flu vaccine will come back in the future when people do not use masks 

  • Kate Bingham, UK Vaccine Taskforce

July 2020, AAV vs mRNA delivery across UK local centers administered both types supply and delivery uplift 

  • Q&A 9:00 AM – 9:15 AM  

9:00 AM – 9:25 AM HOT TOPICS

Benign Blood Disorders

Hemophilia has been and remains a hallmark indication for the CGT. Given its well-defined biology, larger market, and limited need for gene transfer to provide therapeutic benefit, it has been at the forefront of clinical development for years, however, product approval remains elusive. What are the main hurdles to this success? Contrary to many indications that CGT pursues no therapeutic options are available to patients, hemophiliacs have an increasing number of highly efficacious treatment options. How does the competitive landscape impact this field differently than other CGT fields? With many different players pursuing a gene therapy option for hemophilia, what are the main differentiators? Gene therapy for hemophilia seems compelling for low and middle-income countries, given the cost of currently available treatments; does your company see opportunities in this market? Moderator: Nancy Berliner, MD

  • Chief, Division of Hematology, BWH
  • H. Franklin Bunn Professor of Medicine, HMS

Speakers: Theresa Heggie

  • CEO, Freeline Therapeutics

Safety concerns, high burden of treatment CGT has record of safety and risk/benefit adoption of Tx functional cure CGT is potent Tx relative small quantity of protein needs be delivered 

Potency and quality less quantity drug and greater potency

risk of delivery unwanted DNA, capsules are critical 

analytics is critical regulator involvement in potency definition

Close of collaboration is excitingGallia Levy, MD, PhD

  • Chief Medical Officer, Spark Therapeutics

Hemophilia CGT is the highest potential for Global access logistics in underdeveloped countries working with NGOs practicality of the Tx

Roche reached 120 Counties great to be part of the Roche GroupAmir Nashat, PhD

  • Managing Partner, Polaris Ventures

Suneet Varma

  • Global President of Rare Disease, Pfizer

Gene therapy at Pfizer small molecule, large molecule and CGT – spectrum of choice allowing Hemophilia patients to marry 

1/3 internal 1/3 partnership 1/3 acquisitions 

Learning from COVID-19 is applied for other vaccine development

review of protocols and CGT for Hemophelia

You can’t buy Time

With MIT Pfizer is developing a model for Hemopilia CGT treatment

  • Q&A 9:30 AM – 9:45 AM  

9:25 AM – 9:35 AM FIRST LOOK

Treating Rett Syndrome through X-reactivation

Jeannie Lee, MD, PhD

  • Molecular Biologist, MGH
  • Professor of Genetics, HMS

200 disease X chromosome unlock for neurological genetic diseases: Rett Syndromeand other autism spectrum disorders female model vs male mice model

deliver protein to the brain 

restore own missing or dysfunctional protein

Epigenetic not CGT – no exogent intervention Xist ASO drug

Female model

  • Q&A 9:35 AM – 9:55 AM  

9:35 AM – 9:45 AM FIRST LOOK

Rare but mighty: scaling up success in single gene disorders

Florian Eichler, MD

  • Director, Center for Rare Neurological Diseases, MGH
  • Associate Professor, Neurology, HMS

Single gene disorder NGS enable diagnosis, DIagnosis to Treatment How to know whar cell to target, make it available and scale up Address gap: missing components Biomarkers to cell types lipid chemistry cell animal biology 

crosswalk from bone marrow matter 

New gene discovered that causes neurodevelopment of stagnant genes Examining new Biology cell type specific biomarkers 

  • Q&A 9:45 AM – 10:05 AM  

9:50 AM – 10:15 AM HOT TOPICS

Diabetes | Grand Challenge

The American Diabetes Association estimates 30 million Americans have diabetes and 1.5 million are diagnosed annually. GCT offers the prospect of long-sought treatment for this enormous cohort and their chronic requirements. The complexity of the disease and its management constitute a grand challenge and highlight both the potential of GCT and its current limitations.

  •  Islet transplantation for type 1 diabetes has been attempted for decades. Problems like loss of transplanted islet cells due to autoimmunity and graft site factors have been difficult to address. Is there anything different on the horizon for gene and cell therapies to help this be successful?
  • How is the durability of response for gene or cell therapies for diabetes being addressed? For example, what would the profile of an acceptable (vs. optimal) cell therapy look like?

Moderator: Marie McDonnell, MD

  • Chief, Diabetes Section and Director, Diabetes Program, BWH
  • Lecturer on Medicine, HMS

Type 1 Diabetes cost of insulin for continuous delivery of drug

alternative treatments: 

The Future: neuropotent stem cells 

What keeps you up at night  Speakers: Tom Bollenbach, PhD

  • Chief Technology Officer, Advanced Regenerative Manufacturing Institute

Data managment sterility sensors, cell survival after implantation, stem cells manufacturing, process development in manufacturing of complex cells

Data and instrumentation the Process is the Product

Manufacturing tight schedules Manasi Jaiman, MD

  • Vice President, Clinical Development, ViaCyte
  • Pediatric Endocrinologist

continous glucose monitoring Bastiano Sanna, PhD

  • EVP, Chief of Cell & Gene Therapies and VCGT Site Head, Vertex Pharmaceuticals

100 years from discovering Insulin, Insulin is not a cure in 2021 – asking patients to partner more 

Produce large quantities of the Islet cells encapsulation technology been developed 

Scaling up is a challengeRogerio Vivaldi, MD

  • CEO, Sigilon Therapeutics

Advanced made, Patient of Type 1 Outer and Inner compartments of spheres (not capsule) no immune suppression continuous secretion of enzyme Insulin independence without immune suppression 

Volume to have of-the-shelf inventory oxegenation in location lymphatic and vascularization conrol the whole process modular platform learning from others

  • Q&A 10:20 AM – 10:35 AM  

10:20 AM – 10:40 AM FIRESIDE

Building A Unified GCT Strategy

  Introducer: John Fish

  • CEO, Suffolk
  • Chairman of Board Trustees, Brigham Health

Moderator: Meg Tirrell

  • Senior Health and Science Reporter, CNBC

Last year, what was it at Novartis Speaker: Jay Bradner, MD

  • President, NIBR

Keep eyes open, waiting the Pandemic to end and enable working back on all the indications 

Portfolio of MET, Mimi Emerging Therapies 

Learning from the Pandemic – operationalize the practice science, R&D leaders, new collaboratives at NIH, FDA, Novartis

Pursue programs that will yield growth, tropic diseases with Gates Foundation, Rising Tide pods for access CGT within Novartis Partnership with UPenn in Cell Therapy 

Cost to access to IP from Academia to a Biotech CRISPR accessing few translations to Clinic

Protein degradation organization constraint valuation by parties in a partnership 

Novartis: nuclear protein lipid nuclear particles, tamplate for Biotech to collaborate

Game changing: 10% of the Portfolio, New frontiers human genetics in Ophthalmology, CAR-T, CRISPR, Gene Therapy Neurological and payloads of different matter

  • Q&A 10:45 AM – 11:00 AM  

10:40 AM – 10:50 AM

Break

  10:50 AM – 11:00 AM FIRST LOOK

Getting to the Heart of the Matter: Curing Genetic Cardiomyopathy

Christine Seidman, MD

  • Director, Cardiovascular Genetics Center, BWH
  • Smith Professor of Medicine & Genetics, HMS

The Voice of Dr. Seidman – Her abstract is cited below

The ultimate opportunity presented by discovering the genetic basis of human disease is accurate prediction and disease prevention. To enable this achievement, genetic insights must enable the identification of at-risk

individuals prior to end-stage disease manifestations and strategies that delay or prevent clinical expression. Genetic cardiomyopathies provide a paradigm for fulfilling these opportunities. Hypertrophic cardiomyopathy (HCM) is characterized by left ventricular hypertrophy, diastolic dysfunction with normal or enhanced systolic performance and a unique histopathology: myocyte hypertrophy, disarray and fibrosis. Dilated cardiomyopathy (DCM) exhibits enlarged ventricular volumes with depressed systolic performance and nonspecific histopathology. Both HCM and DCM are prevalent clinical conditions that increase risk for arrhythmias, sudden death, and heart failure. Today treatments for HCM and DCM focus on symptoms, but none prevent disease progression. Human molecular genetic studies demonstrated that these pathologies often result from dominant mutations in genes that encode protein components of the sarcomere, the contractile unit in striated muscles. These data combined with the emergence of molecular strategies to specifically modulate gene expression provide unparalleled opportunities to silence or correct mutant genes and to boost healthy gene expression in patients with genetic HCM and DCM. Many challenges remain, but the active and vital efforts of physicians, researchers, and patients are poised to ensure success.

Hypertrophic and Dilated Cardiomyopaies ‘

10% receive heart transplant 12 years survival 

Mutation puterb function

TTN: contribute 20% of dilated cardiomyopaty

Silence gene 

pleuripotential cells deliver therapies 

  • Q&A 11:00 AM – 11:20 AM  

11:00 AM – 11:10 AM FIRST LOOK

Unlocking the secret lives of proteins in health and disease

Anna Greka, MD, PhD

  • Medicine, BWH
  • Associate Professor, Medicine, HMS

Cyprus Island, kidney disease by mutation causing MUC1 accumulation and death BRD4780 molecule that will clear the misfolding proteins from the kidney organoids: pleuripotent stem cells small molecule developed for applications in the other cell types in brain, eye, gene mutation build mechnism for therapy clinical models transition from Academia to biotech 

Q&A

  • 11:10 AM – 11:30 AM  

11:10 AM – 11:35 AM

Rare and Ultra Rare Diseases | GCT Breaks Through

One of the most innovative segments in all of healthcare is the development of GCT driven therapies for rare and ultra-rare diseases. Driven by a series of insights and tools and funded in part by disease focused foundations, philanthropists and abundant venture funding disease after disease is yielding to new GCT technology. These often become platforms to address more prevalent diseases. The goal of making these breakthroughs routine and affordable is challenged by a range of issues including clinical trial design and pricing.

  • What is driving the interest in rare diseases?
  • What are the biggest barriers to making breakthroughs ‘routine and affordable?’
  • What is the role of retrospective and prospective natural history studies in rare disease?  When does the expected value of retrospective disease history studies justify the cost?
  • Related to the first question, what is the FDA expecting as far as controls in clinical trials for rare diseases?  How does this impact the collection of natural history data?

Moderator: Susan Slaugenhaupt, PhD

  • Scientific Director and Elizabeth G. Riley and Daniel E. Smith Jr., Endowed Chair, Mass General Research Institute
  • Professor, Neurology, HMS

Speakers: Leah Bloom, PhD

  • SVP, External Innovation and Strategic Alliances, Novartis Gene Therapies

Ultra rare (less than 100) vs rare difficulty to recruit patients and to follow up after treatment Bobby Gaspar, MD, PhD

  • CEO, Orchard Therapeutics

Study of rare condition have transfer to other larger diseases – delivery of therapeutics genes, like immune disorders 

Patient testimonials just to hear what a treatment can make Emil Kakkis, MD, PhD

  • CEO, Ultragenyx

Do 100 patient study then have information on natural history to develop a clinical trial Stuart Peltz, PhD

  • CEO, PTC Therapeutics

Rare disease, challenge for FDA approval and after market commercialization follow ups

Justification of cost for Rare disease – demonstration of Change is IP in value patients advocacy is helpful

  • Q&A 11:40 AM – 11:55 AM  

11:40 AM – 12:00 PM FIRESIDE

Partnering Across the GCT Spectrum

  Moderator: Erin Harris

  • Chief Editor, Cell & Gene

Perspective & professional tenure

Partnership in manufacturing what are the recommendations?

Hospital systems: Partnership Challenges  Speaker: Marc Casper

  • CEO, ThermoFisher

25 years in Diagnostics last 20 years at ThermoFisher 

products used in the Lab for CAR-T research and manufacture 

CGT Innovations: FDA will have a high level of approval each year

How move from research to clinical trials to manufacturing Quicker process

Best practices in Partnerships: the root cause if acceleration to market service providers to deliver highest standards

Building capacity by acquisition to avoid the waiting time

Accelerate new products been manufactured 

Collaborations with Academic Medical center i.e., UCSF in CGT joint funding to accelerate CGT to clinics’

Customers are extremely knowledgable, scale the capital investment made investment

150MIL a year to improve the Workflow 

  • Q&A 12:05 PM – 12:20 PM  

12:05 PM – 12:30 PM

  • 12:05 PM – 12:20 PM  

12:05 PM – 12:30 PM

CEO Panel | Anticipating Disruption | Planning for Widespread GCT

The power of GCT to cure disease has the prospect of profoundly improving the lives of patients who respond. Planning for a disruption of this magnitude is complex and challenging as it will change care across the spectrum. Leading chief executives shares perspectives on how the industry will change and how this change should be anticipated. Moderator: Meg Tirrell

  • Senior Health and Science Reporter, CNBC

CGT becoming staple therapy what are the disruptors emerging Speakers: Lisa Dechamps

  • SVP & Chief Business Officer, Novartis Gene Therapies

Reimagine medicine with collaboration at MGH, MDM condition in children 

The Science is there, sustainable processes and systems impact is transformational

Value based pricing, risk sharing Payers and Pharma for one time therapy with life span effect

Collaboration with FDAKieran Murphy

  • CEO, GE Healthcare

Diagnosis of disease to be used in CGT

2021 investment in CAR-T platform 

Investment in several CGT frontier

Investment in AI, ML in system design new technologies 

GE: Scale and Global distributions, sponsor companies in software 

Waste in Industry – Healthcare % of GDP, work with MGH to smooth the workflow faster entry into hospital and out of Hospital

Telemedicine during is Pandemic: Radiologist needs to read remotely 

Supply chain disruptions slow down all ecosystem 

Production of ventilators by collaboration with GM – ingenuity 

Scan patients outside of hospital a scanner in a Box Christian Rommel, PhD

  • Head, Pharmaceuticals Research & Development, Bayer AG

CGT – 2016 and in 2020 new leadership and capability 

Disease Biology and therapeutics

Regenerative Medicine: CGT vs repair building pipeline in ophthalmology and cardiovascular 

During Pandemic: Deliver Medicines like Moderna, Pfizer – collaborations between competitors with Government Bayer entered into Vaccines in 5 days, all processes had to change access innovations developed over decades for medical solutions 

  • Q&A 12:35 PM – 12:50 PM  

12:35 PM – 12:55 PM FIRESIDE

Building a GCT Portfolio

GCT represents a large and growing market for novel therapeutics that has several segments. These include Cardiovascular Disease, Cancer, Neurological Diseases, Infectious Disease, Ophthalmology, Benign Blood Disorders, and many others; Manufacturing and Supply Chain including CDMO’s and CMO’s; Stem Cells and Regenerative Medicine; Tools and Platforms (viral vectors, nano delivery, gene editing, etc.). Bayer’s pharma business participates in virtually all of these segments. How does a Company like Bayer approach the development of a portfolio in a space as large and as diverse as this one? How does Bayer approach the support of the production infrastructure with unique demands and significant differences from its historical requirements? Moderator:

Shinichiro Fuse, PhD

  • Managing Partner, MPM Capital

Speaker: Wolfram Carius, PhD

  • EVP, Pharmaceuticals, Head of Cell & Gene Therapy, Bayer AG

CGT will bring treatment to cure, delivery of therapies 

Be a Leader repair, regenerate, cure

Technology and Science for CGT – building a portfolio vs single asset decision criteria development of IP market access patients access acceleration of new products

Bayer strategy: build platform for use by four domains  

Gener augmentation

Autologeneic therapy, analytics

Gene editing

Oncology Cell therapy tumor treatment: What kind of cells – the jury is out

Of 23 product launch at Bayer no prediction is possible some high some lows 

  • Q&A 1:00 PM – 1:15 PM  

12:55 PM – 1:35 PM

Lunch

  1:40 PM – 2:05 PM

GCT Delivery | Perfecting the Technology

Gene delivery uses physical, chemical, or viral means to introduce genetic material into cells. As more genetically modified therapies move closer to the market, challenges involving safety, efficacy, and manufacturing have emerged. Optimizing lipidic and polymer nanoparticles and exosomal delivery is a short-term priority. This panel will examine how the short-term and long-term challenges are being tackled particularly for non-viral delivery modalities. Moderator: Natalie Artzi, PhD

  • Assistant Professor, BWH

Speakers: Geoff McDonough, MD

  • CEO, Generation Bio

Sonya Montgomery

  • CMO, Evox Therapeutics

Laura Sepp-Lorenzino, PhD

  • Chief Scientific Officer, Executive Vice President, Intellia Therapeutics

Doug Williams, PhD

  • CEO, Codiak BioSciences
  • Q&A 2:10 PM – 2:25 PM  

2:05 PM – 2:10 PM

Invention Discovery Grant Announcement

  2:10 PM – 2:20 PM FIRST LOOK

Enhancing vesicles for therapeutic delivery of bioproducts

Xandra Breakefield, PhD

  • Geneticist, MGH, MGH
  • Professor, Neurology, HMS
  • Q&A 2:20 PM – 2:35 PM  

2:20 PM – 2:30 PM FIRST LOOK

Versatile polymer-based nanocarriers for targeted therapy and immunomodulation

Natalie Artzi, PhD

  • Assistant Professor, BWH
  • Q&A 2:30 PM – 2:45 PM  

2:55 PM – 3:20 PM HOT TOPICS

Gene Editing | Achieving Therapeutic Mainstream

Gene editing was recognized by the Nobel Committee as “one of gene technology’s sharpest tools, having a revolutionary impact on life sciences.” Introduced in 2011, gene editing is used to modify DNA. It has applications across almost all categories of disease and is also being used in agriculture and public health.

Today’s panel is made up of pioneers who represent foundational aspects of gene editing.  They will discuss the movement of the technology into the therapeutic mainstream.

  • Successes in gene editing – lessons learned from late-stage assets (sickle cell, ophthalmology)
  • When to use what editing tool – pros and cons of traditional gene-editing v. base editing.  Is prime editing the future? Specific use cases for epigenetic editing.
  • When we reach widespread clinical use – role of off-target editing – is the risk real?  How will we mitigate? How practical is patient-specific off-target evaluation?

Moderator: J. Keith Joung, MD, PhD

  • Robert B. Colvin, M.D. Endowed Chair in Pathology & Pathologist, MGH
  • Professor of Pathology, HMS

Speakers: John Evans

  • CEO, Beam Therapeutics

Lisa Michaels

  • EVP & CMO, Editas Medicine
  • Q&A 3:25 PM – 3:50 PM  

3:25 PM – 3:50 PM HOT TOPICS

Common Blood Disorders | Gene Therapy

There are several dozen companies working to develop gene or cell therapies for Sickle Cell Disease, Beta Thalassemia, and  Fanconi Anemia. In some cases, there are enzyme replacement therapies that are deemed effective and safe. In other cases, the disease is only managed at best. This panel will address a number of questions that are particular to this class of genetic diseases:

  • What are the pros and cons of various strategies for treatment? There are AAV-based editing, non-viral delivery even oligonucleotide recruitment of endogenous editing/repair mechanisms. Which approaches are most appropriate for which disease?
  • How can companies increase the speed of recruitment for clinical trials when other treatments are available? What is the best approach to educate patients on a novel therapeutic?
  • How do we best address ethnic and socio-economic diversity to be more representative of the target patient population?
  • How long do we have to follow up with the patients from the scientific, patient’s community, and payer points of view? What are the current FDA and EMA guidelines for long-term follow-up?
  • Where are we with regards to surrogate endpoints and their application to clinically meaningful endpoints?
  • What are the emerging ethical dilemmas in pediatric gene therapy research? Are there challenges with informed consent and pediatric assent for trial participation?
  • Are there differences in reimbursement policies for these different blood disorders? Clearly durability of response is a big factor. Are there other considerations?

Moderator: David Scadden, MD

  • Director, Center for Regenerative Medicine; Co-Director, Harvard Stem Cell Institute, Director, Hematologic Malignancies & Experimental Hematology, MGH
  • Jordan Professor of Medicine, HMS

Speakers: Samarth Kukarni, PhDNick Leschly

  • Chief Bluebird, Bluebird Bio

Mike McCune, MD, PhD

  • Head, HIV Frontiers, Global Health Innovative Technology Solutions, Bill & Melinda Gates Foundation
  • Q&A 3:55 PM – 4:15 PM  

3:50 PM – 4:00 PM FIRST LOOK

Gene Editing

J. Keith Joung, MD, PhD

  • Robert B. Colvin, M.D. Endowed Chair in Pathology & Pathologist, MGH
  • Professor of Pathology, HMS
  • Q&A 4:00 PM – 4:20 PM  

4:20 PM – 4:45 PM HOT TOPICS

Gene Expression | Modulating with Oligonucleotide-Based Therapies

Oligonucleotide drugs have recently come into their own with approvals from companies such as Biogen, Alnylam, Novartis and others. This panel will address several questions:

How important is the delivery challenge for oligonucleotides? Are technological advancements emerging that will improve the delivery of oligonucleotides to the CNS or skeletal muscle after systemic administration?

  • Will oligonucleotides improve as a class that will make them even more effective?   Are further advancements in backbone chemistry anticipated, for example.
  • Will oligonucleotide based therapies blaze trails for follow-on gene therapy products?
  • Are small molecules a threat to oligonucleotide-based therapies?
  • Beyond exon skipping and knock-down mechanisms, what other roles will oligonucleotide-based therapies take mechanistically — can genes be activating oligonucleotides?  Is there a place for multiple mechanism oligonucleotide medicines?
  • Are there any advantages of RNAi-based oligonucleotides over ASOs, and if so for what use?

Moderator: Jeannie Lee, MD, PhD

  • Molecular Biologist, MGH
  • Professor of Genetics, HMS

Speakers: Bob Brown, PhD

  • CSO, EVP of R&D, Dicerna

Brett Monia, PhD

  • CEO, Ionis

Alfred Sandrock, MD, PhD

  • EVP, R&D and CMO, Biogen
  • Q&A 4:50 PM – 5:05 PM  

4:45 PM – 4:55 PM FIRST LOOK

RNA therapy for brain cancer

Pierpaolo Peruzzi, MD, PhD

  • Nuerosurgery, BWH
  • Assistant Professor of Neurosurgery, HMS
  • Q&A 4:55 PM – 5:15 PM  

Friday, May 21, 2021

8:30 AM – 8:55 AM

Venture Investing | Shaping GCT Translation

What is occurring in the GCT venture capital segment? Which elements are seeing the most activity? Which areas have cooled? How is the investment market segmented between gene therapy, cell therapy and gene editing? What makes a hot GCT company? How long will the market stay frothy? Some review of demographics — # of investments, sizes, etc. Why is the market hot and how long do we expect it to stay that way? Rank the top 5 geographic markets for GCT company creation and investing? Are there academic centers that have been especially adept at accelerating GCT outcomes? Do the business models for the rapid development of coronavirus vaccine have any lessons for how GCT technology can be brought to market more quickly? Moderator: Meredith Fisher, PhD

  • Partner, Mass General Brigham Innovation Fund

Speakers: David Berry, MD, PhD

  • CEO, Valo Health
  • General Partner, Flagship Pioneering

Robert Nelsen

  • Managing Director, Co-founder, ARCH Venture Partners

Kush Parmar, MD, PhD

  • Managing Partner, 5AM Ventures
  • Q&A 9:00 AM – 9:15 AM  

9:00 AM – 9:25 AM

Regenerative Medicine | Stem Cells

The promise of stem cells has been a highlight in the realm of regenerative medicine. Unfortunately, that promise remains largely in the future. Recent breakthroughs have accelerated these potential interventions in particular for treating neurological disease. Among the topics the panel will consider are:

  • Stem cell sourcing
  • Therapeutic indication growth
  • Genetic and other modification in cell production
  • Cell production to final product optimization and challenges
  • How to optimize the final product

Moderator: Ole Isacson, MD, PhD

  • Director, Neuroregeneration Research Institute, McLean
  • Professor, Neurology and Neuroscience, HMS

Speakers: Kapil Bharti, PhD

  • Senior Investigator, Ocular and Stem Cell Translational Research Section, NIH

Joe Burns, PhD

  • VP, Head of Biology, Decibel Therapeutics

Erin Kimbrel, PhD

  • Executive Director, Regenerative Medicine, Astellas

Nabiha Saklayen, PhD

  • CEO and Co-Founder, Cellino
  • Q&A 9:30 AM – 9:45 AM  

9:25 AM – 9:35 AM FIRST LOOK

Stem Cells

Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS
  • Q&A 9:35 AM – 9:55 AM  

9:35 AM – 10:00 AM

Capital Formation ’21-30 | Investing Modes Driving GCT Technology and Timing

The dynamics of venture/PE investing and IPOs are fast evolving. What are the drivers – will the number of investors grow will the size of early rounds continue to grow? How is this reflected in GCT target areas, company design, and biotech overall? Do patients benefit from these trends? Is crossover investing a distinct class or a little of both? Why did it emerge and what are the characteristics of the players?  Will SPACs play a role in the growth of the gene and cell therapy industry. What is the role of corporate investment arms eg NVS, Bayer, GV, etc. – has a category killer emerged?  Are we nearing the limit of what the GCT market can absorb or will investment capital continue to grow unabated? Moderator: Roger Kitterman

  • VP, Venture, Mass General Brigham

Speakers: Ellen Hukkelhoven, PhD

  • Managing Director, Perceptive Advisors

Peter Kolchinsky, PhD

  • Founder and Managing Partner, RA Capital Management

Deep Nishar

  • Senior Managing Partner, SoftBank Investment Advisors

Oleg Nodelman

  • Founder & Managing Partner, EcoR1 Capital
  • Q&A 10:05 AM – 10:20 AM  

10:00 AM – 10:10 AM FIRST LOOK

New scientific and clinical developments for autologous stem cell therapy for Parkinson’s disease patients

Penelope Hallett, PhD

  • NRL, McLean
  • Assistant Professor Psychiatry, HMS
  • Q&A 10:10 AM – 10:30 AM  

10:10 AM – 10:35 AM HOT TOPICS

Neurodegenerative Clinical Outcomes | Achieving GCT Success

Can stem cell-based platforms become successful treatments for neurodegenerative diseases?

  •  What are the commonalities driving GCT success in neurodegenerative disease and non-neurologic disease, what are the key differences?
  • Overcoming treatment administration challenges
  • GCT impact on degenerative stage of disease
  • How difficult will it be to titrate the size of the cell therapy effect in different neurological disorders and for different patients?
  • Demonstrating clinical value to patients and payers
  • Revised clinical trial models to address issues and concerns specific to GCT

Moderator: Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS

Speakers: Erwan Bezard, PhD

  • INSERM Research Director, Institute of Neurodegenerative Diseases

Nikola Kojic, PhD

  • CEO and Co-Founder, Oryon Cell Therapies

Geoff MacKay

  • President & CEO, AVROBIO

Viviane Tabar, MD

  • Founding Investigator, BlueRock Therapeutics
  • Chair of Neurosurgery, Memorial Sloan Kettering
  • Q&A 10:40 AM – 10:55 AM  

10:35 AM – 11:35 AM

Disruptive Dozen: 12 Technologies that Will Reinvent GCT

Nearly one hundred senior Mass General Brigham Harvard faculty contributed to the creation of this group of twelve GCT technologies that they believe will breakthrough in the next two years. The Disruptive Dozen identifies and ranks the GCT technologies that will be available on at least an experimental basis to have the chance of significantly improving health care. 11:35 AM – 11:45 AM

Concluding Remarks

Friday, May 21, 2021

Computer connection to the iCloud of WordPress.com FROZE completely at 10:30AM EST and no file update was possible. COVERAGE OF MAY 21, 2021 IS RECORDED BELOW FOLLOWING THE AGENDA BY COPY AN DPASTE OF ALL THE TWEETS I PRODUCED ON MAY 21, 2021 8:30 AM – 8:55 AM

Venture Investing | Shaping GCT Translation

What is occurring in the GCT venture capital segment? Which elements are seeing the most activity? Which areas have cooled? How is the investment market segmented between gene therapy, cell therapy and gene editing? What makes a hot GCT company? How long will the market stay frothy? Some review of demographics — # of investments, sizes, etc. Why is the market hot and how long do we expect it to stay that way? Rank the top 5 geographic markets for GCT company creation and investing? Are there academic centers that have been especially adept at accelerating GCT outcomes? Do the business models for the rapid development of coronavirus vaccine have any lessons for how GCT technology can be brought to market more quickly? Moderator: Meredith Fisher, PhD

  • Partner, Mass General Brigham Innovation Fund

Speakers: David Berry, MD, PhD

  • CEO, Valo Health
  • General Partner, Flagship Pioneering

Robert Nelsen

  • Managing Director, Co-founder, ARCH Venture Partners

Kush Parmar, MD, PhD

  • Managing Partner, 5AM Ventures
  • Q&A 9:00 AM – 9:15 AM  

9:00 AM – 9:25 AM

Regenerative Medicine | Stem Cells

The promise of stem cells has been a highlight in the realm of regenerative medicine. Unfortunately, that promise remains largely in the future. Recent breakthroughs have accelerated these potential interventions in particular for treating neurological disease. Among the topics the panel will consider are:

  • Stem cell sourcing
  • Therapeutic indication growth
  • Genetic and other modification in cell production
  • Cell production to final product optimization and challenges
  • How to optimize the final product

Moderator: Ole Isacson, MD, PhD

  • Director, Neuroregeneration Research Institute, McLean
  • Professor, Neurology and Neuroscience, HMS

Speakers: Kapil Bharti, PhD

  • Senior Investigator, Ocular and Stem Cell Translational Research Section, NIH

Joe Burns, PhD

  • VP, Head of Biology, Decibel Therapeutics

Erin Kimbrel, PhD

  • Executive Director, Regenerative Medicine, Astellas

Nabiha Saklayen, PhD

  • CEO and Co-Founder, Cellino
  • Q&A 9:30 AM – 9:45 AM  

9:25 AM – 9:35 AM FIRST LOOK

Stem Cells

Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS
  • Q&A 9:35 AM – 9:55 AM  

9:35 AM – 10:00 AM

Capital Formation ’21-30 | Investing Modes Driving GCT Technology and Timing

The dynamics of venture/PE investing and IPOs are fast evolving. What are the drivers – will the number of investors grow will the size of early rounds continue to grow? How is this reflected in GCT target areas, company design, and biotech overall? Do patients benefit from these trends? Is crossover investing a distinct class or a little of both? Why did it emerge and what are the characteristics of the players?  Will SPACs play a role in the growth of the gene and cell therapy industry. What is the role of corporate investment arms eg NVS, Bayer, GV, etc. – has a category killer emerged?  Are we nearing the limit of what the GCT market can absorb or will investment capital continue to grow unabated? Moderator: Roger Kitterman

  • VP, Venture, Mass General Brigham

Speakers: Ellen Hukkelhoven, PhD

  • Managing Director, Perceptive Advisors

Peter Kolchinsky, PhD

  • Founder and Managing Partner, RA Capital Management

Deep Nishar

  • Senior Managing Partner, SoftBank Investment Advisors

Oleg Nodelman

  • Founder & Managing Partner, EcoR1 Capital
  • Q&A 10:05 AM – 10:20 AM  

10:00 AM – 10:10 AM FIRST LOOK

New scientific and clinical developments for autologous stem cell therapy for Parkinson’s disease patients

Penelope Hallett, PhD

  • NRL, McLean
  • Assistant Professor Psychiatry, HMS
  • Q&A 10:10 AM – 10:30 AM  

10:10 AM – 10:35 AM HOT TOPICS

Neurodegenerative Clinical Outcomes | Achieving GCT Success

Can stem cell-based platforms become successful treatments for neurodegenerative diseases?

  •  What are the commonalities driving GCT success in neurodegenerative disease and non-neurologic disease, what are the key differences?
  • Overcoming treatment administration challenges
  • GCT impact on degenerative stage of disease
  • How difficult will it be to titrate the size of the cell therapy effect in different neurological disorders and for different patients?
  • Demonstrating clinical value to patients and payers
  • Revised clinical trial models to address issues and concerns specific to GCT

Moderator: Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS

Speakers: Erwan Bezard, PhD

  • INSERM Research Director, Institute of Neurodegenerative Diseases

Nikola Kojic, PhD

  • CEO and Co-Founder, Oryon Cell Therapies

Geoff MacKay

  • President & CEO, AVROBIO

Viviane Tabar, MD

  • Founding Investigator, BlueRock Therapeutics
  • Chair of Neurosurgery, Memorial Sloan Kettering
  • Q&A 10:40 AM – 10:55 AM  

10:35 AM – 11:35 AM

Disruptive Dozen: 12 Technologies that Will Reinvent GCT

Nearly one hundred senior Mass General Brigham Harvard faculty contributed to the creation of this group of twelve GCT technologies that they believe will breakthrough in the next two years. The Disruptive Dozen identifies and ranks the GCT technologies that will be available on at least an experimental basis to have the chance of significantly improving health care. 11:35 AM – 11:45 AM

Concluding Remarks

The co-chairs convene to reflect on the insights shared over the three days. They will discuss what to expect at the in-person GCT focused May 2-4, 2022 World Medical Innovation Forum.

 

The co-chairs convene to reflect on the insights shared over the three days. They will discuss what to expect at the in-person GCT focused May 2-4, 2022 World Medical Innovation Forum.Christine Seidman, MD

Hypertrophic and Dilated Cardiomyopaies ‘

10% receive heart transplant 12 years survival 

Mutation puterb function

TTN: contribute 20% of dilated cardiomyopaty

Silence gene 

pleuripotential cells deliver therapies 

  • Q&A 11:00 AM – 11:20 AM  

11:00 AM – 11:10 AM FIRST LOOK

Unlocking the secret lives of proteins in health and disease

Anna Greka, MD, PhD

  • Medicine, BWH
  • Associate Professor, Medicine, HMS

Cyprus Island, kidney disease by mutation causing MUC1 accumulation and death BRD4780 molecule that will clear the misfolding proteins from the kidney organoids: pleuripotent stem cells small molecule developed for applications in the other cell types in brain, eye, gene mutation build mechnism for therapy clinical models transition from Academia to biotech 

Q&A

  • 11:10 AM – 11:30 AM  

11:10 AM – 11:35 AM

Rare and Ultra Rare Diseases | GCT Breaks Through

One of the most innovative segments in all of healthcare is the development of GCT driven therapies for rare and ultra-rare diseases. Driven by a series of insights and tools and funded in part by disease focused foundations, philanthropists and abundant venture funding disease after disease is yielding to new GCT technology. These often become platforms to address more prevalent diseases. The goal of making these breakthroughs routine and affordable is challenged by a range of issues including clinical trial design and pricing.

  • What is driving the interest in rare diseases?
  • What are the biggest barriers to making breakthroughs ‘routine and affordable?’
  • What is the role of retrospective and prospective natural history studies in rare disease?  When does the expected value of retrospective disease history studies justify the cost?
  • Related to the first question, what is the FDA expecting as far as controls in clinical trials for rare diseases?  How does this impact the collection of natural history data?

Moderator: Susan Slaugenhaupt, PhD

  • Scientific Director and Elizabeth G. Riley and Daniel E. Smith Jr., Endowed Chair, Mass General Research Institute
  • Professor, Neurology, HMS

Speakers: Leah Bloom, PhD

  • SVP, External Innovation and Strategic Alliances, Novartis Gene Therapies

Ultra rare (less than 100) vs rare difficulty to recruit patients and to follow up after treatment Bobby Gaspar, MD, PhD

  • CEO, Orchard Therapeutics

Study of rare condition have transfer to other larger diseases – delivery of therapeutics genes, like immune disorders 

Patient testimonials just to hear what a treatment can make Emil Kakkis, MD, PhD

  • CEO, Ultragenyx

Do 100 patient study then have information on natural history to develop a clinical trial Stuart Peltz, PhD

  • CEO, PTC Therapeutics

Rare disease, challenge for FDA approval and after market commercialization follow ups

Justification of cost for Rare disease – demonstration of Change is IP in value patients advocacy is helpful

  • Q&A 11:40 AM – 11:55 AM  

11:40 AM – 12:00 PM FIRESIDE

Partnering Across the GCT Spectrum

  Moderator: Erin Harris

  • Chief Editor, Cell & Gene

Perspective & professional tenure

Partnership in manufacturing what are the recommendations?

Hospital systems: Partnership Challenges  Speaker: Marc Casper

  • CEO, ThermoFisher

25 years in Diagnostics last 20 years at ThermoFisher 

products used in the Lab for CAR-T research and manufacture 

CGT Innovations: FDA will have a high level of approval each year

How move from research to clinical trials to manufacturing Quicker process

Best practices in Partnerships: the root cause if acceleration to market service providers to deliver highest standards

Building capacity by acquisition to avoid the waiting time

Accelerate new products been manufactured 

Collaborations with Academic Medical center i.e., UCSF in CGT joint funding to accelerate CGT to clinics’

Customers are extremely knowledgable, scale the capital investment made investment

150MIL a year to improve the Workflow 

  • Q&A 12:05 PM – 12:20 PM  

12:05 PM – 12:30 PM

CEO Panel | Anticipating Disruption | Planning for Widespread GCT

The power of GCT to cure disease has the prospect of profoundly improving the lives of patients who respond. Planning for a disruption of this magnitude is complex and challenging as it will change care across the spectrum. Leading chief executives shares perspectives on how the industry will change and how this change should be anticipated. Moderator: Meg Tirrell

  • Senior Health and Science Reporter, CNBC

CGT becoming staple therapy what are the disruptors emerging Speakers: Lisa Dechamps

  • SVP & Chief Business Officer, Novartis Gene Therapies

Reimagine medicine with collaboration at MGH, MDM condition in children 

The Science is there, sustainable processes and systems impact is transformational

Value based pricing, risk sharing Payers and Pharma for one time therapy with life span effect

Collaboration with FDAKieran Murphy

  • CEO, GE Healthcare

Diagnosis of disease to be used in CGT

2021 investment in CAR-T platform 

Investment in several CGT frontier

Investment in AI, ML in system design new technologies 

GE: Scale and Global distributions, sponsor companies in software 

Waste in Industry – Healthcare % of GDP, work with MGH to smooth the workflow faster entry into hospital and out of Hospital

Telemedicine during is Pandemic: Radiologist needs to read remotely 

Supply chain disruptions slow down all ecosystem 

Production of ventilators by collaboration with GM – ingenuity 

Scan patients outside of hospital a scanner in a Box Christian Rommel, PhD

  • Head, Pharmaceuticals Research & Development, Bayer AG

CGT – 2016 and in 2020 new leadership and capability 

Disease Biology and therapeutics

Regenerative Medicine: CGT vs repair building pipeline in ophthalmology and cardiovascular 

During Pandemic: Deliver Medicines like Moderna, Pfizer – collaborations between competitors with Government Bayer entered into Vaccines in 5 days, all processes had to change access innovations developed over decades for medical solutions 

  • Q&A 12:35 PM – 12:50 PM  

12:35 PM – 12:55 PM FIRESIDE

Building a GCT Portfolio

GCT represents a large and growing market for novel therapeutics that has several segments. These include Cardiovascular Disease, Cancer, Neurological Diseases, Infectious Disease, Ophthalmology, Benign Blood Disorders, and many others; Manufacturing and Supply Chain including CDMO’s and CMO’s; Stem Cells and Regenerative Medicine; Tools and Platforms (viral vectors, nano delivery, gene editing, etc.). Bayer’s pharma business participates in virtually all of these segments. How does a Company like Bayer approach the development of a portfolio in a space as large and as diverse as this one? How does Bayer approach the support of the production infrastructure with unique demands and significant differences from its historical requirements? Moderator:

Shinichiro Fuse, PhD

  • Managing Partner, MPM Capital

Speaker: Wolfram Carius, PhD

  • EVP, Pharmaceuticals, Head of Cell & Gene Therapy, Bayer AG

CGT will bring treatment to cure, delivery of therapies 

Be a Leader repair, regenerate, cure

Technology and Science for CGT – building a portfolio vs single asset decision criteria development of IP market access patients access acceleration of new products

Bayer strategy: build platform for use by four domains  

Gener augmentation

Autologeneic therapy, analytics

Gene editing

Oncology Cell therapy tumor treatment: What kind of cells – the jury is out

Of 23 product launch at Bayer no prediction is possible some high some lows 

  • Q&A 1:00 PM – 1:15 PM  

12:55 PM – 1:35 PM

Lunch

  1:40 PM – 2:05 PM

GCT Delivery | Perfecting the Technology

Gene delivery uses physical, chemical, or viral means to introduce genetic material into cells. As more genetically modified therapies move closer to the market, challenges involving safety, efficacy, and manufacturing have emerged. Optimizing lipidic and polymer nanoparticles and exosomal delivery is a short-term priority. This panel will examine how the short-term and long-term challenges are being tackled particularly for non-viral delivery modalities. Moderator: Natalie Artzi, PhD

  • Assistant Professor, BWH

Speakers: Geoff McDonough, MD

  • CEO, Generation Bio

Sonya Montgomery

  • CMO, Evox Therapeutics

Laura Sepp-Lorenzino, PhD

  • Chief Scientific Officer, Executive Vice President, Intellia Therapeutics

Doug Williams, PhD

  • CEO, Codiak BioSciences
  • Q&A 2:10 PM – 2:25 PM  

2:05 PM – 2:10 PM

Invention Discovery Grant Announcement

  2:10 PM – 2:20 PM FIRST LOOK

Enhancing vesicles for therapeutic delivery of bioproducts

Xandra Breakefield, PhD

  • Geneticist, MGH, MGH
  • Professor, Neurology, HMS
  • Q&A 2:20 PM – 2:35 PM  

2:20 PM – 2:30 PM FIRST LOOK

Versatile polymer-based nanocarriers for targeted therapy and immunomodulation

Natalie Artzi, PhD

  • Assistant Professor, BWH
  • Q&A 2:30 PM – 2:45 PM  

2:55 PM – 3:20 PM HOT TOPICS

Gene Editing | Achieving Therapeutic Mainstream

Gene editing was recognized by the Nobel Committee as “one of gene technology’s sharpest tools, having a revolutionary impact on life sciences.” Introduced in 2011, gene editing is used to modify DNA. It has applications across almost all categories of disease and is also being used in agriculture and public health.

Today’s panel is made up of pioneers who represent foundational aspects of gene editing.  They will discuss the movement of the technology into the therapeutic mainstream.

  • Successes in gene editing – lessons learned from late-stage assets (sickle cell, ophthalmology)
  • When to use what editing tool – pros and cons of traditional gene-editing v. base editing.  Is prime editing the future? Specific use cases for epigenetic editing.
  • When we reach widespread clinical use – role of off-target editing – is the risk real?  How will we mitigate? How practical is patient-specific off-target evaluation?

Moderator: J. Keith Joung, MD, PhD

  • Robert B. Colvin, M.D. Endowed Chair in Pathology & Pathologist, MGH
  • Professor of Pathology, HMS

Speakers: John Evans

  • CEO, Beam Therapeutics

Lisa Michaels

  • EVP & CMO, Editas Medicine
  • Q&A 3:25 PM – 3:50 PM  

3:25 PM – 3:50 PM HOT TOPICS

Common Blood Disorders | Gene Therapy

There are several dozen companies working to develop gene or cell therapies for Sickle Cell Disease, Beta Thalassemia, and  Fanconi Anemia. In some cases, there are enzyme replacement therapies that are deemed effective and safe. In other cases, the disease is only managed at best. This panel will address a number of questions that are particular to this class of genetic diseases:

  • What are the pros and cons of various strategies for treatment? There are AAV-based editing, non-viral delivery even oligonucleotide recruitment of endogenous editing/repair mechanisms. Which approaches are most appropriate for which disease?
  • How can companies increase the speed of recruitment for clinical trials when other treatments are available? What is the best approach to educate patients on a novel therapeutic?
  • How do we best address ethnic and socio-economic diversity to be more representative of the target patient population?
  • How long do we have to follow up with the patients from the scientific, patient’s community, and payer points of view? What are the current FDA and EMA guidelines for long-term follow-up?
  • Where are we with regards to surrogate endpoints and their application to clinically meaningful endpoints?
  • What are the emerging ethical dilemmas in pediatric gene therapy research? Are there challenges with informed consent and pediatric assent for trial participation?
  • Are there differences in reimbursement policies for these different blood disorders? Clearly durability of response is a big factor. Are there other considerations?

Moderator: David Scadden, MD

  • Director, Center for Regenerative Medicine; Co-Director, Harvard Stem Cell Institute, Director, Hematologic Malignancies & Experimental Hematology, MGH
  • Jordan Professor of Medicine, HMS

Speakers: Samarth Kukarni, PhDNick Leschly

  • Chief Bluebird, Bluebird Bio

Mike McCune, MD, PhD

  • Head, HIV Frontiers, Global Health Innovative Technology Solutions, Bill & Melinda Gates Foundation
  • Q&A 3:55 PM – 4:15 PM  

3:50 PM – 4:00 PM FIRST LOOK

Gene Editing

J. Keith Joung, MD, PhD

  • Robert B. Colvin, M.D. Endowed Chair in Pathology & Pathologist, MGH
  • Professor of Pathology, HMS
  • Q&A 4:00 PM – 4:20 PM  

4:20 PM – 4:45 PM HOT TOPICS

Gene Expression | Modulating with Oligonucleotide-Based Therapies

Oligonucleotide drugs have recently come into their own with approvals from companies such as Biogen, Alnylam, Novartis and others. This panel will address several questions:

How important is the delivery challenge for oligonucleotides? Are technological advancements emerging that will improve the delivery of oligonucleotides to the CNS or skeletal muscle after systemic administration?

  • Will oligonucleotides improve as a class that will make them even more effective?   Are further advancements in backbone chemistry anticipated, for example.
  • Will oligonucleotide based therapies blaze trails for follow-on gene therapy products?
  • Are small molecules a threat to oligonucleotide-based therapies?
  • Beyond exon skipping and knock-down mechanisms, what other roles will oligonucleotide-based therapies take mechanistically — can genes be activating oligonucleotides?  Is there a place for multiple mechanism oligonucleotide medicines?
  • Are there any advantages of RNAi-based oligonucleotides over ASOs, and if so for what use?

Moderator: Jeannie Lee, MD, PhD

  • Molecular Biologist, MGH
  • Professor of Genetics, HMS

Speakers: Bob Brown, PhD

  • CSO, EVP of R&D, Dicerna

Brett Monia, PhD

  • CEO, Ionis

Alfred Sandrock, MD, PhD

  • EVP, R&D and CMO, Biogen
  • Q&A 4:50 PM – 5:05 PM  

4:45 PM – 4:55 PM FIRST LOOK

RNA therapy for brain cancer

Pierpaolo Peruzzi, MD, PhD

  • Nuerosurgery, BWH
  • Assistant Professor of Neurosurgery, HMS
  • Q&A 4:55 PM – 5:15 PM  

Friday, May 21, 2021

Computer connection to the iCloud of WordPress.com FROZE completely at 10:30AM EST and no file update was possible. COVERAGE OF MAY 21, 2021 IS RECORDED BELOW FOLLOWING THE AGENDA BY COPY AN DPASTE OF ALL THE TWEETS I PRODUCED ON MAY 21, 2021

8:30 AM – 8:55 AM

Venture Investing | Shaping GCT Translation

What is occurring in the GCT venture capital segment? Which elements are seeing the most activity? Which areas have cooled? How is the investment market segmented between gene therapy, cell therapy and gene editing? What makes a hot GCT company? How long will the market stay frothy? Some review of demographics — # of investments, sizes, etc. Why is the market hot and how long do we expect it to stay that way? Rank the top 5 geographic markets for GCT company creation and investing? Are there academic centers that have been especially adept at accelerating GCT outcomes? Do the business models for the rapid development of coronavirus vaccine have any lessons for how GCT technology can be brought to market more quickly? Moderator:   Meredith Fisher, PhD

  • Partner, Mass General Brigham Innovation Fund

Strategies, success what changes are needed in the drug discovery process   Speakers:  

Bring disruptive frontier as a platform with reliable delivery CGT double knock out disease cure all change efficiency and scope human centric vs mice centered right scale of data converted into therapeutics acceleratetion 

Innovation in drugs 60% fails in trial because of Toxicology system of the future deal with big diseases

Moderna is an example in unlocking what is inside us Microbiome and beyond discover new drugs epigenetics  

  • Robert Nelsen
    • Managing Director, Co-founder, ARCH Venture Partners

Manufacturing change is not a new clinical trial FDA need to be presented with new rethinking for big innovations Drug pricing cheaper requires systematization How to systematically scaling up systematize the discovery and the production regulatory innovations

Responsibility mismatch should be and what is “are”

Long term diseases Stack holders and modalities risk benefir for populations 

  • Q&A 9:00 AM – 9:15 AM  

9:00 AM – 9:25 AM

Regenerative Medicine | Stem Cells

The promise of stem cells has been a highlight in the realm of regenerative medicine. Unfortunately, that promise remains largely in the future. Recent breakthroughs have accelerated these potential interventions in particular for treating neurological disease. Among the topics the panel will consider are:

  • Stem cell sourcing
  • Therapeutic indication growth
  • Genetic and other modification in cell production
  • Cell production to final product optimization and challenges
  • How to optimize the final product
  • Moderator:
    • Ole Isacson, MD, PhD
      • Director, Neuroregeneration Research Institute, McLean
      • Professor, Neurology and Neuroscience, MGH, HMS

Opportunities in the next generation of the tactical level Welcome the oprimism and energy level of all Translational medicine funding stem cells enormous opportunities 

  • Speakers:
  • Kapil Bharti, PhD
    • Senior Investigator, Ocular and Stem Cell Translational Research Section, NIH
    • first drug required to establish the process for that innovations design of animal studies not done before
    • Off-th-shelf one time treatment becoming cure 
    •  Intact tissue in a dish is fragile to maintain metabolism
    Joe Burns, PhD
    • VP, Head of Biology, Decibel Therapeutics
    • Ear inside the scall compartments and receptors responsible for hearing highly differentiated tall ask to identify cell for anticipated differentiation
    • multiple cell types and tissue to follow
    Erin Kimbrel, PhD
    • Executive Director, Regenerative Medicine, Astellas
    • In the ocular space immunogenecity
    • regulatory communication
    • use gene editing for immunogenecity Cas1 and Cas2 autologous cells
    • gene editing and programming big opportunities 
    Nabiha Saklayen, PhD
    • CEO and Co-Founder, Cellino
    • scale production of autologous cells foundry using semiconductor process in building cassettes
    • solution for autologous cells
  • Q&A 9:30 AM – 9:45 AM  

9:25 AM – 9:35 AM FIRST LOOK

Stem Cells

Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS
  • Cell therapy for Parkinson to replace dopamine producing cells lost ability to produce dopamin
  • skin cell to become autologous cells reprograms to become cells producing dopamine
  • transplantation fibroblast cells metabolic driven process lower mutation burden 
  • Quercetin inhibition elimination undifferentiated cells graft survival oxygenation increased 
  • Q&A 9:35 AM – 9:55 AM  

9:35 AM – 10:00 AM

Capital Formation ’21-30 | Investing Modes Driving GCT Technology and Timing

The dynamics of venture/PE investing and IPOs are fast evolving. What are the drivers – will the number of investors grow will the size of early rounds continue to grow? How is this reflected in GCT target areas, company design, and biotech overall? Do patients benefit from these trends? Is crossover investing a distinct class or a little of both? Why did it emerge and what are the characteristics of the players?  Will SPACs play a role in the growth of the gene and cell therapy industry. What is the role of corporate investment arms eg NVS, Bayer, GV, etc. – has a category killer emerged?  Are we nearing the limit of what the GCT market can absorb or will investment capital continue to grow unabated? Moderator: Roger Kitterman

  • VP, Venture, Mass General Brigham
  • Saturation reached or more investment is coming in CGT 

Speakers: Ellen Hukkelhoven, PhD

  • Managing Director, Perceptive Advisors
  • Cardiac area transduct cells
  • matching tools
  • 10% success of phase 1 in drug development next phase matters more 

Peter Kolchinsky, PhD

  • Founder and Managing Partner, RA Capital Management
  • Future proof for new comers disruptors 
  • Ex Vivo gene therapy to improve funding products what tool kit belongs to 
  • company insulation from next instability vs comapny stabilizing themselves along few years
  • Company interested in SPAC 
  • cross over investment vs SPAC
  • Multi Omics in cancer early screening metastatic diseas will be wiped out 

Deep Nishar

  • Senior Managing Partner, SoftBank Investment Advisors
  • Young field vs CGT started in the 80s 
  • high payloads is a challenge
  • cost effective fast delivery to large populations
  • Mission oriented by the team and management  
  • Multi Omics disease modality 

Oleg Nodelman

  • Founder & Managing Partner, EcoR1 Capital
  • Invest in company next round of investment will be IPO
  • Help company raise money cross over investment vs SPAC
  • Innovating ideas from academia in need for funding 
  • Q&A 10:05 AM – 10:20 AM  

10:00 AM – 10:10 AM FIRST LOOK

New scientific and clinical developments for autologous stem cell therapy for Parkinson’s disease patients

Penelope Hallett, PhD

  • NRL, McLean
  • Assistant Professor Psychiatry, HMS
  • Pharmacologic agent in existing cause another disorders locomo-movement related 
  • efficacy Autologous cell therapy transplantation approach program T cells into dopamine generating neurons greater than Allogeneic cell transplantation 
  • Q&A 10:10 AM – 10:30 AM  

10:10 AM – 10:35 AM HOT TOPICS

Neurodegenerative Clinical Outcomes | Achieving GCT Success

Can stem cell-based platforms become successful treatments for neurodegenerative diseases?

  •  What are the commonalities driving GCT success in neurodegenerative disease and non-neurologic disease, what are the key differences?
  • Overcoming treatment administration challenges
  • GCT impact on degenerative stage of disease
  • How difficult will it be to titrate the size of the cell therapy effect in different neurological disorders and for different patients?
  • Demonstrating clinical value to patients and payers
  • Revised clinical trial models to address issues and concerns specific to GCT

Moderator: Bob Carter, MD, PhD

  • Chairman, Department of Neurosurgery, MGH
  • William and Elizabeth Sweet, Professor of Neurosurgery, HMS
  • Neurogeneration REVERSAL or slowing down 

Speakers: Erwan Bezard, PhD

  • INSERM Research Director, Institute of Neurodegenerative Diseases
  • Cautious on reversal 
  • Early intervantion versus late

Nikola Kojic, PhD

  • CEO and Co-Founder, Oryon Cell Therapies
  • Autologus cell therapy placed focal replacing missing synapses reestablishment of neural circuitary

Geoff MacKay

  • President & CEO, AVROBIO
  • Prevent condition to be manifested in the first place 
  • clinical effect durable single infusion preventions of symptoms to manifest 
  • Cerebral edema – stabilization
  • Gene therapy know which is the abnormal gene grafting the corrected one 
  • More than biomarker as end point functional benefit not yet established  

Viviane Tabar, MD

  • Founding Investigator, BlueRock Therapeutics
  • Chair of Neurosurgery, Memorial Sloan Kettering
  • Current market does not have delivery mechanism that a drug-delivery is the solution Trials would fail on DELIVERY
  • Immune suppressed patients during one year to avoid graft rejection Autologous approach of Parkinson patient genetically mutated reprogramed as dopamine generating neuron – unknowns are present
  • Circuitry restoration
  • Microenvironment disease ameliorate symptoms – education of patients on the treatment 
  • Q&A 10:40 AM – 10:55 AM  

10:35 AM – 11:35 AM

Disruptive Dozen: 12 Technologies that Will Reinvent GCT

Nearly one hundred senior Mass General Brigham Harvard faculty contributed to the creation of this group of twelve GCT technologies that they believe will breakthrough in the next two years. The Disruptive Dozen identifies and ranks the GCT technologies that will be available on at least an experimental basis to have the chance of significantly improving health care. 11:35 AM – 11:45 AM

Concluding Remarks

The co-chairs convene to reflect on the insights shared over the three days. They will discuss what to expect at the in-person GCT focused May 2-4, 2022 World Medical Innovation Forum.

ALL THE TWEETS PRODUCED ON MAY 21, 2021 INCLUDE THE FOLLOWING:

Aviva Lev-Ari

@AVIVA1950

  • @AVIVA1950_PIcs

4h

#WMIF2021

@MGBInnovation

Erwan Bezard, PhD INSERM Research Director, Institute of Neurodegenerative Diseases Cautious on reversal

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

  • @AVIVA1950_PIcs

4h

#WMIF2021

@MGBInnovation

Nikola Kojic, PhD CEO and Co-Founder, Oryon Cell Therapies Autologus cell therapy placed focal replacing missing synapses reestablishment of neural circutary

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

4h

#WMIF2021

@MGBInnovation

Bob Carter, MD, PhD Chairman, Department of Neurosurgery, MGH William and Elizabeth Sweet, Professor of Neurosurgery, HMS Neurogeneration REVERSAL or slowing down? 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

4h

#WMIF2021

@MGBInnovation

Penelope Hallett, PhD NRL, McLean Assistant Professor Psychiatry, HMS efficacy Autologous cell therapy transplantation approach program T cells into dopamine genetating cells greater than Allogeneic cell transplantation 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

4h

#WMIF2021

@MGBInnovation

Penelope Hallett, PhD NRL, McLean Assistant Professor Psychiatry, HMS Pharmacologic agent in existing cause another disorders locomo-movement related 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

@AVIVA1950_PIcs

4h

#WMIF2021

@MGBInnovation

Roger Kitterman VP, Venture, Mass General Brigham Saturation reached or more investment is coming in CGT Multi OMICS and academia originated innovations are the most attractive areas

@pharma_BI

@AVIVA1950

1

3

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

@AVIVA1950_PIcs

4h

#WMIF2021

@MGBInnovation

Roger Kitterman VP, Venture, Mass General Brigham Saturation reached or more investment is coming in CGT 

@pharma_BI

@AVIVA1950

1

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

4h

#WMIF2021

@MGBInnovation

Oleg Nodelman Founder & Managing Partner, EcoR1 Capital Invest in company next round of investment will be IPO 20% discount

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

@AVIVA1950_PIcs

4h

#WMIF2021

@MGBInnovation

Peter Kolchinsky, PhD Founder and Managing Partner, RA Capital Management Future proof for new comers disruptors  Ex Vivo gene therapy to improve funding products what tool kit belongs to 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

4h

#WMIF2021

@MGBInnovation

Deep Nishar Senior Managing Partner, SoftBank Investment Advisors Young field vs CGT started in the 80s  high payloads is a challenge 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Bob Carter, MD, PhD MGH, HMS cells producing dopamine transplantation fibroblast cells metabolic driven process lower mutation burden  Quercetin inhibition elimination undifferentiated cells graft survival oxygenation increased 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Chairman, Department of Neurosurgery, MGH, Professor of Neurosurgery, HMS Cell therapy for Parkinson to replace dopamine producing cells lost ability to produce dopamine skin cell to become autologous cells reprogramed  

@pharma_BI

@AVIVA1950

#WMIF2021

@MGBInnovation

Kapil Bharti, PhD Senior Investigator, Ocular and Stem Cell Translational Research Section, NIH Off-th-shelf one time treatment becoming cure  Intact tissue in a dish is fragile to maintain metabolism to become like semiconductors

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

@AVIVA1950_PIcs

5h

#WMIF2021

@MGBInnovation

Ole Isacson, MD, PhD Director, Neuroregeneration Research Institute, McLean Professor, Neurology and Neuroscience, MGH, HMS Opportunities in the next generation of the tactical level Welcome the oprimism and energy level of all

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Erin Kimbrel, PhD Executive Director, Regenerative Medicine, Astellas In the ocular space immunogenecity regulatory communication use gene editing for immunogenecity Cas1 and Cas2 autologous cells

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Nabiha Saklayen, PhD CEO and Co-Founder, Cellino scale production of autologous cells foundry using semiconductor process in building cassettes by optic physicists

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Joe Burns, PhD VP, Head of Biology, Decibel Therapeutics Ear inside the scall compartments and receptors responsible for hearing highly differentiated tall ask to identify cell for anticipated differentiation control by genomics

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Kapil Bharti, PhD Senior Investigator, Ocular and Stem Cell Translational Research Section, NIH first drug required to establish the process for that innovations design of animal studies not done before 

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Meredith Fisher, PhD Partner, Mass General Brigham Innovation Fund Strategies, success what changes are needed in the drug discovery process@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Robert Nelsen Managing Director, Co-founder, ARCH Venture Partners Manufacturing change is not a new clinical trial FDA need to be presented with new rethinking for big innovations Drug pricing cheaper requires systematization

@pharma_BI

@AVIVA1950

1

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

Kush Parmar, MD, PhD Managing Partner, 5AM Ventures Responsibility mismatch should be and what is “are”

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

5h

#WMIF2021

@MGBInnovation

David Berry, MD, PhD CEO, Valo Health GP, Flagship Pioneering Bring disruptive frontier platform reliable delivery CGT double knockout disease cure all change efficiency scope human centric vs mice centered right scale acceleration

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

6h

#WMIF2021

@MGBInnovation

Kush Parmar, MD, PhD Managing Partner, 5AM Ventures build it yourself, benefit for patients FIrst Look at MGB shows MEE innovation on inner ear worthy investment  

@pharma_BI

@AVIVA1950

@AVIVA1950_PIcs

Aviva Lev-Ari

@AVIVA1950

6h

#WMIF2021

@MGBInnovation

Robert Nelsen Managing Director, Co-founder, ARCH Venture Partners Frustration with supply chain during the Pandemic, GMC anticipation in advance CGT rapidly prototype rethink and invest proactive investor .edu and Pharma

@pharma_BI

@AVIVA1950

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Live Conference Coverage AACR 2020 in Real Time: Monday June 22, 2020 Late Day Sessions

 

Reporter: Stephen J. Williams, PhD

 

Follow Live in Real Time using

#AACR20

@pharma_BI

@AACR

 

Register for FREE at https://www.aacr.org/

 

AACR VIRTUAL ANNUAL MEETING II

 

June 22-24: Free Registration for AACR Members, the Cancer Community, and the Public
This virtual meeting will feature more than 120 sessions and 4,000 e-posters, including sessions on cancer health disparities and the impact of COVID-19 on clinical trials

 

This Virtual Meeting is Part II of the AACR Annual Meeting.  Part I was held online in April and was centered only on clinical findings.  This Part II of the virtual meeting will contain all the Sessions and Abstracts pertaining to basic and translational cancer research as well as clinical trial findings.

 

REGISTER NOW

 

 

 

Virtual Educational Session

Prevention Research, Science Policy, Epidemiology, Survivorship

Carcinogens at Home: Science and Pathways to Prevention

Chemicals known to cause cancer are used and released to the environment in large volumes, exposing people where they live, work, play, and go to school. The science establishing an important role for such exposures in the development of cancers continues to strengthen, yet cancer prevention researchers are largely unfamiliar with the data drawn upon in identifying carcinogens and making decisions about their use. Characterizing and reducing harmful exposures and accelerating the devel

Julia Brody, Kathryn Z. Guyton, Polly J. Hoppin, Bill Walsh, Mary H. Ward

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Educational Session

Tumor Biology, Molecular and Cellular Biology/Genetics, Clinical Research Excluding Trials

EMT Still Matters: Let’s Explore! – Dedicated to the Memory of Isaiah J. Fidler

During carcinoma progression, initially benign epithelial cells acquire the ability to invade locally and disseminate to distant tissues by activating epithelial-mesenchymal transition (EMT). EMT is a cellular process during which epithelial cells lose their epithelial features and acquire mesenchymal phenotypes and behavior. Growing evidence supports the notion that EMT programs during tumor progression are usually activated to various extents and often partial and reversible, thus pr

Jean-Paul Thiery, Heide L Ford, Jing Yang, Geert Berx

DETAILS

Monday, June 22

1:30 PM – 3:00 PM EDT

Virtual Educational Session

Tumor Biology, Experimental and Molecular Therapeutics, Molecular and Cellular Biology/Genetics

One of These Things Is Not Like the Other: The Many Faces of Senescence in Cancer

Cellular senescence is a stable cell growth arrest that is broadly recognized to act as a barrier against tumorigenesis. Senescent cells acquire a senescence-associated secretory phenotype (SASP), a transcriptional response involving the secretion of inflammatory cytokines, immune modulators, and proteases that can shape the tumor microenvironment. The SASP can initially stimulate tumor immune surveillance and reinforce growth arrest. However, if senescent cells are not removed by the

Clemens A Schmitt, Andrea Alimonti, René Bernards

DETAILS

Monday, June 22

1:30 PM – 3:00 PM EDT

Virtual Educational Session

Clinical Research Excluding Trials, Molecular and Cellular Biology/Genetics

Recent Advances in Applications of Cell-Free DNA

The focus of this educational session will be on recent developments in cell-free DNA (cfDNA) analysis that have the potential to impact the care of cancer patients. Tumors continually shed DNA into the circulation, where it can be detected as circulating tumor DNA (ctDNA). Analysis of ctDNA has become a routine part of care for a subset of patients with advanced malignancies. However, there are a number of exciting potential applications that have promising preliminary data but that h

Michael R Speicher, Maximilian Diehn, Aparna Parikh

DETAILS

Monday, June 22

1:30 PM – 3:30 PM EDT

Virtual Methods Workshop

Clinical Research Excluding Trials, Clinical Trials, Experimental and Molecular Therapeutics, Molecular and Cellular Biology/Genetics

Translating Genetics and Genomics to the Clinic and Population

This session will describe how advances in understanding cancer genomes and in genetic testing technologies are being translated to the clinic. The speakers will illustrate the clinical impact of genomic discoveries for diagnostics and treatment of common tumor types in adults and in children. Cutting-edge technologies for characterization of patient and tumor genomes will be described. New insights into the importance of patient factors for cancer risk and outcome, including predispos

Heather L. Hampel, Gordana Raca, Jaclyn Biegel, Jeffrey M Trent

DETAILS

Monday, June 22

1:30 PM – 3:22 PM EDT

Virtual Educational Session

Regulatory Science and Policy, Drug Development, Epidemiology

Under-representation in Clinical Trials and the Implications for Drug Development

The U.S. Food and Drug Administration relies on data from clinical trials to determine whether medical products are safe and effective. Ideally, patients enrolled in those trials are representative of the population in which the product will be used if approved, including people of different ages, races, ethnic groups, and genders. Unfortunately, with few patients enrolling in clinical trials, many groups are not well-represented in clinical trials. This session will explore challenges

Ajay K. Nooka, Nicole J. Gormley, Kenneth C Anderson, Ruben A. Mesa, Daniel J. George, Yelak Biru, RADM Richardae Araojo, Lola A. Fashoyin-Aje

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Cancer Chemistry

Targeted Protein Degradation: Target Validation Tools and Therapeutic Opportunity

This educational session will cover the exciting emerging field of targeted protein degradation. Key learning topics will include: 1. an introduction to the technology and its relevance to oncology; 2. PROTACS, degraders, and CELMoDs; 3. enzymology and protein-protein interactions in targeted protein degraders; 4. examples of differentiated biology due to degradation vs. inhibition; 5. how to address questions of specificity; and 6. how the field is approaching challenges in optimizing therapies

George Burslem, Mary Matyskiela, Lyn H. Jones, Stewart L Fisher, Andrew J Phillips

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Bioinformatics and Systems Biology, Experimental and Molecular Therapeutics, Drug Development, Molecular and Cellular Biology/Genetics

Obstacles and opportunities for protein degradation drug discovery

Lyn H. Jones
  • PROTACs ubiquitin mediated by E3 ligases;  first discovered by DeShaies and targeted to specific proteins
  • PROTACs used in drug discovery against a host of types of targets including kinases and membrane receptors
  • PROTACs can be modular but lack molecular structural activity relationships
  • can use chemical probes for target validation
  • four requirements: candidate exposure at site of action (for example lipophilicity for candidates needed to cross membranes and accumulate in lysosomes), target engagement (ternary occupancy as measured by FRET), functional pharmacology, relevant phenotype
  • PROTACs hijack the proteosomal degradation system

Proteolysis-targeting chimeras as therapeutics and tools for biological discovery

George Burslem
  • first PROTAC developed to coopt the VHL ubiquitin ligase system which degrades HIF1alpha but now modified for EREalpha
  • in screen for potential PROTACS there were compounds which bound high affinity but no degradation so phenotypic screening very important
  • when look at molecular dynamics can see where PROTAC can add additional protein protein interaction, verifed by site directed mutagenesis
  • able to target bcr-Abl
  • he says this is a rapidly expanding field because of all the new E3 ligase targets being discovered

Expanding the horizons of cereblon modulators

Mary Matyskiela

Translating cellular targeted protein degradation to in vivo models using an enzymology framework

Stewart L Fisher
  • new targeting compounds have an E3 ligase binding domain, a target binding domain and a linker domain
  • in vivo these compounds are very effective; BRD4 degraders good invitro and in vivo with little effect on body weight
  • degraders are essential activators of E3 ligases as these degraders bring targets in close proximity so activates a catalytic cycle of a multistep process (has now high turnover number)
  • in enzymatic pathway the degraders make a productive complex so instead of a kcat think of measuring a kprod or productivity of degraders linked up an E3 ligase
  • the degraders are also affecting the rebound protein synthesis; so Emax never to zero and see a small rebound of protein synthesis

 

Data-Driven Approaches for Choosing Combinatorial Therapies

Drug combinations remain the gold standard for treating cancer, as they significantly outperform single agents. However, due to the enormous size of drug combination space, it is virtually impossible to interrogate all possible combinations. This session will discuss approaches to identify novel combinations using both experimental and computational approaches. Speakers will discuss i) approaches to drug screening in cell lines, the impact of the microenvironment, and attempts to more

Bence Szalai, James E Korkola, Lisa Tucker-Kellogg, Jeffrey W Tyner

DETAILS

Monday, June 22

3:45 PM – 5:21 PM EDT

Virtual Educational Session

Tumor Biology

Cancer Stem Cells and Therapeutic Resistance

Cancer stem cells are a subpopulation of cells with a high capacity for self-renewal, differentiation and resistance to therapy. In this session, we will define cancer stem cells, discuss cellular plasticity, interactions between cancer stem cells and the tumor microenvironment, and mechanisms that contribute to therapeutic resistance.

Robert S Kerbel, Dolores Hambardzumyan, Jennifer S. Yu

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Drug Development, Experimental and Molecular Therapeutics

Molecular Imaging in Cancer Research

This session will cover the fundamentals as well as the major advances made in the field of molecular imaging. Topics covered will include the basics for optical, nuclear, and ultrasound imaging; the pros and cons of each modality; and the recent translational advancements. Learning objectives include the fundamentals of each imaging modality, recent advances in the technology, the processes involved to translate an imaging agent from bench to bedside, and how molecular imaging can gui

Julie Sutcliffe, Summer L Gibbs, Mark D Pagel, Katherine W Ferrara

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Tumor Biology, Immunology, Experimental and Molecular Therapeutics, Drug Development

Tumor Endothelium: The Gatekeepers of Tumor Immune Surveillance

Tumor-associated endothelium is a gatekeeper that coordinates the entry and egress of innate and adaptive immune cells within the tumor microenvironment. This is achieved, in part, via the coordinated expression of chemokines and cell adhesion molecules on the endothelial cell surface that attract and retain circulating leukocytes. Crosstalk between adaptive immune cells and the tumor endothelium is therefore essential for tumor immune surveillance and the success of immune-based thera

Dai Fukumura, Maria M Steele, Wen Jiang, Andrew C Dudley

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Immunology, Experimental and Molecular Therapeutics

Novel Strategies in Cancer Immunotherapy: The Next Generation of Targets for Anticancer Immunotherapy

T-cell immunotherapy in the form of immune checkpoint blockade or cellular T-cell therapies has been tremendously successful in some types of cancer. This success has opened the door to consider what other modalities or types of immune cells can be harnessed for exert antitumor functions. In this session, experts in their respective fields will discuss topics including novel approaches in immunotherapy, including NK cells, macrophage, and viral oncotherapies.

Evanthia Galanis, Kerry S Campbell, Milan G Chheda, Jennifer L Guerriero

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Tumor Biology, Drug Development, Immunology, Clinical Research Excluding Trials

Benign Cells as Drivers of Cancer Progression: Fat and Beyond

Carcinomas develop metastases and resistance to therapy as a result of interaction with tumor microenvironment, composed of various nonmalignant cell types. Understanding the complexity and origins of tumor stromal cells is a prerequisite for development of effective treatments. The link between obesity and cancer progression has revealed the engagement of adipose stromal cells (ASC) and adipocytes from adjacent fat tissue. However, the molecular mechanisms through which they stimulate

Guojun Wu, Matteo Ligorio, Mikhail Kolonin, Maria T Diaz-Meco

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Educational Session

Clinical Research Excluding Trials, Experimental and Molecular Therapeutics, Tumor Biology

Dharma Master Jiantai Symposium on Lung Cancer: Know Thy Organ – Lessons Learned from Lung and Pancreatic Cancer Research

The term “cancer” encompasses hundreds of distinct disease entities involving almost every possible site in the human body. Effectively interrogating cancer, either in animals models or human specimens, requires a deep understanding of the involved organ. This includes both the normal cellular constituents of the affected tissue as well as unique aspects of tissue-specific tumorigenesis. It is critical to “Know Thy Organ” when studying cancer. This session will focus on two of the most

Trudy G Oliver, Hossein Borghaei, Laura Delong Wood, Howard C Crawford

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Methods Workshop

Clinical Trials

Clinical Trial Design: Part 1: Novel Approaches and Methods in Clinical Trial Design

Good clinical trial design has always had to balance the competing interests of effectively and convincingly answering the question with the limitations imposed by scarce resources, complex logistics, and risks and potential benefits to participants. New targeted therapies, immuno-oncology, and novel combination treatments add new challenges on top of the old ones. This session will introduce these concerns and 1) suggest ways to consider what outcomes are relevant, 2) how we can best

Mary W. Redman, Nolan A. Wages, Susan G Hilsenbeck, Karyn A. Goodman

DETAILS

Monday, June 22

3:45 PM – 5:45 PM EDT

Virtual Methods Workshop

Tumor Biology, Drug Development

High-Throughput Screens for Drivers of Progression and Resistance

The sequencing of human cancers now provides a landscape of the genetic alterations that occur in human cancer, and increasingly knowledge of somatic genetic alterations is becoming part of the evaluation of cancer patients. In some cases, this information leads directly to the selection of particular therapeutic approaches; however, we still lack the ability to decipher the significance of genetic alterations in many cancers. This session will focus on recent developments that permit the identification of molecular targets in specific cancers. This information, coupled with genomic characterization of cancer, will facilitate the development of new therapeutic agents and provide a path to implement precision cancer medicine to all patients.

William C Hahn, Mark A Dawson, Mariella Filbin, Michael Bassik

DETAILS

Monday, June 22

3:45 PM – 5:15 PM EDT

Defining a cancer dependency map

William C Hahn

Introduction

William C Hahn

Genome-scale CRISPR screens in 3D spheroids identify cancer vulnerabilities

Michael Bassik

Utilizing single-cell RNAseq and CRISPR screens to target cancer stem cells in pediatric brain tumors

Mariella Filbin
  • many gliomas are defined by discreet mutational spectra that also discriminates based on age and site as well (for example many cortical tumors have mainly V600E Braf mutations while thalamus will be FGFR1
  • they did single cell RNAseq on needle biopsy from 7 gliomas which gave about 3500 high quality single cells; obtained full length RNA
  • tumors clustered mainly where the patient it came from but had stromal cell contamination probably so did a deconvolution?  Copy number variation showed which were tumor cells and did principle component analysis
  • it seems they used a human glioma model as training set
  • identified a stem cell like glioma cell so concentrated on the genes altered in these for translational studies
  • developed multiple PDX models from patients
  • PDX transcriptome closest to patient transcriptome but organoid grown in serum free very close while organoids grown in serum very distinct transcriptome
  • developed a CRISPR barcoded library to determine genes for survival genes
  • pulled out BMI1  and EZH2 (polycomb complex proteins) as good targets

Virtual Methods Workshop

Prevention Research, Survivorship, Clinical Research Excluding Trials, Epidemiology

Implementation Science Methods for Cancer Prevention and Control in Diverse Populations: Integration of Implementation Science Methods in Care Settings

Through this Education Session we will use examples from ongoing research to provide an overview of implementation science approaches to cancer prevention and control research. We draw on examples to highlight study design approaches, research methods, and real-world solutions when applying implementation science to achieve health equity. Approaches to defining change in the care setting and measuring sustained changes are also emphasized. Using real examples of patient navigation prog

Graham A Colditz, Sanja Percac-Lima, Nathalie Huguet

DETAILS

Monday, June 22

3:45 PM – 5:30 PM EDT

Virtual Educational Session

Regulatory Science and Policy, Epidemiology

COVID-19 and Cancer: Guidance for Clinical Trial Conduct and Considerations for RWE

This session will consider the use of real-world evidence in the context of oncology clinical trials affected by the COVID-19 pandemic. Key aspects of the FDA’s recent “Guidance on Conduct of Clinical Trials of Medical Products of Medical Products during COVID-19 Public Health Emergency” will be discussed, including telemedicine, accounting for missing data, obtaining laboratory tests and images locally, using remote informed consent procedures, and additional considerations for contin

Wendy Rubinstein, Paul G. Kluetz, Amy P. Abernethy, Jonathan Hirsch, C.K. Wang

 

 

Read Full Post »


Cyclic Dinucleotides and Histone deacetylase inhibitors

Curators: Larry H. Bernsten, MD, FCAP and Aviva Lev-Ari, PhD, RN

LPBI

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

https://pharmaceuticalintelligence.com/2016/04/24/new-class-of-immune-system-stimulants-cyclic-di-nucleotides-cdn-shrink-tumors-and-bolster-vaccines-re-arm-the-immune-systems-natural-killer-cells-which-attack-cancer-cells-and-virus-inf/

A new class of immune system stimulants called cyclic di-nucleotides have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer. Allison’s technique uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This has been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

 

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted). David Raulet, professor of molecular and cell biology and director of IVRI has contributed to making these cells a new focus of cancer research. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

 

Histone deacetylase inhibitors: molecular mechanisms of action

W S Xu1,2, R B Parmigiani1,2 and P A Marks1

Oncogene (2007) 26, 5541–5552; http://dx.doi.org:/10.1038/sj.onc.1210620

This review focuses on the mechanisms of action of histone deacetylase (HDAC) inhibitors (HDACi), a group of recently discovered ‘targeted’ anticancer agents. There are 18 HDACs, which are generally divided into four classes, based on sequence homology to yeast counterparts. Classical HDACi such as the hydroxamic acid-based vorinostat (also known as SAHA and Zolinza) inhibits classes I, II and IV, but not the NAD+-dependent class III enzymes. In clinical trials, vorinostat has activity against hematologic and solid cancers at doses well tolerated by patients. In addition to histones, HDACs have many other protein substrates involved in regulation of gene expression, cell proliferation and cell death. Inhibition of HDACs causes accumulation of acetylated forms of these proteins, altering their function. Thus, HDACs are more properly called ‘lysine deacetylases.’ HDACi induces different phenotypes in various transformed cells, including growth arrest, activation of the extrinsic and/or intrinsic apoptotic pathways, autophagic cell death, reactive oxygen species (ROS)-induced cell death, mitotic cell death and senescence. In comparison, normal cells are relatively more resistant to HDACi-induced cell death. The plurality of mechanisms of HDACi-induced cell death reflects both the multiple substrates of HDACs and the heterogeneous patterns of molecular alterations present in different cancer cells.

histone deacetylase, histone deacetylase inhibitor, apoptosis, mitotic cell death, senescence, angiogenesis

Acetylation and deacetylation of histones play an important role in transcription regulation of eukaryotic cells (Lehrmann et al., 2002;Mai et al., 2005). The acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004). Further, the HDACs have many non-histone proteins substrates such as hormone receptors, chaperone proteins and cytoskeleton proteins, which regulate cell proliferation and cell death (Table 1). Thus, HDACi-induced transformed cell death involves transcription-dependent and transcription-independent mechanisms (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006;Minucci and Pelicci, 2006).

Table 1 – Nonhistone protein substrates of HDACs (partial list).   Full table

http://www.nature.com/common/images/table_thumb.gif

In humans, 18 HDAC enzymes have been identified and classified, based on homology to yeast HDACs (Blander and Guarente, 2004;Bhalla, 2005Marks and Dokmanovic, 2005). Class I HDACs include HDAC1, 2, 3 and 8, which are related to yeast RPD3 deacetylase and have high homology in their catalytic sites. Recent phylogenetic analyses suggest that this class can be divided into classes Ia (HDAC1 and -2), Ib (HDAC3) and Ic (HDAC8) (Gregoretti et al., 2004). Class II HDACs are related to yeast Hda1 (histone deacetylase 1) and include HDAC4, -5, -6, -7, -9 and -10 (Bhalla, 2005Marks and Dokmanovic, 2005). This class is divided into class IIa, consisting of HDAC4, -5, -7 and -9, and class IIb, consisting of HDAC6 and -10, which contain two catalytic sites. All class I and II HDACs are zinc-dependent enzymes. Members of a third class, sirtuins, require NAD+ for their enzymatic activity (Blander and Guarente, 2004) (see review by E Verdin, in this issue). Among them, SIRT1 is orthologous to yeast silent information regulator 2. The enzymatic activity of class III HDACs is not inhibited by compounds such as vorinostat or trichostatin A (TSA), that inhibit class I and II HDACs. Class IV HDAC is represented by HDAC11, which, like yeast Hda 1 similar 3, has conserved residues in the catalytic core region shared by both class I and II enzymes (Gao et al., 2002).

HDACs are not redundant in function (Marks and Dokmanovic, 2005Rosato and Grant, 2005Bolden et al., 2006). Class I HDACs are primarily nuclear in localization and ubiquitously expressed, while class II HDACs can be primarily cytoplasmic and/or migrate between the cytoplasm and nucleus and are tissue-restricted in expression.

The structural details of the HDAC–HDACi interaction has been elucidated in studies of a histone deacetylase-like protein from an anerobic bacterium with TSA and vorinostat (Finnin et al., 1999). More recently, the crystal structure of HDAC8–hydroxamate interaction has been solved (Somoza et al., 2004Vannini et al., 2004). These studies provide an insight into the mechanism of deacetylation of acetylated substrates. The hydroxamic acid moiety of the inhibitor directly interacts with the zinc ion at the base of the catalytic pocket.

This review focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death. HDACi, for example, the hydroxamic acid-based vorinostat (SAHA, Zolinza), are promising drugs for cancer treatment (Richon et al., 1998Marks and Breslow, 2007). Several HDACi are in phase I and II clinical trials, being tested in different tumor types, such as cutaneous T-cell lymphoma, acute myeloid leukemia, cervical cancer, etc (Bug et al., 2005Chavez-Blanco et al., 2005Kelly and Marks, 2005;Duvic and Zhang, 2006) (Table 2). Although considerable progress has been made in elucidating the role of HDACs and the effects of HDACi, these areas are still in early stages of discovery.

Table 2 – HDACi in clinical trials.  Full table

http://www.nature.com/common/images/table_thumb.gif

Recent phylogenetic analyses of bacterial HDACs suggest that all four HDAC classes preceded the evolution of histone proteins (Gregoretti et al., 2004). This suggests that the primary activity of HDACs may be directed against non-histone substrates. At least 50 non-histone proteins of known biological function have been identified, which may be acetylated and substrates of HDACs (Table 1) (Glozak et al., 2005Marks and Dokmanovic, 2005;Rosato and Grant, 2005Bolden et al., 2006Minucci and Pelicci, 2006Zhao et al., 2006). In addition, two recent proteomic studies identified many lysine-acetylated substrates (Iwabata et al., 2005Kim et al., 2006). In view of all these findings, HDACs may be better called ‘N-epsilon-lysine deacetylase’. This designation would also distinguish them from the enzymes that catalyse other types of deacetylation in biological reactions, such as acylases that catalyse the deacetylation of a range of N-acetyl amino acids (Anders and Dekant, 1994).

Non-histone protein targets of HDACs include transcription factors, transcription regulators, signal transduction mediators, DNA repair enzymes, nuclear import regulators, chaperone proteins, structural proteins, inflammation mediators and viral proteins (Table 1). Acetylation can either increase or decrease the function or stability of the proteins, or protein–protein interaction (Glozak et al., 2005). These HDAC substrates are directly or indirectly involved in many biological processes, such as gene expression and regulation of pathways of proliferation, differentiation and cell death. These data suggest that HDACi could have multiple mechanisms of inducing cell growth arrest and cell death (Figure 1).

Figure 1.  Full figure and legend (90K)

Multiple HDACi-activated antitumor pathways. See text for detailed explanation of each pathway. HDAC6, histone deacetylase 6; HIF-1, hypoxia-induced factor-1; HSP90, heat-shock protein 90; PP1, protein phosphatase 1; ROS, reactive oxygen species; TBP2, thioredoxin binding protein 2; Trx, thioredoxin; VEGF, vascular endothelial growth factor.

http://www.nature.com/onc/journal/v26/n37/images/1210620f1.jpg

HDACi have been discovered with different structural characteristics, including hydroximates, cyclic peptides, aliphatic acids and benzamides (Table 2) (Miller et al., 2003Yoshida et al., 2003Marks and Breslow, 2007). Certain HDACi may selectively inhibit different HDACs. For example, MS-275 preferentially inhibits HDAC1 with IC50, at 0.3 m, compared to HDAC3 with an IC50 of about 8 m, and has little or no inhibitory effect against HDAC6 and HDAC8 (Hu et al., 2003). Two novel synthetic compounds, SK7041 and SK7068, preferentially target HDAC1 and 2 and exhibit growth inhibitory effects in human cancer cell lines and tumor xenograft models (Kim et al., 2003a). A small molecule, tubacin, selectively inhibits HDAC6 activity and causes an accumulation of acetylated -tubulin, but does not affect acetylation of histones, and does not inhibit cell cycle progression (Haggarty et al., 2003). No other HDACi for a specific HDAC has been reported.

HDACi selectively alters gene expression

HDACi-induced antitumor pathways

  • HDACi induces cell cycle arrest
  • HDACi activates the extrinsic apoptotic pathways
  • HDACi activates the intrinsic apoptotic pathways
  • HDACi induces mitotic cell death
  • HDACi induces autophagic cell death and senescence
  • ROS, thioredoxin and Trx binding protein 2 in HDACi-induced cell death
  • Antitumor effects of HDAC6 inhibition
  • Activation of protein phosphatase 1
  • Disruption of the function of chaperonin HSP90
  • Disruption of the aggresome pathway
  • HDACi inhibits angiogenesis

HDACi can block tumor angiogenesis by inhibition of hypoxia inducible factors (HIF) (Liang et al., 2006). HIF-1 and HIF-2 are transcription factors for angiogenic genes (Brown and Wilson, 2004). The oxygen level can control HIF activity through two mechanisms. First, under normoxic conditions, HIF-1 binds to von Hippel–Lindau protein (pVHL) and is degraded by the ubiquitination–proteasome system. Second, HIF activity depends on its transactivation potential (TAP), which is affected by the interaction with the coactivator p300/CBP among others. This complex can be disrupted by Factor Inhibiting HIF (FIH). Hypoxic conditions activate HIF through repression of the hydroxylases responsible for HIF degradation and loss of function.

 

Combination of HDACi with other antitumor agents

The HDACi have shown synergistic or additive antitumor effects with a wide range of antitumor reagents, including chemotherapeutic drugs, new targeted therapeutic reagents and radiation, by various mechanisms, some unique for particular combinations (Rosato and Grant, 2004Bhalla, 2005Marks and Dokmanovic, 2005Bolden et al., 2006).

Clinical development of HDACi

At least 14 different HDACi are in some phase of clinical trials as monotherapy or in combination with retinoids, taxols, gemcitabine, radiation, etc, in patients with hematologic and solid tumors, including cancer of lung, breast, pancreas, renal and bladder, melanoma, glioblastoma, leukemias, lymphomas, multiple myeloma (see National Cancer Institute website for CTEP clinical trials, ctep.cancer.gov or clinicaltrials.gov, and website of companies developing HDACi; Table 2).

The resistance to HDACi

Conclusions and perspectives

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi. This is reflected in the different inducer-activated antitumor pathways in transformed cells (Figure 1). The functions of HDACs are not redundant. Thus, a pan-HDAC inhibitor such as vorinostat may activate more antitumor pathways and have therapeutic advantages compared to HDAC isotype-specific inhibitors.

Almost all cancers have multiple defects in the expression and/or structure of proteins that regulate cell proliferation and death. Compared to other antitumor reagents, the plurality of action of HDACi potentially confers efficacy in a wide spectrum of cancers, which have heterogeneity and multiple defects, both among different types of cancer and within different individual tumors of the same type. The multiple defects in a cancer cell may be the reason for transformed cells being more sensitive than normal cells to HDACi. Thus, given the relatively rapid reversibility of vorinostat inhibition of HDACs, normal cells may be able to compensate for HDACi-induced changes more effectively than cancer cells.

HDACi have synergistic or additive antitumor effects with many other antitumor reagents – suggesting that combination of HDACi and other anticancer agents may be very attractive therapeutic strategies for using these agents. Complete understanding of the mechanisms underlying the resistance and sensitivity to HDACi has obvious therapeutic importance. Targeting resistant factors will enhance the antitumor efficacy of HDACi. Identifying markers that can predict response to HDACi is a high priority for expanding the efficacy of these novel anticancer agents.

References  ….

NEWS AND VIEWS   Blocking HDACs boosts regulatory T cells

Nature Medicine News and Views (01 Nov 2007)

RESEARCH   

Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug

Nature Biotechnology Research (01 Jan 2007)

Comments of reviewer:

 

The complexity of cancer has been known for almost a century, in large part from the seminal work of Otto Warburg in the 1920s using manometry, and following the work of Louis Pasteur 60 years earlier with fungi.

 

The volume of work and our unlocking of mitotic activity, apoptosis, mitochondria, and the cytoskeleton has taken us further into the cell interior, cell function, metabolic regulation, and pathophysiology.  Despite the enormous contributions to our knowledge of genomics, there is a large body of work in pathways of cell function that resides in no small part in activity of protein catalysts and enzymes.

 

The work that has been described covers only cyclic dinucleotides and HDACi’s.  Some of the activities described have relevance to microorganisms as well as cancer.  As we have seen, blocking HDACs boosts the activity of regulatory T-cells. There are many specific functional alterations elucidated above.

 

The first presentation is of an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1. This also involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

 

The second is resident in acetylation status of histones and non-histone proteins is determined by histone deacetylases (HDACs) and histone acetyl-transferases (HATs). HATs add acetyl groups to lysine residues, while HDACs remove the acetyl groups. In general, acetylation of histone promotes a more relaxed chromatin structure, allowing transcriptional activation. HDACs can act as transcription repressors, due to histone deacetylation, and consequently promote chromatin condensation. HDAC inhibitors (HDACi) selectively alter gene transcription, in part, by chromatin remodeling and by changes in the structure of proteins in transcription factor complexes (Gui et al., 2004).  The description focuses on the molecular mechanisms triggered by inhibitors of zinc-dependent HDACs in tumor cells that explain in part: (I) the effects of these compounds in inducing transformed cell death and (II) the relative resistance of normal and certain cancer cells to HDACi induced cell death.

 

HDACs have multiple substrates involved in many biological processes, including proliferation, differentiation, apoptosis and other forms of cell death. Indeed, the fact that HDACs have histone and multiple nonhistone protein substrates suggests these enzymes should be referred to as ‘lysine deacetylases’. HDACi can cause transformed cells to undergo growth arrest, differentiation and/or cell death. Normal cells are relatively resistant to HDACi. HDACi are selective in altering gene expression, which may reflect, in part, the proteins composing the transcription factor complex to which HDACs are recruited. Both altered gene expression and changes in non-histone proteins caused by HDACi-induced acetylation play a role in the antitumor activity of HDACi.

 

 

 

 

 

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Effect of mitochondrial stress on epigenetic modifiers

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Early Mitochondrial Stress Alters Epigenetics, Secures Lifelong Health Benefits

GEN 5/3/2016  http://www.genengnews.com/gen-news-highlights/early-mitochondrial-stress-alters-epigenetics-secures-lifelong-health-benefits/81252685/

A little adversity builds character, or so the saying goes. True or not, the saying does seem an apt description of a developmental phenomenon that shapes gene expression. While it knows nothing of character, the gene expression apparatus appears to respond well to short-term mitochondrial stress that occurs early in development. In fact, transient stress seems to result in lasting benefits. These benefits, which include improved metabolic function and increased longevity, have been observed in both worms and mice, and may even occur—or be made to occur—in humans.

Gene expression is known to be subject to reprogramming by epigenetic modifiers, but such modifiers generally affect metabolism or lifespan, not both. A new set of epigenetic modifiers, however, has been found to trigger changes that do just that—both improve metabolism and extend lifespan.

Scientists based at the University of California, Berkeley, and the École Polytechnique Fédérale de Lausanne (EPFL) have discovered enzymes that are ramped up after mild stress during early development and continue to affect the expression of genes throughout the animal’s life. When the scientists looked at strains of inbred mice that have radically different lifespans, those with the longest lifespans had significantly higher expression of these enzymes than did the short-lived mice.

“Two of the enzymes we discovered are highly, highly correlated with lifespan; it is the biggest genetic correlation that has ever been found for lifespan in mice, and they’re both naturally occurring variants,” said Andrew Dillin, a UC Berkeley professor of molecular and cell biology. “Based on what we see in worms, boosting these enzymes could reprogram your metabolism to create better health, with a possible side effect of altering lifespan.”

Details of the work, which appeared online April 29 in the journal Cell, are presented in a pair of papers. One paper (“Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity”) resulted from an effort led by Dillin and the EPFL’s Johan Auwerx. The other paper (“Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt”) resulted from an effort led by Dillin and his UC Berkeley colleague Barbara Meyer.

According to these papers, mitochondrial stress activates enzymes in the brain that affect DNA folding, exposing a segment of DNA that contains the 1500 genes involved in the work of the mitochondria. A second set of enzymes then tags these genes, affecting their activation for much or all of the lifetime of the animal and causing permanent changes in how the mitochondria generates energy.

The first set of enzymes—methylases, in particular LIN-65—add methyl groups to the DNA, which can silence promoters and thus suppress gene expression. By also opening up the mitochondrial genes, these methylases set the stage for the second set of enzymes—demethylases, in this case jmjd-1.2 and jmjd-3.1—to ramp up transcription of the mitochondrial genes. When the researchers artificially increased production of the demethylases in worms, all the worms lived longer, a result identical to what is observed after mitochondrial stress.

“By changing the epigenetic state, these enzymes are able to switch genes on and off,” Dillin noted. This happens only in the brain of the worm, however, in areas that sense hunger or satiety. “These genes are expressed in neurons that are sensing the nutritional status of the animal, and these signals emanate out to the periphery to change peripheral metabolism,” he continued.

When the scientists profiled enzymes in short- and long-lived mice, they found upregulation of these genes in the brains of long-lived mice, but not in other tissues or in the brains of short-lived mice. “These genes are expressed in the hypothalamus, exactly where, when you eat, the signals are generated that tell you that you are full. And when you are hungry, signals in that region tell you to go and eat,” Dillin explained said. “These genes are all involved in peripheral feedback.”

Among the mitochondrial genes activated by these enzymes are those involved in the body’s response to proteins that unfold, which is a sign of stress. Increased activity of the proteins that refold other proteins is another hallmark of longer life.

These observations suggest that the reversal of aging by epigenetic enzymes could also take place in humans.

“It seems that, while extreme metabolic stress can lead to problems later in life, mild stress early in development says to the body, ‘Whoa, things are a little bit off-kilter here, let’s try to repair this and make it better.’ These epigenetic switches keep this up for the rest of the animal’s life,” Dillin stated.

 

Two Conserved Histone Demethylases Regulate Mitochondrial Stress-Induced Longevity

Carsten Merkwirth6, Virginija Jovaisaite6, Jenni Durieux,…., Reuben J. Shaw, Johan Auwerx, Andrew Dillin

Highlights
  • H3K27 demethylases jmjd-1.2 and jmjd-3.1 are required for ETC-mediated longevity
  • jmjd-1.2 and jmjd-3.1 extend lifespan and are sufficient for UPRmt activation
  • UPRmt is required for increased lifespan due to jmjd-1.2 or jmjd-3.1 overexpression
  • JMJD expression is correlated with UPRmt and murine lifespan in inbred BXD lines

Across eukaryotic species, mild mitochondrial stress can have beneficial effects on the lifespan of organisms. Mitochondrial dysfunction activates an unfolded protein response (UPRmt), a stress signaling mechanism designed to ensure mitochondrial homeostasis. Perturbation of mitochondria during larval development in C. elegans not only delays aging but also maintains UPRmt signaling, suggesting an epigenetic mechanism that modulates both longevity and mitochondrial proteostasis throughout life. We identify the conserved histone lysine demethylases jmjd-1.2/PHF8 and jmjd-3.1/JMJD3 as positive regulators of lifespan in response to mitochondrial dysfunction across species. Reduction of function of the demethylases potently suppresses longevity and UPRmt induction, while gain of function is sufficient to extend lifespan in a UPRmt-dependent manner. A systems genetics approach in the BXD mouse reference population further indicates conserved roles of the mammalian orthologs in longevity and UPRmt signaling. These findings illustrate an evolutionary conserved epigenetic mechanism that determines the rate of aging downstream of mitochondrial perturbations.

Figure thumbnail fx1

 

Mitochondrial Stress Induces Chromatin Reorganization to Promote Longevity and UPRmt
Ye Tian, Gilberto Garcia, Qian Bian, Kristan K. Steffen, Larry Joe, Suzanne Wolff, Barbara J. Meyer, Andrew Dillincorrespondence
http://dx.doi.org/10.1016/j.cell.2016.04.011             Publication stage: In Press Corrected Proof
Highlights
  • LIN-65 accumulates in the nucleus in response to mitochondrial stress
  • Mitochondrial stress-induced chromatin changes depend on MET-2 and LIN-65
  • LIN-65 and DVE-1 exhibit interdependence in nuclear accumulation
  • met-2 and atfs-1 act in parallel to affect mitochondrial stress-induced longevity

Organisms respond to mitochondrial stress through the upregulation of an array of protective genes, often perpetuating an early response to metabolic dysfunction across a lifetime. We find that mitochondrial stress causes widespread changes in chromatin structure through histone H3K9 di-methylation marks traditionally associated with gene silencing. Mitochondrial stress response activation requires the di-methylation of histone H3K9 through the activity of the histone methyltransferase met-2 and the nuclear co-factor lin-65. While globally the chromatin becomes silenced by these marks, remaining portions of the chromatin open up, at which point the binding of canonical stress responsive factors such as DVE-1 occurs. Thus, a metabolic stress response is established and propagated into adulthood of animals through specific epigenetic modifications that allow for selective gene expression and lifespan extension

 Siddharta Mukherjee’s Writing Career Just Got Dealt a Sucker Punch
Author: Theral Timpson

Siddharha Mukherjee won the 2011 Pulitzer Prize in non-fiction for his book, The Emperor of All Maladies.  The book has received widespread acclaim among lay audience, physicians, and scientists alike.  Last year the book was turned into a special PBS series.  But, according to a slew of scientists, we should all be skeptical of his next book scheduled to hit book shelves this month, The Gene, An Intimate History.

Publishing an article on epigenetics in the New Yorker this week–perhaps a selection from his new book–Mukherjee has waltzed into one of the most active scientific debates in all of biology: that of gene regulation, or epigenetics.

Jerry Coyne, the evolutionary biologist known for keeping journalists honest, has published a two part critique of Mukherjee’s New Yorker piece.  The first part–wildly tweeted yesterday–is a list of quotes from Coyne’s colleagues and those who have written in to the New Yorker, including two Nobel prize winners, Wally Gilbert and Sidney Altman, offering some very unfriendly sentences.

Wally Gilbert: “The New Yorker article is so wildly wrong that it defies rational analysis.”

Sidney Altman:  “I am not aware that there is such a thing as an epigenetic code.  It is unfortunate to inflict this article, without proper scientific review, on the audience of the New Yorker.”

The second part is a thorough scientific rebuttal of the Mukherjee piece.  It all serves as a great drama about one of the most contested ideas in biology and also as a cautionary tale to journalists, even experienced writers such as Mukherjee, about the dangers of wading into scientific arguments.  Readers may remember that a few years ago, science writer, David Dobbs, similarly skated into the same topic with his piece, Die, Selfish Gene, Die, and which raised a similar shitstorm, much of it from Coyne.

Mukherjee’s mistake is in giving credence to only one side of a very fierce debate–that the environment causes changes in the genome which can be passed on; another kind of evolution–as though it were settled science.   Either Mukherjee, a physicisan coming off from a successful book and PBS miniseries on cancer, is setting himself up as a scientist, or he has been a truly naive science reporter.   If he got this chapter so wrong, what does it mean about an entire book on the gene?

Coyne quotes one of his colleagues who raised some questions about the New Yorker’s science reporting, one particular question we’ve been asking here at Mendelspod.  How do we know what we know?  Does science now have an edge on any other discipline for being able to create knowledge?

Coyne’s colleague is troubled by science coverage in the New Yorker, and goes so far as to write that the New Yorker has been waging a “war on behalf of cultural critics and literary intellectuals against scientists and technologists.”

From my experience, it’s not quite that tidy.  First of all, the New Yorker is the best writing I read each week.  Period.  Second, I haven’t found their science writing to have the slant claimed in the quote above.  For example, most other mainstream outlets–including the New York Times with the Amy Harmon pieces–have given the anti-GMO crowd an equal say in the mistaken search for a “balance” on whether GMOs are harmful.  (Remember John Stewart’s criticism of Fox News?  That they give a false equivalent between two sides even when there is no equivalent on the other side?)

But the New Yorker has not fallen into this trap on GMOs and most of their pieces on the topic–mainly by Michael Specter–have been decidedly pro science and therefore decided pro GMO.

So what led Mukherjee to play scientist as well as journalist?  There’s no question about whether I enjoy his prose.  His writing beautifully whisks me away so that I don’t feel that I’m really working to understand.  There is a poetic complexity that constantly brings different threads effortlessly together, weaving them into the same light.  At one point he uses the metaphor of a web for the genome, with the epigenome being the stuff that sticks to the web.  He borrows the metaphor from the Hindu notion of “being”, or jaal.

“Genes form the threads of the web; the detritus that adheres to it transforms every web into a singular being.”

There have been a few writers on Twitter defending Mukherjee’s piece.  Tech Review’s Antonio Regalado called Coyne and his colleagues “tedious literalists” who have an “issue with epigenetic poetry.”

At his best, Mukherjee can take us down the sweet alleys of his metaphors and family stories with a new curiosity for the scientific truth.  He can hold a mirror up to scientists, or put the spotlight on their work.   At their worst, Coyne and his scientific colleagues can reek of a fear of language and therefore metaphor.  The always outspoken scientist and author, Richard Dawkins, who made his name by personifying the gene, was quick to personify epigentics in a tweet:   “It’s high time the 15 minutes of underserved fame for “epigenetics” came to an overdue end.”  Dawkins is that rare scientist who has consistently been as comfortable with rhetoric and language as he is with data.

Hats off to Coyne who reminds us that a metaphor–however lovely–does not some science make. If Mukherjee wants to play scientist, let him create and gather data. If it’s the role of science journalist he wants, let him collect all the science he can before he begins to pour it into his poetry.

 

Same but Different  

How epigenetics can blur the line between nature and nurture.

Annals of Science MAY 2, 2016 ISSUE     BY

The author’s mother (right) and her twin are a study in difference and identity. CREDIT: PHOTOGRAPH BY DAYANITA SINGH FOR THE NEW YORKER

October 6, 1942, my mother was born twice in Delhi. Bulu, her identical twin, came first, placid and beautiful. My mother, Tulu, emerged several minutes later, squirming and squalling. The midwife must have known enough about infants to recognize that the beautiful are often the damned: the quiet twin, on the edge of listlessness, was severely undernourished and had to be swaddled in blankets and revived.

The first few days of my aunt’s life were the most tenuous. She could not suckle at the breast, the story runs, and there were no infant bottles to be found in Delhi in the forties, so she was fed through a cotton wick dipped in milk, and then from a cowrie shell shaped like a spoon. When the breast milk began to run dry, at seven months, my mother was quickly weaned so that her sister could have the last remnants.
Tulu and Bulu grew up looking strikingly similar: they had the same freckled skin, almond-shaped face, and high cheekbones, unusual among Bengalis, and a slight downward tilt of the outer edge of the eye, something that Italian painters used to make Madonnas exude a mysterious empathy. They shared an inner language, as so often happens with twins; they had jokes that only the other twin understood. They even smelled the same: when I was four or five and Bulu came to visit us, my mother, in a bait-and-switch trick that amused her endlessly, would send her sister to put me to bed; eventually, searching in the half-light for identity and difference—for the precise map of freckles on her face—I would realize that I had been fooled.

But the differences were striking, too. My mother was boisterous. She had a mercurial temper that rose fast and died suddenly, like a gust of wind in a tunnel. Bulu was physically timid yet intellectually more adventurous. Her mind was more agile, her tongue sharper, her wit more lancing. Tulu was gregarious. She made friends easily. She was impervious to insults. Bulu was reserved, quieter, and more brittle. Tulu liked theatre and dancing. Bulu was a poet, a writer, a dreamer.

….. more

Why are identical twins alike? In the late nineteen-seventies, a team of scientists in Minnesota set out to determine how much these similarities arose from genes, rather than environments—from “nature,” rather than “nurture.” Scouring thousands of adoption records and news clips, the researchers gleaned a rare cohort of fifty-six identical twins who had been separated at birth. Reared in different families and different cities, often in vastly dissimilar circumstances, these twins shared only their genomes. Yet on tests designed to measure personality, attitudes, temperaments, and anxieties, they converged astonishingly. Social and political attitudes were powerfully correlated: liberals clustered with liberals, and orthodoxy was twinned with orthodoxy. The same went for religiosity (or its absence), even for the ability to be transported by an aesthetic experience. Two brothers, separated by geographic and economic continents, might be brought to tears by the same Chopin nocturne, as if responding to some subtle, common chord struck by their genomes.

One pair of twins both suffered crippling migraines, owned dogs that they had named Toy, married women named Linda, and had sons named James Allan (although one spelled the middle name with a single “l”). Another pair—one brought up Jewish, in Trinidad, and the other Catholic, in Nazi Germany, where he joined the Hitler Youth—wore blue shirts with epaulets and four pockets, and shared peculiar obsessive behaviors, such as flushing the toilet before using it. Both had invented fake sneezes to diffuse tense moments. Two sisters—separated long before the development of language—had invented the same word to describe the way they scrunched up their noses: “squidging.” Another pair confessed that they had been haunted by nightmares of being suffocated by various metallic objects—doorknobs, fishhooks, and the like.

The Minnesota twin study raised questions about the depth and pervasiveness of qualities specified by genes: Where in the genome, exactly, might one find the locus of recurrent nightmares or of fake sneezes? Yet it provoked an equally puzzling converse question: Why are identical twins different? Because, you might answer, fate impinges differently on their bodies. One twin falls down the crumbling stairs of her Calcutta house and breaks her ankle; the other scalds her thigh on a tipped cup of coffee in a European station. Each acquires the wounds, calluses, and memories of chance and fate. But how are these changes recorded, so that they persist over the years? We know that the genome can manufacture identity; the trickier question is how it gives rise to difference.

….. more

But what turns those genes on and off, and keeps them turned on or off? Why doesn’t a liver cell wake up one morning and find itself transformed into a neuron? Allis unpacked the problem further: suppose he could find an organism with two distinct sets of genes—an active set and an inactive set—between which it regularly toggled. If he could identify the molecular switches that maintain one state, or toggle between the two states, he might be able to identify the mechanism responsible for cellular memory. “What I really needed, then, was a cell with these properties,” he recalled when we spoke at his office a few weeks ago. “Two sets of genes, turned ‘on’ or ‘off’ by some signal.”

more…

“Histones had been known as part of the inner scaffold for DNA for decades,” Allis went on. “But most biologists thought of these proteins merely as packaging, or stuffing, for genes.” When Allis gave scientific seminars in the early nineties, he recalled, skeptics asked him why he was so obsessed with the packing material, the stuff in between the DNA.  …. A skein of silk tangled into a ball has very different properties from that same skein extended; might the coiling or uncoiling of DNA change the activity of genes?

In 1996, Allis and his research group deepened this theory with a seminal discovery. “We became interested in the process of histone modification,” he said. “What is the signal that changes the structure of the histone so that DNA can be packed into such radically different states? We finally found a protein that makes a specific chemical change in the histone, possibly forcing the DNA coil to open. And when we studied the properties of this protein it became quite clear that it was also changing the activity of genes.” The coils of DNA seemed to open and close in response to histone modifications—inhaling, exhaling, inhaling, like life.

Allis walked me to his lab, a fluorescent-lit space overlooking the East River, divided by wide, polished-stone benches. A mechanical stirrer, whirring in a corner, clinked on the edge of a glass beaker. “Two features of histone modifications are notable,” Allis said. “First, changing histones can change the activity of a gene without affecting the sequence of the DNA.” It is, in short, formally epi-genetic, just as Waddington had imagined. “And, second, the histone modifications are passed from a parent cell to its daughter cells when cells divide. A cell can thus record ‘memory,’ and not just for itself but for all its daughter cells.”

…..

 

 

The New Yorker screws up big time with science: researchers criticize the Mukherjee piece on epigenetics

Jerry Coyne
https://whyevolutionistrue.wordpress.com/2016/05/05/the-new-yorker-screws-up-big-time-with-science-researchers-criticize-the-mukherjee-piece-on-epigenetics/

Abstract: This is a two part-post about a science piece on gene regulation that just appeared in the New Yorker. Today I give quotes from scientists criticizing that piece; tomorrow I’ll present a semi-formal critique of the piece by two experts in the field.

esterday I gave readers an assignment: read the new New Yorkerpiece by Siddhartha Mukherjee about epigenetics. The piece, called “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”) was brought to my attention by two readers, both of whom praised it.  Mukherjee, a physician, is well known for writing the Pulitzer-Prize-winning book (2011) The Emperor of All Maladies: A Biography of Cancer. (I haven’t read it yet, but it’s on my list.)  Mukherjee has a new book that will be published in May: The Gene: An Intimate History. As I haven’t seen it, the New Yorker piece may be an excerpt from this book.

Everyone I know who has read The Emperor of All Maladies gives it high praise. I wish I could say the same for Mukherjee’s New Yorker piece. When I read it at the behest of the two readers, I found his analysis of gene regulation incomplete and superficial. Although I’m not an expert in that area, I knew that there was a lot of evidence that regulatory proteins called “transcription factors”, and not “epigenetic markers” (see discussion of this term tomorrow) or modified histones—the factors emphasized by Mukherjee—played hugely important roles in gene regulation. The speculations at the end of the piece about “Lamarckian evolution” via environmentally induced epigenetic changes in the genome were also unfounded, for we have no evidence for that kind of adaptive evolution. Mukherjee does, however, mention that lack of evidence, though I wish he’d done so more strongly given that environmental modification of DNA bases is constantly touted as an important and neglected factor in evolution.

Unbeknownst to me, there was a bit of a kerfuffle going on in the community of scientists who study gene regulation, with many of them finding serious mistakes and omissions in Mukherjee’s piece.  There appears to have been some back-and-forth emailing among them, and several wrote letters to the New Yorker, urging them to correct the misconceptions, omissions, and scientific errors in “Same but different.” As I understand it, both Mukherjee and the New Yorker simply batted these criticisms away, and, as far as I know, will not publish any corrections.  So today and tomorrow I’ll present the criticisms here, just so they’ll be on the record.

Because Mukherjee writes very well, and because even educated laypeople won’t know the story of gene regulation revealed over the last few decades,  they may not see the big lacunae in his piece. It is, then,  important to set matters straight, for at least we should know what science has told us about how genes are turned on and off. The criticism of Mukherjee’s piece, coming from scientists who really are experts in gene regulation, shows a lack of care on the part of Mukherjee and theNew Yorker: both a superficial and misleading treatment of the state of the science, and a failure of the magazine to properly vet this piece (I have no idea whether they had it “refereed” not just by editors but by scientists not mentioned in the piece).

Let me add one thing about science and the New Yorker. I believe I’ve said this before, but the way the New Yorker treats science is symptomatic of the “two cultures” problem. This is summarized in an email sent me a while back by a colleague, which I quote with permission:

The New Yorker is fine with science that either serves a literary purpose (doctors’ portraits of interesting patients) or a political purpose (environmental writing with its implicit critique of modern technology and capitalism). But the subtext of most of its coverage (there are exceptions) is that scientists are just a self-interested tribe with their own narrative and no claim to finding the truth, and that science must concede the supremacy of literary culture when it comes to anything human, and never try to submit human affairs to quantification or consilience with biology. Because the magazine is undoubtedly sophisticated in its writing and editing they don’t flaunt their postmodernism or their literary-intellectual proprietariness, but once you notice it you can make sense of a lot of their material.

. . . Obviously there are exceptions – Atul Gawande is consistently superb – but as soon as you notice it, their guild war on behalf of cultural critics and literary intellectuals against scientists, technologists, and analytic scholars becomes apparent.

…. more

Researchers criticize the Mukherjee piece on epigenetics: Part 2

Trigger warning: Long science post!

Yesterday I provided a bunch of scientists’ reactions—and these were big names in the field of gene regulation—to Siddhartha Mukherjee’s ill-informed piece in The New Yorker, “Same but different” (subtitle: “How epigenetics can blur the line between nature and nurture”). Today, in part 2, I provide a sentence-by-sentence analysis and reaction by two renowned researchers in that area. We’ll start with a set of definitions (provided by the authors) that we need to understand the debate, and then proceed to the critique.

Let me add one thing to avoid confusion: everything below the line, including the definition (except for my one comment at the end) was written by Ptashne and Greally.

by Mark Ptashne and John Greally

Introduction

Ptashne is The Ludwig Professor of Molecular Biology at the Memorial Sloan Kettering Cancer Center in New York. He wrote A Genetic Switch, now in its third edition, which describes the principles of gene regulation and the workings of a ‘switch’; and, with Alex Gann, Genes and Signals, which extends these principles and ideas to higher organisms and to other cellular processes as well.  John Greally is the Director of the Center for Epigenomics at the Albert Einstein College of Medicine in New York.

 

The New Yorker  (May 2, 2016) published an article entitled “Same But Different” written by Siddhartha Mukherjee.  As readers will have gathered from the letters posted yesterday, there is a concern that the article is misleading, especially for a non-scientific audience. The issue concerns our current understanding of “gene regulation” and how that understanding has been arrived at.

First some definitions/concepts:

Gene regulation refers to the “turning on and off of genes”.  The primary event in turning a gene “on” is to transcribe (copy) it into messenger RNA (mRNA). That mRNA is then decoded, usually, into a specific protein.  Genes are transcribed by the enzyme called RNA polymerase.

Development:  the process in which a fertilized egg (e.g., a human egg) divides many times and eventually forms an organism.  During this process, many of the roughly 23,000 genes of a human are turned “on” or “off” in different combinations, at different times and places in the developing organism. The process produces many different cell types in different organs (e.g. liver and brain), but all retain the original set of genes.

Transcription factors: proteins that bind to specific DNA sequences near specific genes and turn transcription of those genes on and off. A transcriptional ‘activator’, for example, bears two surfaces: one binds a specific sequence in DNA, and the other binds to, and thereby recruits to the gene, protein complexes that include RNA polymerase. It is widely acknowledged that the identity of a cell in the body depends on the array of transcription factors present in the cell, and the cell’s history.  RNA molecules can also recognize specific genomic sequences, and they too sometimes work as regulators.  Neither transcription factors nor these kinds of RNA molecules – the fundamental regulators of gene expression and development – are mentioned in the New Yorker article.

Signals:  these come in many forms (small molecules like estrogen, larger molecules (often proteins such as cytokines) that determine the ability of transcription factors to work.  For example, estrogen binds directly to a transcription factor (the estrogen receptor) and, by changing its shape, permits it to bind DNA and activate transcription.

Memory”:  a dividing cell can (often does) produce daughters that are identical, and that express identical genes as does the mother cell.  This occurs because the transcription factors present in the mother cell are passively transmitted to the daughters as the cell divides, and they go to work in their new contexts as before.  To make two different daughters, the cell must distribute its transcription factors asymmetrically.

Positive Feedback: An activator can maintain its own expression by  positive feedback.  This requires, simply, that a copy of the DNA sequence to which the activator binds is  present  near its own gene. Expression of the activator  then becomes self-perpetuating.  The activator (of which there now are many copies in the cell) activates  other target genes as it maintains its own expression. This kind of ‘memory circuit’, first described  in  bacteria, is found in higher organisms as well.  Positive feedback can explain how a fully differentiated cell (that is, a cell that has reached its developmental endpoint) maintains its identity.

Nucleosomes:  DNA in higher organisms (eukaryotes) is wrapped, like beads on a string, around certain proteins (called histones), to form nucleosomes.  The histones are subject to enzymatic modifications: e.g., acetyl, methyl, phosphate, etc. groups can be added to these structures. In bacteria there are no nucleosomes, and the DNA is more or less ‘naked’.

“Epigenetic modifications: please don’t worry about the word ”epigenetic”; it is misused in any case. What Mukherjee refers to by this term are the histone modifications mentioned above, and a modification to DNA itself: the addition of methyl groups. Keep in mind that the organisms that have taught us the most about development – flies (Drosophila) and worms (C. elegans)—do not have the enzymes required for DNA methylation. That does not mean that DNA methylation cannot do interesting things in humans, for example, but it is obviously not at the heart of gene regulation.

Specificity Development requires the highly specific sequential turning on and off of sets of genes.  Transcription factors and RNA supply this specificity, but   enzymes that impart modifications to histones  cannot: every nucleosome (and hence every gene) appears the same to the enzyme.  Thus such enzymes cannot pick out particular nucleosomes associated with particular genes to modify.  Histone modifications might be imagined to convey ‘memory’ as cells divide – but there are no convincing indications that this happens, nor are there molecular models that might explain why they would have the imputed effects.

Analysis and critique of Mukherjee’s article

The picture we have just sketched has taken the combined efforts of many scientists over 50 years to develop.  So what, then, is the problem with the New Yorker article?

There are two: first, the picture we have just sketched, emphasizing the primary role of transcription factors and RNA, is absent.  Second, that picture is replaced by highly dubious speculations, some of which don’t make sense, and none of which has been shown to work as imagined in the article.

(Quotes from the Mukherjee article are indented and in plain text; they are followed by comments, flush left and in bold, by Ptashne and Greally.)

In 1978, having obtained a Ph.D. in biology at Indiana University, Allis began to tackle a problem that had long troubled geneticists and cell biologists: if all the cells in the body have the same genome, how does one become a nerve cell, say, and another a blood cell, which looks and functions very differently?

The problems referred to were recognized long before 1978.  In fact, these were exactly the problems that the great French scientists François Jacob and Jacques Monod took on in the 1950s-60s.  In a series of brilliant experiments, Jacob and Monod showed that in bacteria, certain genes encode products that regulate (turn on and off) specific other genes.  Those regulatory molecules turned out to be proteins, some of which respond to signals from the environment.  Much of the story of modern biology has been figuring out how these proteins – in bacteria and in higher organisms  – bind to and regulate specific genes.  Of note is that in higher organisms, the regulatory proteins look and act like those in bacteria, despite the fact that eukaryotic DNA is wrapped in nucleosomes  whereas bacterial DNA is not.   We have also learned that certain RNA molecules can play a regulatory role, a phenomenon made possible by the fact that RNA molecules, like regulatory proteins, can recognize specific genomic sequences.

In the nineteen-forties, Conrad Waddington, an English embryologist, had proposed an ingenious answer: cells acquired their identities just as humans do—by letting nurture (environmental signals) modify nature (genes). For that to happen, Waddington concluded, an additional layer of information must exist within a cell—a layer that hovered, ghostlike, above the genome. This layer would carry the “memory” of the cell, recording its past and establishing its future, marking its identity and its destiny but permitting that identity to be changed, if needed. He termed the phenomenon “epigenetics”—“above genetics.”

This description greatly misrepresents the original concept.  Waddington argued that development proceeds not by the loss (or gain) of genes, which would be a “genetic” process, but rather that some genes would be selectively expressed in specific and complex cellular patterns as development proceeds.  He referred to this intersection of embryology (then called “epigenesis”) and genetics as “epigenetic”.We now understand that regulatory proteins work in combinations to turn on and off genes, including their own genes, and that sometimes the regulatory proteins respond to signals sent by other cells.  It should be emphasized that Waddington never proposed any “ghost-like” layer of additional information hovering above the gene.  This is a later misinterpretation of a literal translation of the term epigenetics, with “epi-“ meaning “above/upon” the genetic information encoded in DNA sequence.  Unfortunately, this new and pervasive definition encompasses all of transcriptional regulation and is of no practical value.

…..more

By 2000, Allis and his colleagues around the world had identified a gamut of proteins that could modify histones, and so modulate the activity of genes. Other systems, too, that could scratch different kinds of code on the genome were identified (some of these discoveries predating the identification of histone modifications). One involved the addition of a chemical side chain, called a methyl group, to DNA. The methyl groups hang off the DNA string like Christmas ornaments, and specific proteins add and remove the ornaments, in effect “decorating” the genome. The most heavily methylated parts of the genome tend to be dampened in their activity.

It is true that enzymes that modify histones have been found—lots of them.  A striking problem is that, after all this time, it is not at all clear what the vast majority of these modifications do.  When these enzymatic activities are eliminated by mutation of their active sites (a task substantially easier to accomplish in yeast than in higher organisms) they mostly have little or no effect on transcription.  It is not even clear that histones are the biologically relevant substrates of most of these enzymes.  

 In the ensuing decade, Allis wrote enormous, magisterial papers in which a rich cast of histone-modifying proteins appear and reappear through various roles, mapping out a hatchwork of complexity. . . These protein systems, overlaying information on the genome, interacted with one another, reinforcing or attenuating their signals. Together, they generated the bewildering intricacy necessary for a cell to build a constellation of other cells out of the same genes, and for the cells to add “memories” to their genomes and transmit these memories to their progeny. “There’s an epigenetic code, just like there’s a genetic code,” Allis said. “There are codes to make parts of the genome more active, and codes to make them inactive.”

By ‘epigenetic code’ the author seems to mean specific arrays of nucleosome modifications, imparted over time and cell divisions, marking genes for expression.  This idea has been tested in many experiments and has been found not to hold.

….. and more

 

Larry H. Bernstein, MD, FCAP

I hope that this piece brings greater clarity to the discussion.  I have heard the use of the term “epigenetics” for over a decade.  The term was never so clear.  I think that the New Yorker article was a reasonable article for the intended audience.  It was not intended to clarify debates about a mechanism for epigenetic based changes in evolutionary science.  I think it actually punctures the “classic model” of the cell depending only on double stranded DNA and transcription, which deflates our concept of the living cell.  The concept of epigenetics was never really formulated as far as I have seen, and I have done serious work in enzymology and proteins at a time that we did not have the technology that exists today.  I have considered with the critics that protein folding, protein misfolding, protein interactions with proximity of polar and nonpolar groups, and the regulatory role of microRNAs that are not involved in translation, and the evolving concept of what is “dark (noncoding) DNA” lend credence to the complexity of this discussion.  Even more interesting is the fact that enzymes (and isoforms of enzymes) have a huge role in cellular metabolic differences and in the function of metabolic pathways.  What is less understood is the extremely fast reactions involved in these cellular reactions.  These reactions are in my view critical drivers.  This is brought out by Erwin Schroedinger in the book What is Life? which infers that there can be no mathematical expression of life processes.

 

 

 

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published 

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included.  However, there is a substantial amount of material in the following categories:

  1. The systemic inflammatory response
    https://pharmaceuticalintelligence.com/2014/11/08/introduction-to-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2014/11/09/summary-and-perspectives-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2015/12/19/neutrophil-serine-proteases-in-disease-and-therapeutic-considerations/
    https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/
    https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/
    https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/
    https://pharmaceuticalintelligence.com/2012/07/08/zebrafish-provide-insights-into-causes-and-treatment-of-human-diseases/
    https://pharmaceuticalintelligence.com/2016/01/25/ibd-immunomodulatory-effect-of-retinoic-acid-il-23il-17a-axis-correlates-with-the-nitric-oxide-pathway/
    https://pharmaceuticalintelligence.com/2015/11/29/role-of-inflammation-in-disease/
    https://pharmaceuticalintelligence.com/2013/03/06/can-resolvins-suppress-acute-lung-injury/
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
  2. sepsis
    https://pharmaceuticalintelligence.com/2012/10/20/nitric-oxide-and-sepsis-hemodynamic-collapse-and-the-search-for-therapeutic-options/
  3. vasculitis
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
    https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
    https://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/
  4. neurodegenerative disease
    https://pharmaceuticalintelligence.com/2013/02/27/ustekinumab-new-drug-therapy-for-cognitive-decline-resulting-from-neuroinflammatory-cytokine-signaling-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/01/26/amyloid-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/02/15/alzheimers-disease-tau-art-thou-or-amyloid/
    https://pharmaceuticalintelligence.com/2016/01/26/beyond-tau-and-amyloid/
    https://pharmaceuticalintelligence.com/2015/12/10/remyelination-of-axon-requires-gli1-inhibition/
    https://pharmaceuticalintelligence.com/2015/11/28/neurovascular-pathways-to-neurodegeneration/
    https://pharmaceuticalintelligence.com/2015/11/13/new-alzheimers-protein-aicd-2/
    https://pharmaceuticalintelligence.com/2015/10/31/impairment-of-cognitive-function-and-neurogenesis/
    https://pharmaceuticalintelligence.com/2014/05/06/bwh-researchers-genetic-variations-can-influence-immune-cell-function-risk-factors-for-alzheimers-diseasedm-and-ms-later-in-life/
  5. cancer immunology
    https://pharmaceuticalintelligence.com/2013/04/12/innovations-in-tumor-immunology/
    https://pharmaceuticalintelligence.com/2016/01/09/signaling-of-immune-response-in-colon-cancer/
    https://pharmaceuticalintelligence.com/2015/05/12/vaccines-small-peptides-aptamers-and-immunotherapy-9/
    https://pharmaceuticalintelligence.com/2015/01/30/viruses-vaccines-and-immunotherapy/
    https://pharmaceuticalintelligence.com/2015/10/20/gene-expression-and-adaptive-immune-resistance-mechanisms-in-lymphoma/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
  6. autoimmune diseases: rheumatoid arthritis, colitis, ileitis, …
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2016/01/07/two-new-drugs-for-inflammatory-bowel-syndrome-are-giving-patients-hope/
    https://pharmaceuticalintelligence.com/2015/12/16/contribution-to-inflammatory-bowel-disease-ibd-of-bacterial-overgrowth-in-gut-on-a-chip/
    https://pharmaceuticalintelligence.com/2016/02/13/cytokines-in-ibd/
    https://pharmaceuticalintelligence.com/2016/01/23/autoimmune-inflammtory-bowl-diseases-crohns-disease-ulcerative-colitis-potential-roles-for-modulation-of-interleukins-17-and-23-signaling-for-therapeutics/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2015/03/01/diarrheas-bacterial-and-nonbacterial/
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2014/01/28/biologics-for-autoimmune-diseases-cambridge-healthtech-institutes-inaugural-may-5-6-2014-seaport-world-trade-center-boston-ma/
    https://pharmaceuticalintelligence.com/2015/11/19/rheumatoid-arthritis-update/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2012/09/13/tofacitinib-an-oral-janus-kinase-inhibitor-in-active-ulcerative-colitis/
    https://pharmaceuticalintelligence.com/2013/03/05/approach-to-controlling-pathogenic-inflammation-in-arthritis/
    https://pharmaceuticalintelligence.com/2013/03/05/rheumatoid-arthritis-risk/
    https://pharmaceuticalintelligence.com/2012/07/08/the-mechanism-of-action-of-the-drug-acthar-for-systemic-lupus-erythematosus-sle/
  7. T cells in immunity
    https://pharmaceuticalintelligence.com/2015/09/07/t-cell-mediated-immune-responses-signaling-pathways-activated-by-tlrs/
    https://pharmaceuticalintelligence.com/2015/05/14/allogeneic-stem-cell-transplantation-9-2/
    https://pharmaceuticalintelligence.com/2015/02/19/graft-versus-host-disease/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2014/05/27/immunity-and-host-defense-a-bibliography-of-research-technion/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2013/04/14/immune-regulation-news/

Proteomics, metabolomics and diabetes

https://pharmaceuticalintelligence.com/2015/11/16/reducing-obesity-related-inflammation/

https://pharmaceuticalintelligence.com/2015/10/25/the-relationship-of-stress-hypermetabolism-to-essential-protein-needs/

https://pharmaceuticalintelligence.com/2015/10/24/the-relationship-of-s-amino-acids-to-marasmic-and-kwashiorkor-pem/

https://pharmaceuticalintelligence.com/2015/10/24/the-significant-burden-of-childhood-malnutrition-and-stunting/

https://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

https://pharmaceuticalintelligence.com/2015/03/07/transthyretin-and-the-stressful-condition/

https://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

https://pharmaceuticalintelligence.com/2015/01/31/proteomics/

https://pharmaceuticalintelligence.com/2015/01/17/proteins-an-evolutionary-record-of-diversity-and-adaptation/

https://pharmaceuticalintelligence.com/2014/11/01/summary-of-signaling-and-signaling-pathways/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/10/24/diabetes-mellitus/

https://pharmaceuticalintelligence.com/2014/10/16/metabolomics-summary-and-perspective/

https://pharmaceuticalintelligence.com/2014/10/14/metabolic-reactions-need-just-enough/

https://pharmaceuticalintelligence.com/2014/11/03/introduction-to-protein-synthesis-and-degradation/

https://pharmaceuticalintelligence.com/2015/09/25/proceedings-of-the-nyas/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

https://pharmaceuticalintelligence.com/2013/03/05/irf-1-deficiency-skews-the-differentiation-of-dendritic-cells/

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Neutrophil Serine Proteases in Disease and Therapeutic Considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

SERPINB1 Regulates the activity of the neutrophil proteases elastase, cathepsin G, proteinase-3, chymase,
chymotrypsin, and kallikrein-3. Belongs to the serpin family. Ov-serpin subfamily. Note: This description may
include information from UniProtKB.
Chromosomal Location of Human Ortholog: 6p25
Cellular Component: extracellular space; membrane; cytoplasm
Molecular Function: serine-type endopeptidase inhibitor activity
Reference #:  P30740 (UniProtKB)
Alt. Names/Synonyms: anti-elastase; EI; ELANH2; ILEU; LEI; Leukocyte elastase inhibitor; M/NEI; MNEI; Monocyte/neutrophil elastase inhibitor; Peptidase inhibitor 2; PI-2; PI2; protease inhibitor 2 (anti-elastase), monocyte/neutrophil derived; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; Serpin B1; serpin peptidase inhibitor, clade B (ovalbumin), member 1; SERPINB1
Gene Symbols: SERPINB1
Molecular weight: 42,742 Da
 

SERPIN PEPTIDASE INHIBITOR, CLADE B (OVALBUMIN), MEMBER 1; SERPINB1

Alternative titles; symbols
PROTEASE INHIBITOR 2, MONOCYTE/NEUTROPHIL DERIVED; ELANH2
ELASTASE INHIBITOR, MONOCYTE/NEUTROPHIL; EI
HGNC Approved Gene Symbol: SERPINB1
Cloning and Expression
Monocyte/neutrophil elastase inhibitor (EI) is a protein of approximately 42,000 Mr with serpin-like functional properties.
Remold-O’Donnell et al. (1992) cloned EI cDNA and identified 3 EI mRNA species of 1.5, 1.9, and 2.6 kb in monocyte-like cells
and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI enzymatic activity. The cDNA open reading frame encoded
a 379-amino acid protein. Its sequence established EI as a member of the serpin superfamily. Sequence alignment indicated that
the reactive center P1 residue is cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making
EI a naturally occurring cys-serpin.
 

 

Mapping

In the course of studying 4 closely linked genes encoding members of the ovalbumin family of serine proteinase inhibitors
(Ov-serpins) located on 18q21.3, Schneider et al. (1995) investigated the mapping of elastase inhibitor. They prepared PCR
primer sets of the gene, and by using the NIGMS monochromosomal somatic cell hybrid panel, showed that the EI gene maps
to chromosome 6.

By amplifying DNA of a somatic cell hybrid panel, Evans et al. (1995) unambiguously localized ELANH2 to chromosome 6.
With the use of a panel of radiation and somatic cell hybrids specific for chromosome 6, they refined the localization to
the short arm telomeric of D6S89, F13A (134570), and D6S202 at 6pter-p24.

http://www.phosphosite.org/getImageAction.do?id=27292293

 

 

REFERENCES
Evans, E., Cooley, J., Remold-O’Donnell, E. Characterization and chromosomal localization of ELANH2, the gene encoding human
monocyte/neutrophil elastase inhibitor. Genomics 28: 235-240, 1995. [PubMed: 8530031related citations] [Full Text]
Remold-O’Donnell, E., Chin, J., Alberts, M. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Proc. Nat. Acad. Sci. 89: 5635-5639, 1992. [PubMed: 1376927related citations][Full Text]
Schneider, S. S., Schick, C., Fish, K. E., Miller, E., Pena, J. C., Treter, S. D., Hui, S. M., Silverman, G. A. A serine proteinase inhibitor locus at
18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Nat. Acad. Sci. 92: 3147-3151, 1995.
[PubMed: 7724531,related citations] [Full Text]

 

Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Short name: Serpin_B1

Family relationships

  • Serpin family (IPR000215)
    • Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Description

Leukocyte elastase inhibitor is also known as serpin B1. Serpins (SERine Proteinase INhibitors) belong to MEROPS inhibitor family I4 (clan ID)
[PMID: 14705960].

Serpin B1 regulates the activity of neutrophil serine proteases such as elastase, cathepsin G and proteinase-3 and may play a regulatory role to
limit inflammatory damage due to proteases of cellular origin [PMID: 11747453]. It also functions as a potent intracellular inhibitor of granzyme
H [PMID: 23269243]. In mouse, four different homologues of human serpin B1 have been described [PMID: 12189154].

 

The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection

Dapeng Gong1,2, Charaf Benarafa1,2, Kevan L Hartshorn3 and Eileen Remold-O’Donnell1,2
J Immunol April 2009; 182(Meeting Abstract Supplement) 43.10
http://www.jimmunol.org/cgi/content/meeting_abstract/182/1_MeetingAbstracts/43.10

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFN, TNF, IL-17, IFN, G-CSF) and chemokines (MIP-1, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection.

 

The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosainfection

Charaf Benarafa 1 , 2 Gregory P. Priebe 3 , 4 , and Eileen Remold-O’Donnell 1 , 2
JEM July 30, 2007; 204(8): 1901-1909   http://dx.doi.org:/10.1084/jem.20070494

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance inserpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.

 

Neutrophils are the first and most abundant phagocytes mobilized to clear pathogenic bacteria during acute lung infection. Prominent among their antimicrobial weapons, neutrophils carry high concentrations of a unique set of serine proteases in their granules, including neu trophil elastase (NE), cathepsin G (CG), and proteinase-3. These neutrophil serine proteases (NSPs) are required to kill phagocytosed bacteria and fungi (12). Indeed, neutrophils lacking NE fail to kill phagocytosed pathogens, and mice deficient for NE and/or CG have increased mortality after infection with pulmonary pathogens (34). However, NSPs in the lung airspace can have a detrimental effect in severe inflammatory lung disease through degradation of host defense and matrix proteins (57). Thus, understanding of the mechanisms that regulate NSP actions during lung infections associated with neutrophilia will help identify strategies to balance host defense and prevent infection-induced tissue injury.

 

SERPINB1, also known as monocyte NE inhibitor (8), is an ancestral serpin super-family protein and one of the most efficient inhibitors of NE, CG, and proteinase-3 (910). SERPINB1 is broadly expressed and is at particularly high levels in the cytoplasm of neutrophils (1112). SERPINB1 has been found complexed to neutro phil proteases in lung fluids of cystic fibrosis patients and in a baboon model of bronchopulmonary dysplasia (1314). Although these studies suggest a role for SERPINB1 in regulating NSP activity, it is unclear whether these complexes reflect an important physiological role for SERPINB1 in the lung air space.

RESULTS

To define the physiological importance of SERPINB1 in shaping the outcome of bacterial lung infection, we generated mice deficient for serpinb1 (serpinb1−/−) by targeted mutagenesis in embryonic stem (ES) cells (Fig. 1, A–C). Crossings of heterozygous mice produced WT (+/+), heterozygous (+/−), and KO (−/−) mice for serpinb1 at expected Mendelian ratios (25% +/+, 51% +/−, and 24% −/−; n = 225; Fig. 1 D), indicating no embryonic lethality. Bone marrow neutrophils of serpinb1−/− mice lacked expression of the protein, whereas heterozygous serpinb1+/− mice had reduced levels compared with WT mice (Fig. 1 E). Importantly, levels of the cognate neutrophil proteases NE and CG, measured as antigenic units, were not altered by deletion of serpinb1 (Fig. 1 F). When maintained in a specific pathogen-free environment, serpinb1−/− mice did not differ from WT littermates in growth, litter size, or life span (followed up to 12 mo), and no gross or histopathological defects were observed at necropsy in 8-wk-old mice.

6–8-wk-old animals were intranasally inoculated with the nonmucoid Pseudomonas aeruginosa strain PAO1. Using two infection doses (3 × 106 and 7 × 106 CFU/mouse),serpinb1−/− mice had a significantly lower survival probability and a shorter median survival time compared with WT mice (Fig. 2 A). Further groups of infected mice were used to evaluate bacterial clearance. At 6 h after infection, the bacteria were similarly restricted in mice of the two genotypes, suggesting that the serpinb1−/− mice have a normal initial response to infection. At 24 h, the median bacterial count in the lungs of serpinb1−/− mice was five logs higher than that of the WT mice (P < 0.001), and the infection had spread systemically in serpinb1−/− mice but not in WT mice, as shown by high median CFU counts in the spleen (Fig. 2 B). Histological examination at 24 h after infection revealed abundant neutrophil infiltration in the lungs of both WT and serpinb1−/− mice, and consistent with the bacteriological findings, numerous foci of bacterial colonies and large areas of alveolar exudates were found in serpinb1−/− mice only (Fig. 2 C). When challenged with the mucoid P. aeruginosa clinical strain PA M57-15 isolated from a cystic fibrosis patient, WT mice cleared >99.9% of the inoculum within 24 h, whereas serpinb1-deficient mice failed to clear the infection (Fig. 2 D). Thus, the NSP inhibitor serpinb1 is essential for maximal protection against pneumonia induced by mucoid and nonmucoid strains of P. aeruginosa.

Figure 2.

Serpinb1−/− mice fail to clear P. aeruginosalung infection. (A) Kaplan-Meier survival curves of WT (+/+) and serpinb1-deficient (−/−) mice intranasally inoculated with nonmucoid P. aeruginosa strain PAO1. Increased mortality of serpinb1−/− mice was statistically significant (P = 0.03 at 3 × 106CFU/mouse; P < 0.0001 at 7 × 106CFU/mouse). (B) CFUs per milligram of lung (left) and splenic (right) tissue determined 6 and 24 h after inoculation with 3 × 106 CFUP. aeruginosa PAO1 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Each symbol represents a value for an individual mouse. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test. Data below the limit of detection (dotted line) are plotted as 0.5 CFU × dilution factor. (C) Lung sections stained with hematoxylin and eosin show bacterial colonies (arrowheads) and alveolar exudate in lungs of serpinb1−/− mice 24 h after infection with P. aeruginosa PAO1. Bars, 50 μm. (D) Total CFUs in the lung and spleen 24 h after inoculation with 2 × 108 CFU of the mucoid P. aeruginosa strain PA M57-15 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test.

To verify specificity of the gene deletion, we tested whether delivering rSERPINB1 would correct the defective phenotype. Indeed, intranasal instillation of rSERPINB1 to serpinb1−/− mice at the time of inoculation significantly improved clearance of P. aeruginosa PAO1 from the lungs assessed at 24 h and reduced bacteremia compared with infectedserpinb1−/− mice that received PBS instead of the recombinant protein (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20070494/DC1). We have previously demonstrated that rSERPINB1 has no effect on the growth of P. aeruginosa in vitro (15) and does not induce bacterial aggrega tion (16). Also, rSERPINB1 mixed with PAO1 had no effect on adherence of the bacteria to human bronchial epithelial and corneal epithelial cell lines (unpublished data). Therefore, the improved bacterial clearance in treated serpinb1−/− mice is not related to a direct antibacterial role for rSERPINB1 but rather to reducing injury induced by excess neutrophil proteases. In addition, previous in vivo studies in WT rats showed that rSERPINB1 can protect against elastase-induced lung injury (17) and accelerate bacterial clearance two- to threefold in the Pseudomonas agar bead model (15).

Evidence of excess NSP action was examined in the lungs of infected serpinb1−/− mice by measuring surfactant protein–D (SP-D). SP-D, a multimeric collagenous C-type lectin produced by alveolar epithelial cells, is highly relevant as a host defense molecule, because it functions as an opsonin in microbial clearance (18) and acts on alveolar macrophages to regulate pro- and antiinflammatory cytokine production (19). SP-D is also relevant as an NSP target because it is degraded in vitro by trace levels of each of the NSPs (1620). SP-D levels in lung homogenates of WT and serpinb1−/− mice were similar 6 h after P. aeruginosa infection. At 24 h, SP-D levels were reduced in the lungs ofserpinb1−/− mice compared with WT mice, as indicated by immunoblots. A lower molecular mass band indicative of proteolytic degradation is also apparent (Fig. 3 A). Densitometry analysis of the 43-kD SP-D band relative to β-actin indicated that the reduction of SP-D level was statistically significant (+/+, 45 ± 6 [n = 8]; −/−, 10 ± 2 [n = 8]; P < 0.0001 according to the Student’s t test). Furthermore, rSERPINB1 treatment ofP. aeruginosa–infected serpinb1−/− mice partly prevented the degradation of SP-D in lung homogenates compared with nontreated mice (Fig. S1 B). As a further test of the impact of serpinb1 deletion on NSP activity, isolated neutrophils of serpinb1−/− mice were treated with LPS and FMLP and tested for their ability to cleave recombinant rat SP-D (rrSP-D) in vitro. The extent of rrSP-D cleavage by serpinb1−/− neutrophils was fourfold greater than by WT neutrophils, as determined by densitometry. The cleavage was specific for NSPs because it was abrogated by rSERPINB1 and diisopropyl fluorophosphate (Fig. 3 B). Collectively, these findings indicate a direct role for serpinb1 in regulating NSP activity released by neutrophils and in preserving SP-D, an important-host defense molecule.

Efficient clearance of P. aeruginosa infection requires an early cytokine and chemokine response coordinated by both resident alveolar macrophages and lung parenchymal cells (2122). The IL-8 homologue keratinocyte-derived chemokine (KC) and the cytokines TNF-α, IL-1β, and G-CSF were measured in cell-free bronchoalveolar (BAL) samples. Although the tested cytokines were undetectable in sham-infected mice of both genotypes (unpublished data), comparable induc tion of these cytokines was observed in BAL of WT and serpinb1−/− mice at 6 h after infection, demonstrating that there is no early defect in cytokine production in serpinb1−/− mice. At 24 h, levels of TNF-α, KC, and IL-1β were sustained or increased in serpinb1−/− mice and significantly higher than cytokine levels in WT mice. G-CSF levels at 24 h were elevated to a similar extent in BAL of WT and KO mice (Fig. 3 C). However, G-CSF levels were significantly higher in the serum of serpinb1−/− mice (WT, 336 ± 80 ng/ml; KO, 601 ± 13 ng/ml; n = 6 of each genotype; P < 0.01). In addition, serpinb1−/− mice that were treated at the time of infection with rSERPINB1 had cytokine levels in 24-h lung homogenates that were indistinguishable from those of infected WT mice (Fig. S1 C). The increased cytokine production in the lungs of infected serpinb1−/− mice may be caused by failed bacterial clearance but also by excess NSPs, which directly induce cytokine and neutrophil chemokine production in pulmonary parenchymal cells and alveolar macrophages (2324).

Neutrophil recruitment to the lungs was next examined as a pivotal event of the response to P. aeruginosa infection (25). Lung homogenates were assayed for the neutrophil-specific enzyme myeloperoxidase (MPO) to quantify marginating, interstitial, and alveolar neutrophils. Neutrophils in BAL fluid were directly counted as a measure of neutrophil accumulation in the alveolar and airway lumen. MPO in lung homo genates was undetectable in uninfected mice and was comparably increased in mice of both genotypes at 6 h, suggesting normal early serpinb1−/− neutrophil margination and migration into the interstitium. However, by 24 h after infection, MPO levels in lung homogenates remained high in WT mice but were significantly decreased in serpinb1−/− mice (Fig. 4 A). Importantly, the content of MPO per cell was the same for isolated neutrophils of WT andserpinb1−/− mice (+/+, 369 ± 33 mU/106 cells; −/−, 396 ± 27 mU/106 cells). The numbers of neutrophils in BAL were negligible in uninfected mice and were similarly increased in WT and serpinb1−/− mice at 6 h after infection. Neutrophil counts in BAL further increased at 24 h, but the mean BAL neutrophil numbers were significantly lower in serpinb1−/− mice compared with WT mice (Fig. 4 B). The evidence from the 6-h quantitation of MPO in homogenates and neutrophils in BAL strongly suggests that neutrophil recruitment is not defective in infected serpinb1−/− mice. Moreover, the high levels of cytokines and neutrophil chemoattractant KC in serpinb1−/− mice at 24 h (Fig. 3 C) also suggest that, potentially, more neutrophils should be recruited. Therefore, to examine neutrophil recruitment in serpinb1−/− mice, we used a noninfectious model in which neutrophils are mobilized to migrate to the lung after intranasal delivery of P. aeruginosa LPS. MPO levels in lung homogenate and neutrophil numbers in BAL were not statistically different in WT and serpinb1−/− mice 24 h after LPS instillation (Fig. 4, C and D). Furthermore, the number of circulating blood neutrophils and recruited peritoneal neutrophils after injection of sterile irritants glycogen and thioglycollate did not differ in WT and serpinb1−/− mice (unpublished data). Alveolar macrophage numbers were similar in uninfected mice of both genotypes (∼5 × 105 cells/mouse) and did not substantially change upon infection. Collectively, these findings show that neutrophil recruitment to the lungs in response to P. aeruginosa infection is not defective in serpinb1−/− mice, and therefore, the recovery of lower numbers of serpinb1−/− neutrophils at 24 h after infection suggests their decreased survival.

To examine the putative increased death of serpinb1−/− neutrophils in the lungs after P. aeruginosa infection, lung sections were analyzed by immunohistochemistry. Caspase-3–positive leukocytes were more relevant in the alveolar space of serpinb1−/− mice compared with WT mice at 24 h after infection, suggesting increased neutrophil apoptosis (Fig. 5 A). The positive cells were counted in 50 high power fields (hpf’s), and mean numbers of caspase-3–stained cells were increased in the lungs of serpinb1/− mice (1.8 ± 0.2 cells/hpf) compared with WT mice (0.4 ± 0.1 cells/hpf; P < 0.0001). To characterize neutrophils in the alveoli and airways, neutrophils in BAL were identified in flow cytometry by forward scatter (FSC) and side scatter and were stained with annexin V (AnV) and propidium iodide (PI). At 24 h after infection, the proportion of late apoptotic/necrotic neutrophils (AnV+PI+) was increased at the expense of viable neutrophils (AnVPI) in the BAL of serpinb1−/− mice compared with WT mice (Fig. 5 B). Neutrophil fragments in BAL were also identified in flow cytometry by low FSC (FSClow) within the neutrophil population defined by the neutrophil marker Gr-1. The number of neutrophil fragments (FSClow, Gr-1+) relative to intact neutrophils was increased two- to threefold at 24 h after infection for serpinb1−/− compared with WT mice (Fig. 5 C). Moreover, free MPO in BAL supernatants was increased in serpinb1−/− mice compared with WT mice at 24 h after infection, indicating increased PMN lysis or degranulation (Fig. 5 D).

Finally, we questioned whether the enhanced death of serpinb1−/− pulmonary neutrophils was a primary effect of gene deletion or a secondary effect caused by, for example, bacteria or components of inflammation. To address this, neutrophils were collected using the noninfectious LPS recruitment model and were cultured in vitro to allow for spontaneous cell death. After 24 h, the percentages of apoptotic and necrotic neutrophils evaluated by microscopy were increased in serpinb1−/− neutrophils compared with WT neutrophils (Fig. 6, A–C). A similar increase in apoptotic cells was observed using AnV/PI staining and measurements of hypodiploid DNA (unpublished data). Moreover, live cell numbers from serpinb1−/− mice remaining in culture after 24 h were significantly decreased compared with WT mice (Fig. 6 D). The in vitro findings indicate that enhanced death of pulmonary neutrophils of infected serpinb1−/− mice is at least in part a cell-autonomous defect likely mediated by unchecked NSP actions.

 

In this paper, we have demonstrated that serpinb1, an intracellular serpin family member, regulates the innate immune response and protects the host during lung bacterial infection. Serpinb1 is among the most potent inhibitors of NSPs and is carried at high levels within neutrophils. Serpinb1-deficient mice fail to clear P. aeruginosa PAO1 lung infection and succumb from systemic bacterial spreading. The defective immune function in serpinb1−/− mice stems at least in part from an increased rate of neutrophil necrosis, reducing the number of phagocytes and leading to increased NSP activity in the lungs with proteolysis of SP-D. In addition, serpinb1-deficient mice also have impaired clearance of the mucoid clinical strain PA M57-15. Interestingly, mucoid strains of P. aeruginosa are cleared with a very high efficiency from the lungs of WT and cystic fibrosis transmembrane conductance regulator–deficient mice (26). The phenotype of serpinb1−/− mice reproduces major pathologic features of human pulmonary diseases characterized by excessive inflammation, massive neutrophil recruitment to the air space, and destruction of cellular and molecular protective mechanisms. Importantly, serpinb1 deficiency may be helpful as an alternative or additional model of the inflammatory lung pathology of cystic fibrosis.

The present study documents a key protective role for serpinb1 in regulating NSP actions in the lung. This role has previously been attributed to the NSP inhibitors α1-antitrypsin and secretory leukocyte protease inhibitor, which are found in the airway and alveolar lining fluid (2728). However, patients with α1-antitrypsin deficiency do not present with pulmonary infection secondary to innate immune defects despite increased NSP activity that leads to reduced lung elasticity and emphysema. Moreover, there is so far no evidence that deficiency in secretory leukocyte protease inhibitor results in failure to clear pulmonary infection. Because synthesis and storage of NSPs in granules is an event that exclusively takes place in bone marrow promyelocytes (29), the regulation of NSPs in the lung relies entirely on NSP inhibitors. Thus, the extent of the innate immune defect inserpinb1−/− mice and the normalization of bacterial clearance with topical rSERPINB1 treatment indicate that serpinb1 is required to regulate NSP activity in the airway fluids and that, during acute lung infection associated with high neutrophilic recruitment, there is insufficient compensation by other NSP inhibitors. The devastating effects of NSPs when released in the lungs by degranulating and necrotic neutrophils are well documented in human pulmonary diseases (5630). Therefore, our findings clearly establish a physiological and nonredundant role for serpinb1 in regulating NSPs during pulmonary infection.

NSPs also cleave molecules involved in apoptotic cell clearance, including the surfactant protein SP-D and the phosphatidylserine receptor on macrophages (3132), thereby tipping the balance further toward a detrimental outcome. The increased numbers of leukocytes with active caspase-3 in the alveolar space of P. aeruginosa–infectedserpinb1−/− mice suggest that the removal of apoptotic cells may be inadequate during infection. SP-D has been shown to stimulate phagocytosis of P. aeruginosa by alveolar macrophages in vitro (33), and SP-D–deficient mice were found to have defective early (6-h) clearance of P. aeruginosa from the lung (34). Although the destruction of SP-D alone may not entirely account for the defective phenotype of serpinb1−/− mice, loss of SP-D likely diminishes bacterial clearance and removal of apop totic neutrophils.

Given that NSPs also mediate bacterial killing, why would NSP excess lead to a failed bacterial clearance? In the NE KO mice, the decreased killing activity of neutrophils is a direct consequence of the loss of the bactericidal activity of NE. The absence of an early bacterial clearance defect at 6 h after infection in serpinb1−/− mice suggests that there is initially normal bacterial killing. The current understanding is that the compartmentalization of the NSPs is crucial to the outcome of their actions: on the one hand, NSPs are protective when killing microbes within phagosomes, and on the other hand, extracellular NSPs destroy innate immune defense molecules such as lung collectins, immunoglobulins, and complement receptors. We have shown that the regulation of NSP activity is essential and that cytoplasmic serpinb1 provides this crucial shield. Neutrophils undergoing cell death gradually transition from apoptosis, characterized by a nonpermeable plasma membrane, to necrosis and lysis, where cellular and granule contents, including NSPs, are released. The increased pace of serpinb1−/− neutrophil cell death strongly suggests that unopposed NSPs may precipitate neutrophil demise and, therefore, reduce the neutrophil numbers leading to a late-onset innate immune defect. High levels of G-CSF, a prosurvival cytokine for neutrophils, also indicate that increased cell death is likely independent or downstream of G-CSF.

In conclusion, serpinb1 deficiency unleashes unbridled proteolytic activity during inflammation and thereby disables two critical components of the host response to bacterial infection, the neutrophil and the collectin SP-D. The phenotype of the infectedserpinb1-deficient mouse, characterized by a normal early antibacterial response that degenerates over time, highlights the delicate balance of protease–antiprotease systems that protect the host against its own defenses as well as invading microbes during infection-induced inflammation.

 

 

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

K Kessenbrock,1 LFröhlich,2 M Sixt,3 …., A Belaaouaj,5 J Ring,6,7 M Ollert,6 R Fässler,3 and DE. Jenne1
J Clin Invest. 2008 Jul 1; 118(7): 2438–2447.   http://dx.doi.org:/10.1172/JCI34694

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

 

Neutrophils belong to the body’s first line of cellular defense and respond quickly to tissue injury and invading microorganisms (1). In a variety of human diseases, like autoimmune disorders, infections, or hypersensitivity reactions, the underlying pathogenic mechanism is the formation of antigen-antibody complexes, so-called immune complexes (ICs), which trigger an inflammatory response by inducing the infiltration of neutrophils (2). The subsequent stimulation of neutrophils by C3b-opsonized ICs results in the generation of ROS and the release of intracellularly stored proteases leading to tissue damage and inflammation (3). It is therefore important to identify the mechanisms that control the activation of infiltrating neutrophils.

Neutrophils abundantly express a unique set of neutrophil serine proteases (NSPs), namely cathepsin G (CG), proteinase 3 (PR3; encoded by Prtn3), and neutrophil elastase (NE; encoded by Ela2), which are stored in the cytoplasmic, azurophilic granules. PR3 and NE are closely related enzymes, with overlapping and potentially redundant substrate specificities different from those of CG. All 3 NSPs are implicated in antimicrobial defense by degrading engulfed microorganisms inside the phagolysosomes of neutrophils (48). Among many other functions ascribed to these enzymes, PR3 and NE were also suggested to play a fundamental role in granulocyte development in the bone marrow (911).

While the vast majority of the enzymes is stored intracellularly, minor quantities of PR3 and NE are externalized early during neutrophil activation and remain bound to the cell surface, where they are protected against protease inhibitors (1213). These membrane presented proteases were suggested to act as path clearers for neutrophil migration by degrading components of the extracellular matrix (14). This notion has been addressed in a number of studies, which yielded conflicting results (1517). Thus, the role of PR3 and NE in leukocyte extravasation and interstitial migration still remains controversial.

Emerging data suggest that externalized NSPs can contribute to inflammatory processes in a more complex way than by simple proteolytic tissue degradation (18). For instance, recent observations using mice double-deficient for CG and NE indicate that pericellular CG enhances IC-mediated neutrophil activation and inflammation by modulating integrin clustering on the neutrophil cell surface (1920). Because to our knowledge no Prtn3–/– mice have previously been generated, the role of this NSP in inflammatory processes has not been deciphered. Moreover, NE-dependent functions that can be compensated by PR3 in Ela2–/–animals are still elusive.

One mechanism by which NSPs could upregulate the inflammatory response has recently been proposed. The ubiquitously expressed progranulin (PGRN) is a growth factor implicated in tissue regeneration, tumorigenesis, and inflammation (2123). PGRN was previously shown to directly inhibit adhesion-dependent neutrophil activation by suppressing the production of ROS and the release of neutrophil proteases in vitro (23). This antiinflammatory activity was degraded by NE-mediated proteolysis of PGRN to granulin (GRN) peptides (23). In contrast, GRN peptides may enhance inflammation (23) and have been detected in neutrophil-rich peritoneal exudates (24). In short, recent studies proposed PGRN as a regulator of the innate immune response, but the factors that control PGRN function are still poorly defined and its relevance to inflammation needs to be elucidated in vivo.

In the present study, we generated double-deficient Prtn3–/–Ela2–/– mice to investigate the role of these highly similar serine proteases in noninfectious neutrophilic inflammation. We established that PR3 and NE are required for acute inflammation in response to subcutaneous IC formation. The proteases were found to be directly involved in early neutrophil activation events, because isolated Prtn3–/–Ela2–/– neutrophils were poorly activated by ICs in vitro. These defects in Prtn3–/–Ela2–/– mice were accompanied by accumulation of PGRN. We demonstrated that PGRN represents a potent inflammation-suppressing factor that is cleaved by both PR3 and NE. Our data delineate what we believe to be a previously unknown proinflammatory role for PR3 and NE, which is accomplished via the local inactivation of antiinflammatory PGRN.

 

Generation of Prtn3–/–Ela2–/– mice.

To analyze the role of PR3 and NE in neutrophilic inflammation, we generated a Prtn3–/–Ela2–/– mouse line by targeted gene disruption in embryonic stem cells (see Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34694DS1). Positive recombination of the Prtn3/Ela2locus was proven by Southern blotting of embryonic stem cell clones (Figure ​(Figure1A).1A). Prtn3–/–Ela2–/– mice showed no expression of mRNA for PR3 and NE in bone marrow cells, as assessed by RT-PCR (Figure ​(Figure1B).1B). The successful elimination of PR3 and NE was confirmed at the level of proteolytic activity in neutrophil lysates using a PR3/NE-specific chromogenic substrate (Supplemental Figure 3) as well as by casein zymography (Figure ​(Figure1C).1C). The substantially reduced casein degradation by heterozygous neutrophils indicates gene-dosage dependence of PR3/NE activities. Furthermore, PR3 and NE deficiency was proven by Western blotting using cell lysates from bone marrow–derived neutrophils, while other enzymes stored in azurophilic granula, such as CG and myeloperoxidase (MPO), were normally detected (Figure ​(Figure1D).1D). Crossing of heterozygous Prtn3+/–Ela2+/– mice resulted in regular offspring of WT, heterozygous, and homozygous genotype according to the Mendelian ratio. Despite the absence of 2 abundant serine proteases, and in contrast to expectations based on previous reports (911), we found unchanged neutrophil morphology (Figure ​(Figure1E)1E) and regular neutrophil populations in the peripheral blood of the mutant mice, the latter as assessed via flow cytometry to determine the differentiation markers CD11b and Gr-1 (Figure ​(Figure1F)1F) (2526). Moreover, Prtn3–/–Ela2–/– mice demonstrated normal percentages of the leukocyte subpopulations in the peripheral blood, as determined by the Diff-Quick staining protocol and by hemocytometric counting (Supplemental Figure 2, A and B). Hence, the proteases are not crucially involved in granulopoiesis, and ablating PR3 and NE in the germ line represents a valid approach to assess their biological significance in vivo.

 

Figure 1

Generation and characterization of Prtn3–/–Ela2–/– mice.

PR3 and NE are dispensable for neutrophil extravasation and interstitial migration.

To examine neutrophil infiltration into the perivascular tissue, we applied phorbol esters (croton oil) to the mouse ears. At 4 h after stimulation, we assessed the neutrophil distribution in relation to the extravascular basement membrane (EBM) by immunofluorescence microscopy of fixed whole-mount specimens (Figure ​(Figure2A).2A). We found that Prtn3–/–Ela2–/– neutrophils transmigrated into the interstitium without retention at the EBM (Figure ​(Figure2B),2B), resulting in quantitatively normal and widespread neutrophil influx compared with WT mice (Figure ​(Figure2C).2C). Moreover, we analyzed chemotactic migration of isolated neutrophils through a 3-dimensional collagen meshwork in vitro (Supplemental Video 1) and found unhampered chemotaxis toward a C5a gradient, based on the directionality (Figure ​(Figure2D)2D) and velocity (Figure ​(Figure2E)2E) of Prtn3–/–Ela2–/–neutrophils. These findings led us to conclude that PR3 and NE are not principally required for neutrophil extravasation or interstitial migration.

 

Figure 2

PR3 and NE are not principally required for neutrophil extravasation and interstitial migration.

Reduced inflammatory response to ICs in Prtn3–/–Ela2–/– mice.

The formation of ICs represents an important trigger of neutrophil-dependent inflammation in many human diseases (2). To determine the role of PR3 and NE in this context, we induced a classic model of subcutaneous IC-mediated inflammation, namely the reverse passive Arthus reaction (RPA) (27). At 4 h after RPA induction, we assessed the cellular inflammatory infiltrates by histology using H&E-stained skin sections (Figure ​(Figure3A).3A). Neutrophils, which were additionally identified by Gr-1 immunohistochemistry, made up the vast majority of all cellular infiltrates (Figure ​(Figure3A).3A). We found that neutrophil infiltration to the sites of IC formation was severely diminished in Prtn3–/–Ela2–/– mice. Indeed, histological quantification revealed significantly reduced neutrophil influx in Prtn3–/–Ela2–/– mice compared with WT mice, while Ela2–/– mice showed marginally reduced neutrophil counts (Figure ​(Figure3B).3B). These results indicate that PR3 and NE fulfill an important proinflammatory function during IC-mediated inflammation.

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Figure 3

Impaired inflammatory response to locally formed ICs inPrtn3–/–Ela2–/– mice.

(A) Representative photomicrographs of inflamed skin sections 4 h after IC formation. Neutrophils were identified morphologically (polymorphic nucleus) in H&E stainings and by Gr-1 staining (red). The cellular infiltrates were located to the adipose tissue next to the panniculus carnosus muscle (asterisks) and were primarily composed of neutrophil granulocytes. Scale bars: 200 μm. (B) Neutrophil infiltrates in lesions from Prtn3–/–Ela2–/– mice were significantly diminished compared with Ela2–/– mice and WT mice. Neutrophil influx in Ela2–/–mice was slightly, but not significantly, diminished compared with WT mice. Results are mean ± SEM infiltrated neutrophils per HPF. *P < 0.05.

PR3 and NE enhance neutrophil activation by ICs in vitro.

PR3 and NE enhance neutrophil activation by ICs in vitro.

Because PR3 and NE were required for the inflammatory response to IC (Figure ​(Figure3),3), but not to phorbol esters (Figure ​(Figure2),2), we considered the enzymes as enhancers of the neutrophil response to IC. We therefore assessed the oxidative burst using dihydrorhodamine as a readout for cellular activation of isolated, TNF-α–primed neutrophils in the presence of ICs in vitro. While both WT and Prtn3–/–Ela2–/– neutrophils showed a similar, approximately 20-min lag phase before the oxidative burst commenced, the ROS production over time was markedly reduced, by 30%–40%, in the absence of PR3 and NE (Figure ​(Figure4A).4A). In contrast, oxidative burst triggered by 25 nM PMA was not hindered in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure4B),4B), which indicated no general defect in producing ROS. We also performed a titration series ranging from 0.1 to 50 nM PMA and found no reduction in oxidative burst activity in Prtn3–/–Ela2–/– neutrophils at any PMA concentration used (Supplemental Figure 4). These data are consistent with our in vivo experiments showing that neutrophil influx to ICs was impaired (Figure ​(Figure3),3), whereas the inflammatory response to phorbol esters was normal (Figure ​(Figure2,2, A–C), in Prtn3–/–Ela2–/– mice. To compare neutrophil priming in WT and Prtn3–/–Ela2–/–neutrophils, we analyzed cell surface expression of CD11b after 30 min of incubation at various concentrations of TNF-α and found no difference (Supplemental Figure 5). Moreover, we observed normal neutrophil adhesion to IC-coated surfaces (Supplemental Figure 6A) and unaltered phagocytosis of opsonized, fluorescently labeled E. coli bacteria (Supplemental Figure 6, B and C) in the absence of both proteases. We therefore hypothesized that PR3 and NE enhance early events of adhesion-dependent neutrophil activation after TNF-α priming and binding of ICs. It is important to note that Ela2–/– neutrophils were previously shown to react normally in the same setup (20). Regarding the highly similar cleavage specificities of both proteases, we suggested that PR3 and NE complemented each other during the process of neutrophil activation and inflammation.

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Figure 4

Impaired oxidative burst and PGRN degradation by IC-activatedPrtn3–/–Ela2–/– neutrophils.

Oxidative burst as the readout for neutrophil activation by ICs was measured over time. (A) While no difference was observed during the initial 20-min lag phase of the oxidative burst, Prtn3–/–Ela2–/– neutrophils exhibited diminished ROS production over time compared with WT neutrophils. (B) Bypassing receptor-mediated activation using 25 nM PMA restored the diminished oxidative burst of Prtn3–/–Ela2–/–neutrophils. Results are presented as normalized fluorescence in AU (relative to maximum fluorescence produced by WT cells). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate. (C) Isolated mouse neutrophils were activated by ICs in vitro and tested for PGRN degradation by IB. In the cellular fraction, the PGRN (~80 kDa) signal was markedly increased in Prtn3–/–Ela2–/–cells compared with WT and Ela2–/– neutrophils. Intact PGRN was present in the supernatant (SN) of IC-activated Prtn3–/–Ela2–/–neutrophils only, not of WT or Ela2–/– cells. (D and E) Exogenous administration of 100 nM PGRN significantly reduced ROS production of neutrophils activated by ICs (D), but not when activated by PMA (E). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate.

Antiinflammatory PGRN is degraded by PR3 and NE during IC-mediated neutrophil activation.

PGRN inhibits neutrophil activation by ICs in vitro.

Both PR3 and NE process PGRN in vitro.

Figure 5

PR3 and NE are major PGRN processing enzymes of neutrophils.

PGRN inhibits IC-mediated inflammation in vivo.

Figure 6

PGRN is a potent inhibitor of IC-stimulated inflammation in vivo.

PR3 and NE cleave PGRN during inflammation in vivo.

Finally, we aimed to demonstrate defective PGRN degradation in Prtn3–/–Ela2–/– mice during neutrophilic inflammation in vivo. For practical reasons, we harvested infiltrated neutrophils from the inflamed peritoneum 4 h after casein injection and subjected the lysates of these cells to anti-PGRN Western blot. Intact, inhibitory PGRN was detected in Prtn3–/–Ela2–/– neutrophils, but not in WT cells (Figure ​(Figure6D).6D). These data prove that neutrophilic inflammation is accompanied by proteolytic removal of antiinflammatory PGRN and that the process of PGRN degradation is essentially impaired in vivo in the absence of PR3 and NE.

 

Chronic inflammatory and autoimmune diseases are often perpetuated by continuous neutrophil infiltration and activation. According to the current view, the role of NSPs in these diseases is mainly associated with proteolytic tissue degradation after their release from activated or dying neutrophils. However, recent observations suggest that NSPs such as CG may contribute to noninfectious diseases in a more complex manner, namely as specific regulators of inflammation (18). Here, we demonstrate that PR3 and NE cooperatively fulfilled an important proinflammatory role during neutrophilic inflammation. PR3 and NE directly enhanced neutrophil activation by degrading oxidative burst–suppressing PGRN. These findings support the use of specific serine protease inhibitors as antiinflammatory agents.

Much attention has been paid to the degradation of extracellular matrix components by NSPs. We therefore expected that ablation of both PR3 and NE would cause impaired neutrophil extravasation and interstitial migration. Surprisingly, we found that the proteases were principally dispensable for these processes:Prtn3–/–Ela2–/– neutrophils migrated normally through a dense, 3-dimensional collagen matrix in vitro and demonstrated regular extravasation in vivo when phorbol esters were applied (Figure ​(Figure2).2). This finding is in agreement with recent reports that neutrophils preferentially and readily cross the EBM through regions of low matrix density in the absence of NE (28).

Conversely, we observed that PR3 and NE were required for the inflammatory response to locally formed ICs (Figure ​(Figure3).3). Even isolated Prtn3–/–Ela2–/– neutrophils were challenged in performing oxidative burst after IC stimulation in vitro (Figure ​(Figure4A),4A), showing that the proteases directly enhanced the activation of neutrophils also in the absence of extracellular matrix. However, when receptor-mediated signal transduction was bypassed by means of PMA, neutrophils from Prtn3–/–Ela2–/– mice performed normal oxidative burst (Figure ​(Figure4B),4B), indicating that the function of the phagocyte oxidase (phox) complex was not altered in the absence of PR3 and NE. These findings substantiate what we believe to be a novel paradigm: that all 3 serine proteases of azurophilic granules (CG, PR3, and NE), after their release in response to IC encounter, potentiate a positive autocrine feedback on neutrophil activation.

In contrast to CG, the highly related proteases PR3 and NE cooperate in the effacement of antiinflammatory PGRN, leading to enhanced neutrophil activation. Previous studies already demonstrated that PGRN is a potent inhibitor of the adhesion-dependent oxidative burst of neutrophils in vitro, which can be degraded by NE (23). Here, we showed that PR3 and NE play an equally important role in the regulation of PGRN function. Ela2–/– neutrophils were sufficiently able to degrade PGRN. Only in the absence of both PR3 and NE was PGRN degradation substantially impaired, resulting in the accumulation of antiinflammatory PGRN during neutrophil activation in vitro (Figure ​(Figure4C)4C) and neutrophilic inflammation in vivo (Figure ​(Figure6D).6D). Moreover, we provided in vivo evidence for the crucial role of PGRN as an inflammation-suppressing mediator, because administration of recombinant PGRN potently inhibited the neutrophil influx to sites of IC formation (Figure ​(Figure6,6, A–C). Hence, the cooperative degradation of PGRN by PR3 and NE is a decisive step for the establishment of neutrophilic inflammation.

The molecular mechanism of PGRN function is not yet completely understood, but it seems to interfere with integrin (CD11b/CD18) outside-in signaling by blocking the function of pyk2 and thus dampens adhesion-related oxidative burst even when added after the initial lag phase of oxidase activation (23). PGRN is produced by neutrophils and stored in highly mobile secretory granules (29). It was recently shown that PGRN can bind to heparan-sulfated proteoglycans (30), which are abundant components of the EBM and various cell surfaces, including those of neutrophils. Also, PR3 and NE are known to interact with heparan sulfates on the outer membrane of neutrophils, where the enzymes appear to be protected against protease inhibitors (121331). These circumstantial observations support the notion that PGRN cleavage by PR3 and NE takes place at the pericellular microenvironment of the neutrophil cell surface.

Impaired outside-in signaling most likely reduced the oxidative burst in Prtn3–/–Ela2–/– neutrophils adhering to ICs. In support of this hypothesis, we excluded an altered response to TNF-α priming (Supplemental Figure 5) as well as reduced adhesion to immobilized ICs and defective endocytosis of serum-opsonized E. coli in Prtn3–/–Ela2–/– neutrophils (Supplemental Figure 6). MPO content and processing was also unchanged in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure1D);1D); hence, the previously discussed inhibitory effect of MPO on phox activity (3233) does not appear to be stronger in neutrophils lacking PR3 and NE. Because there was no difference in the lag phase of the oxidative burst, initial IC-triggered receptor activation was probably not affected by either PRGN or PR3/NE. Our concept is consistent with all these observations and takes into account that PGRN unfolds its suppressing effects in the second phase, when additional membrane receptors, endogenous PGRN, and some PR3/NE from highly mobile intracellular pools are translocated to the cell surface. The decline and cessation of ROS production suggested to us that outside-in signaling was not sustained and that active oxidase complexes were no longer replenished in the absence of PR3 and NE. Our present findings, however, do not allow us to exclude other potential mechanisms, such as accelerated disassembly of the active oxidase complex.

 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2430496/bin/JCI0834694.f7.jpg

Proposed function of PR3 and NE in IC-mediated inflammation.

TNF-α–primed neutrophils extravasate from blood vessels, translocate PR3/NE to the cellular surface, and discharge PGRN to the pericellular environment (i). During transmigration of interstitial tissues (ii), neutrophil activation is initially suppressed by relatively high pericellular levels of antiinflammatory PGRN (green shading), which is also produced locally by keratinocytes and epithelial cells of the skin. Until IC depots are reached, neutrophil activation is inhibited by PGRN. Surface receptors (e.g., Mac-1) recognize ICs, which results in signal transduction (black dotted arrow) and activation of the phox. The molecular pathway of PGRN-mediated inhibition is not completely understood, but it may interfere with integrin signaling after IC encounter (green dotted line inside the cell). Adherence of neutrophils to ICs (iii) further increases pericellular PR3 and NE activity. PR3 and NE cooperatively degrade PGRN in the early stage of neutrophilic activation to facilitate optimal neutrophil activation (red shading), resulting in sustained integrin signaling (red arrow) and robust production of ROS by the phox system. Subsequently, neutrophils release ROS together with other proinflammatory mediators and chemotactic agents, thereby enhancing the recruitment of further neutrophils and establishing inflammation (iv). In the absence of PR3/NE, the switch from inflammation-suppressing (ii) to inflammation-enhancing (iii) conditions is substantially delayed, resulting in diminished inflammation in response to ICs (iv).

 

NSPs are strongly implicated as effector molecules in a large number of destructive diseases, such as emphysema or the autoimmune blistering skin disease bullous pemphigoid (143537). Normally, PR3/NE activity is tightly controlled by high plasma levels of α1-antitrypsin. This balance between proteases and protease inhibitors is disrupted in patients with genetic α1-antitrypsin deficiency, which represents a high risk factor for the development of emphysema and certain autoimmune disorders (38). The pathogenic effects of NSPs in these diseases have so far been associated with tissue destruction by the proteases after their release from dying neutrophils. Our findings showed that PR3 and NE were already involved in much earlier events of the inflammatory process, because the enzymes directly regulated cellular activation of infiltrating neutrophils by degrading inflammation-suppressing PGRN. This concept is further supported by previous studies showing increased inflammation in mice lacking serine protease inhibitors such as SERPINB1 or SLPI (3940). Blocking PR3/NE activity using specific inhibitors therefore represents a promising therapeutic strategy to treat chronic, noninfectious inflammation. Serine protease inhibitors as antiinflammatory agents can interfere with the disease process at 2 different stages, because they attenuate both early events of neutrophil activation and proteolytic tissue injury caused by released NSPs.

 

 

 

 

Editorial: Serine proteases, serpins, and neutropenia

David C. Dale

J Leuko Biol July 2011;  90(1): 3-4   http://dx.doi.org:/10.1189/jlb.1010592

Cyclic neutropenia and severe congenital neutropenia are autosomal-dominant diseases usually attributable to mutations in the gene for neutrophil elastase orELANE. Patients with these diseases are predisposed to recurrent and life-threatening infections [1]. Neutrophil elastase, the product of the ELANE gene, is a serine protease that is synthesized and packaged in the primary granules of neutrophils. These granules are formed at the promyelocytes stage of neutrophil development. Synthesis of mutant neutrophil elastase in promyelocytes triggers the unfolded protein response and a cascade of intracellular events, which culminates in death of neutrophil precursors through apoptosis [2]. This loss of cells causes the marrow abnormality often referred to as “maturation arrest” [34].

Neutrophil elastase is one of the serine proteases normally inhibited by serpinB1. In this issue of JLB, Benarafa and coauthors [5] present their intriguing studies of serpinB1 expression in human myeloid cells and their extensive investigations ofSERPINB1−/− mice. They observed that serpinB1 expression parallels protease expression. The peak of serpinB1 expression occurs in promyelocytes. Benarafa et al. [5] found that SERPINB1−/− mice have a deficiency of postmitotic neutrophils in the bone marrow. This change was accompanied by an increase in the plasma levels of G-CSF. The decreased supply of marrow neutrophils reduced the number of neutrophils that could be mobilized to an inflammatory site. Using colony-forming cell assays, they determined that the early myeloid progenitor pool was intact. Separate assays showed that maturing myeloid cells were being lost through accelerated apoptosis of maturing neutrophils in the marrow. The authors concluded that serpinB1 is required for maintenance of a healthy reserve of marrow neutrophils and a normal acute immune response [5].

This paper provides new and fascinating insights for understanding the mechanism for neutropenia. It also suggests opportunities to investigate potential therapies for patients with neutropenia and prompts several questions. As inhibition of the activity of intracellular serine proteases is the only known function of serpinB1, the findings reported by Benarafa et al. [5] suggest that uninhibited serine proteases perturbed neutrophil production severely. The SERPINB1−/− mice used in their work have accelerated apoptosis of myeloid cells, a finding suggesting that uninhibited serine proteases or mutant neutrophil elastase perturb myelopoiesis by similar mechanisms. It is now important to determine whether the defect in the SERPINB1−/− mice is, indeed, attributable to uninhibited activity of normal neutrophil elastase, other neutrophil proteases, or another mechanism. ″Double-knockout″ studies in mice deficient in neutrophil elastase and serpinB1 might provide an answer.

This report provides evidence regarding the intracellular mechanisms for the apoptosis of myeloid cells and indicates that other studies are ongoing. The key antiapoptotic proteins, Mcl-1, Bcl-XL, and A1/Bfl-I, are apparently not involved. A more precise understanding of the mechanisms of cell death is important for development of targeted therapies for neutropenia. It is also important to discover whether only cells of the neutrophil lineage are involved or whether monocytes are also affected. In cyclic and congenital neutropenia, patients failed to produce neutrophils, but they can produce monocytes; in fact, they overproduce monocytes and have significantly elevated blood monocyte counts. Neutropenia with monocytosis is probably attributable to differences in the expression of ELANE in the two lineages. Benarafa et al. [5] reported that human bone marrow monocytes contain substantially less serpinB1 than marrow neutrophils, suggesting that the expression of serpinB1 and the serine proteases are closely coordinated.

This report shows the importance of the marrow neutrophil reserves in the normal response to infections. Compared with humans, healthy mice are always neutropenic, but they have a bigger marrow neutrophil reserve, and their mature neutrophils in the marrow and blood look like human band neutrophils. These differences are well known, but they are critical for considering the clinical inferences that can be made from this report. For example, although theSERPINB1−/− mice were not neutropenic, human SERPINB1−/− might cause neutropenia because of physiological differences between the species. If some but not all mutations in SERPINB1 cause neutropenia, we might gain a better understanding about how serpinB1 normally inhibits the neutrophil’s serine proteases.

We do not know if some or all of the mutant neutrophil elastases can be inhibited by serpinB1. We do not know whether cyclic or congenital neutropenia are attributable to defects in this interaction. However, we do know that there are chemical inhibitors of neutrophil elastase that can abrogate apoptosis of myeloid cells in a cellular model for congenital neutropenia [6]. It would be interesting to see if these chemical inhibitors can replace the natural inhibitor and normalize neutrophil production in the SERPINB1−/− mice. This would provide evidence to support use of chemical protease inhibitors as a treatment for cyclic and congenital neutropenia.

Concerns with the use of G-CSF for the treatment of cyclic and congenital neutropenia are how and why some of these patients are at risk of developing leukemia. Are the SERPINB1−/− mice with a hyperproliferative marrow and high G-CSF levels also at risk of developing myeloid leukemia?

This is a very provocative paper, and much will be learned from further studies of the SERPINB1−/− mice.

 

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Mathias Baumann1,2, Christine T. N. Pham3, and Charaf Benarafa1

Blood May 9, 2013; 121(19)   http://www.bloodjournal.org/content/121/19/3900

Key Points

  • Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.
  • Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.

Abstract

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1−/−) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia ofsB1−/− mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1−/− mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1−/− PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.

Introduction

Polymorphonuclear neutrophil (PMN) granulocytes are essential components of the innate immune response to infection. PMNs are relatively short-lived leukocytes that originate from hematopoietic stem cells in the bone marrow (BM) in a process called granulopoiesis. Granulopoiesis proceeds through a proliferative phase followed by a maturation phase. After maturation, the BM retains a large reserve of mature PMNs, which includes over 90% of the mature PMNs in the body while only a small proportion (1%-5%) is in the blood.1,2 Even in noninflammatory conditions, granulopoiesis is remarkable as >1011 PMNs are produced daily in an adult human, only to be disposed of, largely unused, a few hours later.3 There is evidence that the majority of PMNs produced never reach circulation and die within the BM.4 Congenital or acquired forms of neutropenia are associated with the highest risks of bacterial and fungal infection,5 indicating a strong evolutionary pressure to maintain granulopoiesis at high levels and sustain a large mobilizable pool of PMNs in the BM.

In steady state, PMNs die by apoptosis, a form of programmed cell death that allows for the safe disposal of aging PMNs and their potentially toxic cargo. Like in other cells, caspases participate in the initiation, amplification, and execution steps of apoptosis in PMNs.6,7 Interestingly, noncaspase cysteine proteases calpain and cathepsin D were reported to induce PMN apoptosis through activation of caspases.811 In addition, PMNs carry a unique set of serine proteases (neutrophil serine proteases [NSPs]) including elastase (NE), cathepsin G (CG), and proteinase-3 (PR3) stored active in primary granules. There is strong evidence for a role of NSPs in killing pathogens and inducing tissue injury when released extracellularly.1214 In contrast, the function of NSPs in PMN homeostasis and cell death remains elusive. In particular, no defects in granulopoiesis or PMN homeostasis have been reported in mice deficient in cathepsin G (CG−/−),15 neutrophil elastase (NE−/−),16,17 or dipeptidylpeptidase I (DPPI−/−), which lack active NSPs.18 We have recently shown that mice lacking the serine protease inhibitor serpinB1 (sB1−/−) have reduced PMN survival in the lungs following Pseudomonas infection and that these mice have a profound reduction in mature PMN numbers in the BM.19,20SerpinB1, also known as monocyte NE inhibitor, is expressed at high levels in the cytoplasm of PMNs and is one of the most potent inhibitors of NE, CG, and PR3.21,22 In this study, we tested the hypothesis that serpinB1 promotes PMN survival by inhibiting 1 or several NSPs, and we discovered a novel regulatory pathway in PMN homeostasis in vivo.

 

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Figure 1

Defective PMN reserve in BM chimera depends on serpinB1 deficiency in the hematopoietic compartment. Flow cytometry analysis of major BM leukocyte subsets of lethally irradiated mice was performed 8 to 10 weeks after BM transfer. (A) Irradiated WT (CD45.1) mice were transferred with WT (●) or sB1−/− (○) BM cells. (B) Irradiated WT (●) andsB1−/− (○) mice both CD45.2 were transferred with WT (CD45.1) BM cells. Each circle represents leukocyte numbers for 1 mouse and horizontal line indicates the median. Median subsets numbers were compared by the Mann-Whitney test (*P < .05; ***P < .001).

CG regulates neutrophil numbers in the BM

Because serpinB1 is an efficient inhibitor of NE, CG, and PR3, we then examined PMN numbers in mice deficient in 1 or several NSPs in combination with serpinB1 deletion. As expected, sB1−/− mice had significantly reduced numbers and percentage of mature PMNs in the BM compared with WT and heterozygous sB1+/− mice. In addition, PMN numbers were normal in mice deficient in either DPPI, NE, or CG (Figure 2A). DPPI is not inhibited by serpinB1 but is required for the activation of all NSPs, and no NSP activity is detectable in DPPI−/− mice.18,23 PMN counts in DPPI−/−.sB1−/− BM were significantly higher than in sB1−/− BM, suggesting that 1 or several NSPs contribute to the PMN survival defect. To examine the role of NSPs in this process, we crossed several NSP-deficient strains with sB1−/− mice. We found that NE.CG.sB1−/− mice had normal PMN numbers indicating that these NSPs play a key role in the defective phenotype of sB1−/− PMNs (Figure 2A). Furthermore, CG.sB1−/− mice showed normal PMN numbers whereasNE.sB1−/− mice retained the BM neutropenia phenotype indicating that CG, but not NE, plays a significant role in the death of sB1−/− PMNs (Figure 2A). In addition, the double-deficient NE.sB1−/− mice had significantly lower BM myelocyte numbers than sB1−/− mice while the myelocyte numbers in singly deficient NE−/− and sB1−/− BM were normal (Figure 2B). These results suggest that NE may promote myeloid cell proliferation, an activity that is revealed only when serpinB1 is absent. This complex interaction between sB1 and NE requires further investigation. On the other hand, B-cell and monocyte numbers and relative percentage in the BM were largely similar in all genotypes (supplemental Figure 2). Total numbers of blood leukocytes, erythrocytes, and platelets were normal in mice deficient in NSPs and/or serpinB1 (supplemental Figure 3). PMN numbers in blood were normal insB1−/− mice in steady state and combined deficiency of NSPs did not significantly alter these numbers (Figure 2C). Taken together, our results indicate that serpinB1 likely sustains the survival of postmitotic PMNs through its interaction with CG.

Figure 2

PMN and myelocyte numbers in BM and blood of mice deficient in NSPs and serpinB1.

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CG-mediated PMN death proceeds independent of caspase activity

Figure 4

sB1−/− PMN death mediated by CG does not require caspase activity

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Granule membrane permeabilization induces CG-mediated death in PMNs

To test whether granule disruption contributes to the serpinB1-regulated CG-dependent cell death, BM cells were treated with the lysosomotropic agent LLME. LLME accumulates in lysosomes where the acyl transferase activity of DPPI generates hydrophobic (Leu-Leu)n-OMe polymers that induce lysosomal membrane permeabilization (LMP) and cytotoxicity in granule-bearing cells such as cytotoxic T lymphocytes, NK cells, and myeloid cells.29,30

Figure 5

LMP induces CG-mediated death in PMNs

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G-CSF therapy increases sB1−/− PMN numbers via enhanced granulopoiesis

G-CSF therapy is an effective long-term treatment in many cases of severe congenital neutropenia and it is also used to prevent chemotherapy-induced febrile neutropenia by enhancing PMN production. In addition, G-CSF delays neutrophil apoptosis by differentially regulating proapoptotic and antiapoptotic factors.10 To test whether G-CSF could rescue sB1−/− PMN survival defect, WT and sB1−/− mice were treated with therapeutic doses of G-CSF or saline for 5 days and BM and blood PMNs were analyzed 24 hours after the last injection. Total counts of myelocytes and PMNs were significantly increased in the BM of treated mice compared with their respective untreated genotype controls (Figure 6A-B). The increase in myelocyte numbers was identical in G-CSF–treated WT and sB1−/− mice, indicating that G-CSF–induced granulopoiesis proceeds normally in sB1−/−myeloid progenitors (Figure 6B).

Figure 6

In vivo G-CSF therapy increases PMN numbers in BM of sB1−/− mice.

 

SerpinB1 is a member of the clade B serpins, a subfamily composed of leaderless proteins with nucleocytoplasmic localization. Clade B serpins are often expressed in cells that also carry target proteases, which led to the hypothesis that intracellular serpins protect against misdirected granule proteases and/or protect bystander cells from released proteases.31 We previously reported that deficiency in serpinB1 is associated with reduced PMN survival in the BM and at inflammatory sites.19,20 The evidence presented here demonstrates that the cytoprotective function of serpinB1 in PMNs is based on the inhibition of granule protease CG. Deficiency in CG was sufficient to rescue the defect of sB1−/− mice as illustrated by normal PMN counts in the BM of double knockout CG.sB1−/− mice. We also showed that the protease-serpin interaction occurred within PMNs. Indeed, WT PMNs had a greater survival over sB1−/− PMNs in mixed BM chimera, whereas the survival of CG.sB1−/− PMNs was similar to WT PMNs after BM transfer. SerpinB1 is an ancestral clade B serpin with a conserved specificity determining reactive center loop in all vertebrates.32 Furthermore, human and mouse serpinB1 have the same specificity for chymotrypsin-like and elastase-like serine proteases.21,22 Likewise, human and mouse CG have identical substrate specificities and the phenotype of CG−/− murine PMN can be rescued by human CG.33 Therefore, it is highly likely that the antagonistic functions of CG and serpinB1 in cellular homeostasis observed in mice can be extended to other species.

Extracellular CG was previously reported to promote detachment-induced apoptosis (anoikis) in human and mouse cardiomyocytes.34 This activity is mediated through the shedding and transactivation of epidermal growth factor receptor and downregulation of focal adhesion signaling.35,36 In our study, exogenous human CG also induced PMN death in vitro but these effects were not enhanced in sB1−/− PMNs and the neutropenia associated with serpinB1 deficiency was principally cell intrinsic. How intracellular CG induces PMN death remains to be fully investigated. However, our studies provide some indications on the potential pathways. Like other NSPs, the expression of CG is transcriptionally restricted to the promyelocyte stage during PMN development and NSPs are then stored in active form in primary azurophil granules.37 Because serpinB1 is equally efficient at inhibiting NE, CG, and PR3, it was surprising that deletion of CG alone was sufficient to achieve a complete reversal of the PMN survival defect in CG.sB1−/− mice. A possible explanation would be that CG gains access to targets more readily than other granule proteases. There is evidence that binding to serglycin proteoglycans differs between NE and CG resulting in altered sorting of NE but not CG into granules of serglycin-deficient PMNs.38 Different interactions with granule matrix may thus contribute to differential release of CG from the granules compared with other NSPs. However, because sB1−/− PMNs have similar levels of CG and NE as WT PMNs20 and because LLME-induced granule permeabilization likely releases all granule contents equally, we favor an alternative interpretation where CG specifically targets essential cellular components that are not cleaved by the other serpinB1-inhibitable granule proteases. Upon granule permeabilization, we found that CG can induce cell death upstream of caspases as well as independent of caspases. CG was previously shown to activate caspase-7 in vitro and it functions at neutral pH, which is consistent with a physiological role in the nucleocytoplasmic environment.39 Cell death induced by lysosomal/granule membrane permeabilization has previously been linked to cysteine cathepsins in other cell types. However, these proteases appear to depend on caspase activation to trigger apoptosis and they function poorly at neutral pH, questioning their potential role as regulators of cell death.40 In contrast, CG-mediated cell death is not completely blocked by caspase inhibition, which is a property reminiscent of granzymes in cytotoxic T cells.41 In fact, CG is phylogenetically most closely related to serine proteases granzyme B and H.42 Granzymes have numerous nuclear, mitochondrial, and cytoplasmic target proteins leading to cell death41 and we anticipate that this may also be the case for CG.

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G-CSF therapy is successfully used to treat most congenital and acquired neutropenia through increased granulopoiesis, mobilization from the BM, and increased survival of PMNs. Prosurvival effects of G-CSF include the upregulation of antiapoptotic Bcl-2 family members, which act upstream of the mitochondria and the activation of effector caspases. In sB1−/− mice, G-CSF levels in serum are fourfold higher than in WT mice in steady state and this is accompanied by an upregulation of the antiapoptotic Bcl-2 family member Mcl-1 in sB1−/− PMNs.19 Here, G-CSF therapy significantly increased granulopoiesis in both WT and sB1−/− mice. However, the PMN numbers in treated sB1−/− BM and blood were significantly lower than those of treated WT mice, indicating only a partial rescue of the survival defect. This is consistent with our findings that CG-mediated death can proceed independent of caspases and can thus bypass antiapoptotic effects mediated by G-CSF.

CG has largely been studied in association with antimicrobial and inflammatory functions due to its presence in PMNs.1214,49 In this context, we have previously shown that serpinB1 contributes to prevent increased mortality and morbidity associated with production of inflammatory cytokines upon infection with Pseudomonas aeruginosa and influenza A virus.20,50 In this study, we demonstrate that serpinB1 inhibition of the primary granule protease CG in PMNs is essential for PMN survival and this ultimately regulates PMN numbers in vivo. Our findings also extend the roles of CG from antimicrobial and immunoregulatory functions to a novel role in inducing cell death.

 

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Brice KorkmazMarshall S. HorwitzDieter E. Jenne and Francis Gauthier
Pharma Rev Dec 2010; 62(4):726-759  http://dx.doi.org:/10.1124/pr.110.002733

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

 

Human polymorphonuclear neutrophils represent 35 to 75% of the population of circulating leukocytes and are the most abundant type of white blood cell in mammals (Borregaard et al., 2005). They are classified as granulocytes because of their intracytoplasmic granule content and are characterized by a multilobular nucleus. Neutrophils develop from pluripotent stem cells in the bone marrow and are released into the bloodstream where they reach a concentration of 1.5 to 5 × 109 cells/liter. Their half-life in the circulation is only on the order of a few hours. They play an essential role in innate immune defense against invading pathogens and are among the primary mediators of inflammatory response. During the acute phase of inflammation, neutrophils are the first inflammatory cells to leave the vasculature, where they migrate toward sites of inflammation, following a gradient of inflammatory stimuli. They are responsible for short-term phagocytosis during the initial stages of infection (Borregaard and Cowland, 1997Hampton et al., 1998Segal, 2005). Neutrophils use complementary oxidative and nonoxidative pathways to defend the host against invading pathogens (Kobayashi et al., 2005).

The three serine proteases neutrophil elastase (NE1), proteinase 3 (PR3), and cathepsin G (CG) are major components of neutrophil azurophilic granules and participate in the nonoxidative pathway of intracellular and extracellular pathogen destruction. These neutrophil serine proteases (NSPs) act intracellularly within phagolysosomes to digest phagocytized microorganisms in combination with microbicidal peptides and the membrane-associated NADPH oxidase system, which produces reactive oxygen metabolites (Segal, 2005). An additional extracellular antimicrobial mechanism, neutrophil extracellular traps (NET), has been described that is made of a web-like structure of DNA secreted by activated neutrophils (Papayannopoulos and Zychlinsky, 2009) (Fig. 1). NETs are composed of chromatin bound to positively charged molecules, such as histones and NSPs, and serve as physical barriers that kill pathogens extracellularly, thus preventing further spreading. NET-associated NSPs participate in pathogen killing by degrading bacterial virulence factors extracellularly (Brinkmann et al., 2004;Papayannopoulos and Zychlinsky, 2009).

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Fig. 1.

Polymorphonuclear neutrophil. Quiescent (A) and chemically activated (B) neutrophils purified from peripheral blood. C, PMA-activated neutrophils embedded within NET and neutrophil spreading on insoluble elastin.

In addition to their involvement in pathogen destruction and the regulation of proinflammatory processes, NSPs are also involved in a variety of inflammatory human conditions, including chronic lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and acute respiratory distress syndrome) (Lee and Downey, 2001Shapiro, 2002Moraes et al., 2003Owen, 2008b). In these disorders, accumulation and activation of neutrophils in the airways result in excessive secretion of active NSPs, thus causing lung matrix destruction and inflammation. NSPs are also involved in other human disorders as a consequence of gene mutations, altered cellular trafficking, or, for PR3, autoimmune disease. Mutations in the ELA2/ELANE gene encoding HNE are the cause of human cyclic neutropenia and severe congenital neutropenia (Horwitz et al., 19992007). Neutrophil membrane-bound proteinase 3 (mPR3) is the major target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA), which are associated with Wegener granulomatosis (Jenne et al., 1990). All three proteases are affected by mutation of the gene (CTSC) encoding dipeptidyl peptidase I (DPPI), which activates several granular hematopoietic serine proteases (Pham and Ley, 1999Adkison et al., 2002). Mutations of CTSC cause Papillon-Lefèvre syndrome and palmoplantar keratosis (Hart et al., 1999Toomes et al., 1999).

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Fully processed mature HNE, PR3, and CG isolated from azurophilic granules contain, respectively, 218 (Bode et al., 1986Sinha et al., 1987), 222 (Campanelli et al., 1990b), and 235 (Salvesen et al., 1987Hof et al., 1996) residues. They are present in several isoforms depending on their carbohydrate content, with apparent mass of 29 to 33 kDa upon SDS-polyacrylamide gel electrophoresis (Twumasi and Liener, 1977Watorek et al., 1993). HNE and PR3 display two sites of N-glycosylation, whereas CG possesses only one. NSPs are stored mainly in neutrophil azurophilic granules, but HNE is also localized in the nuclear envelope, as revealed by immunostaining and electron microscopy (Clark et al., 1980;Benson et al., 2003), whereas PR3 is also found in secretory vesicles (Witko-Sarsat et al., 1999a). Upon neutrophil activation, granular HNE, PR3, and CG are secreted extracellularly, although some molecules nevertheless remain at the cell surface (Owen and Campbell, 1999Owen, 2008a). The mechanism through which NSPs are sorted from the trans-Golgi network to the granules has not been completely defined, even though an intracellular proteoglycan, serglycin, has been identified as playing a role in elastase sorting and packaging into azurophilic granules (Niemann et al., 2007). Unlike HNE and CG, PR3 is constitutively expressed on the membranes of freshly isolated neutrophils (Csernok et al., 1990Halbwachs-Mecarelli et al., 1995). Stimulation of neutrophils at inflammatory sites triggers intracytoplasmic granules to translocate to the phagosomes and plasma membrane, thereby liberating their contents. The first step of the translocation to the target membrane depends on cytoskeleton remodeling and microtubule assembly (Burgoyne and Morgan, 2003). This is followed by a second step of granule tethering and docking, which are dependent on the sequential intervention of SNARE proteins (Jog et al., 2007).

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Exposure of neutrophils to cytokines (TNF-α), chemoattractants (platelet-activating factor, formyl-Met-Leu-Phe, or IL-8), or bacterial lipopolysaccharide leads to rapid granule translocation to the cell surface with secretion of HNE, PR3, and CG into the extracellular medium (Owen and Campbell, 1999). A fraction of secreted HNE, PR3, and CG is detected at the surface of activated neutrophils (Owen et al., 1995a1997Campbell et al., 2000). Resting purified neutrophils from peripheral blood express variable amounts of PR3 on their surface. A bimodal, apparently genetically determined, distribution has been observed with two populations of quiescent neutrophils that express or do not express the protease at their surface (Halbwachs-Mecarelli et al., 1995Schreiber et al., 2003). The percentage of mPR3-positive neutrophils ranges from 0 to 100% of the total neutrophil population within individuals. Furthermore, the percentage of mPR3-positive neutrophils remains stable over time and is not affected by neutrophil activation (Halbwachs-Mecarelli et al., 1995).

The mechanism through which HNE and CG are associated with the outer surface of the plasma membrane of neutrophils mainly involves electrostatic interactions with the sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans (Campbell and Owen, 2007). These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al., 1995b1997;Korkmaz et al., 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al., 1999aKorkmaz et al., 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al., 1999Hajjar et al., 2008) (Fig. 3B). Several membrane partners of PR3 have been identified, including CD16/FcγRIIIb (David et al., 2005Fridlich et al., 2006), phospholipid scramblase-1, a myristoylated membrane protein with translocase activity present in lipid rafts (Kantari et al., 2007), CD11b/CD18 (David et al., 2003), and human neutrophil antigen NB1/CD177 (von Vietinghoff et al., 2007Hu et al., 2009), a 58- to 64-kDa glycosyl-phosphatidylinositol anchored surface receptor belonging to the urokinase plasminogen activator receptor superfamily (Stroncek, 2007). NB1 shows a bimodal distribution that superimposes with that of PR3 on purified blood neutrophils (Bauer et al., 2007). Active, mature forms of PR3 but not pro-PR3 can bind to the surface of NB1-transfected human embryonic kidney 293 cells (von Vietinghoff et al., 2008) and Chinese hamster ovary cells (Korkmaz et al., 2008b). Interaction involves the hydrophobic patch of PR3 because specific amino acid substitutions disrupting this patch in the closely related gibbon PR3 prevent binding to NB1-transfected cells (Korkmaz et al., 2008b). Decreased interaction of pro-PR3 with NB1-transfected cells is explained by the topological changes affecting the activation domain containing the hydrophobic patch residues. Together, these results support the hydrophobic nature of PR3-membrane interaction.

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Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001Pham, 2006Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.

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Processing of Cytokines, Chemokines, and Growth Factors.

Processing and Activation of Cellular Receptors.

Induction of Apoptosis by Proteinase 3.

Physiological Inhibitors of Elastase, Proteinase 3, and Cathepsin G

During phagocytosis and neutrophil turnover, HNE, PR3, and CG are released into the extracellular space as active proteases. The proteolytic activity of HNE, PR3, and CG seems to be tightly regulated in the extracellular and pericellular space to avoid degradation of connective tissue proteins including elastin, collagen, and proteoglycans (Janoff, 1985). Protein inhibitors that belong to three main families, the serpins, the chelonianins, and the macroglobulins, ultimately control proteolytic activity of HNE, PR3, and CG activities. The individual contributions of these families depend on their tissue localization and that of their target proteases. The main characteristics of HNE, PR3, and CG physiological inhibitors are presented in Table 2.

 

Serine Protease Inhibitors

Serpins are the largest and most diverse family of protease inhibitors; more than 1000 members have been identified in human, plant, fungi, bacteria, archaea, and certain viruses (Silverman et al., 2001Mangan et al., 2008). They share a similar highly conserved tertiary structure and similar molecular weight of approximately 50 kDa. Human serpins belong to the first nine clades (A–I) of the 16 that have been described based on phylogenic relationships (Irving et al., 2000Silverman et al., 2001Mangan et al., 2008). For historical reasons, α1-protease inhibitor (α1-PI) was assigned to the first clade. Clade B, also known as the ov-serpin clan because of the similarity of its members to ovalbumin (a protein that belongs to the serpin family but lacks inhibitory activity), is the second largest clan in humans, with 15 members identified so far. Ov-serpin clan members are generally located in the cytoplasm and, to a lesser extent, on the cell surface and nucleus (Remold-O’Donnell, 1993).

Serpins play important regulatory functions in intracellular and extracellular proteolytic events, including blood coagulation, complement activation, fibrinolysis, cell migration, angiogenesis, and apoptosis (Potempa et al., 1994). Serpin dysfunction is known to contribute to diseases such as emphysema, thrombosis, angioedema, and cancer (Carrell and Lomas, 1997Lomas and Carrell, 2002). Most inhibitory serpins target trypsin-/chymotrypsin-like serine proteases, but some, termed “cross-class inhibitors,” have been shown to target cysteine proteases (Annand et al., 1999). The crystal structure of the prototype plasma inhibitor α1-PI revealed the archetype native serpin fold (Loebermann et al., 1984). All serpins typically have three β-sheets (termed A, B, and C) and eight or nine α-helices (hA–hI) arranged in a stressed configuration. The so-called reactive center loop (RCL) of inhibitory molecules determines specificity and forms the initial encounter complex with the target protease (Potempa et al., 1994Silverman et al., 2001). Serpins inhibit proteases by a suicide substrate inhibition mechanism. The protease initially recognizes the serpin as a potential substrate using residues of the reactive center loop and cleaves it between P1 and P1′ This cleavage allows insertion of the cleaved RCL into the β-sheet A of the serpin, dragging the protease with it and moving it over 71 Å to the distal end of the serpin to form a 1:1 stoichiometric covalent inhibitory complex (Huntington et al., 2000). Such cleavage generates a ∼4-kDa C-terminal fragment that remains noncovalently bound to the cleaved serpin. Displacement of the covalently attached active site serine residue from its catalytic partner histidine explains the loss of catalytic function in the covalent complex. The distortion of the catalytic site structure prevents the release of the protease from the complex, and the structural disorder induces its proteolytic inactivation (Huntington et al., 2000). Covalent complex formation between serpin and serine proteases triggers a number of conformational changes, particularly in the activation domain loops of the bound protease (Dementiev et al., 2006).

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Pathophysiology of Elastase, Proteinase 3 and Cathepsin G in Human Diseases

In many instances, the initiation and propagation of lung damage is a consequence of an exaggerated inappropriate inflammatory response, which includes the release of proteases and leukocyte-derived cytotoxic products (Owen, 2008b;Roghanian and Sallenave, 2008). Inflammation is a physiological protective response to injury or infection consisting of endothelial activation, leukocyte recruitment and activation, vasodilation, and increased vascular permeability. Although designed to curtail tissue injury and facilitate repair, the inflammatory response sometimes results in further injury and organ dysfunction. Inflammatory chronic lung diseases, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis are syndromes of severe pulmonary dysfunction resulting from a massive inflammatory response and affecting millions of people worldwide. The histological hallmark of these chronic inflammatory lung diseases is the accumulation of neutrophils in the microvasculature of the lung. Neutrophils are crucial to the innate immune response, and their activation leads to the release of multiple cytotoxic products, including reactive oxygen species and proteases (serine, cysteine, and metalloproteases). The physiological balance between proteases and antiproteases is required for the maintenance of the lung’s connective tissue, and an imbalance in favor of proteases results in lung injury (Umeki et al., 1988Tetley, 1993). A number of studies in animal and cell culture models have demonstrated a contribution of HNE and related NSPs to the development of chronic inflammatory lung diseases. Available preclinical and clinical data suggest that inhibition of NSP in lung diseases suppresses or attenuates the contribution of NSP to pathogenesis (Chughtai and O’Riordan, 2004Voynow et al., 2008Quinn et al., 2010). HNE could also participate in fibrotic lung remodeling by playing a focused role in the conversion of latent transforming growth factor-β into its biologically active form (Chua and Laurent, 2006Lungarella et al., 2008).

Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

ANCA-associated vasculitides encompasses a variety of diseases characterized by inflammation of blood vessels and by the presence of autoantibodies directed against neutrophil constituents. These autoantibodies are known as ANCAs (Kallenberg et al., 2006). In Wegener granulomatosis (WG), antibodies are mostly directed against PR3. WG is a relatively uncommon chronic inflammatory disorder first described in 1931 by Heinz Karl Ernst Klinger as a variant of polyarteritis nodosa (Klinger, 1931). In 1936, the German pathologist Friedrich Wegener described the disease as a distinct pathological entity (Wegener, 19361939). WG is characterized by necrotizing granulomatous inflammation and vasculitis of small vessels and can affect any organ (Fauci and Wolff, 1973Sarraf and Sneller, 2005). The most common sites of involvement are the upper and lower respiratory tract and the kidneys. WG affects approximately 1 in 20,000 people; it can occur in persons of any age but most often affects those aged 40 to 60 years (Walton, 1958Cotch et al., 1996). Approximately 90% of patients have cold or sinusitis symptoms that fail to respond to the usual therapeutic measures and that last considerably longer than the usual upper respiratory tract infection. Lung involvement occurs in approximately 85% of the patients. Other symptoms include nasal membrane ulcerations and crusting, saddle-nose deformity, inflammation of the ear with hearing problems, inflammation of the eye with sight problems, and cough (with or without hemoptysis).

Hereditary Neutropenias

Neutropenia is a hematological disorder characterized by an abnormally low number of neutrophils (Horwitz et al., 2007). The normal neutrophil count fluctuates across human populations and within individual patients in response to infection but typically lies in the range of 1.5 to 5 × 109 cells/liter. Neutropenia is categorized as severe when the cell count falls below 0.5 × 109 cells/liter. Hence, patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening. Common causes of neutropenia include cancer chemotherapy, drug reactions, autoimmune diseases, and hereditary disorders (Berliner et al., 2004Schwartzberg, 2006).

Papillon-Lefèvre Syndrome

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New Strategies for Fighting Neutrophil Serine Protease-Related Human Diseases

Administration of therapeutic inhibitors to control unwanted proteolysis at inflammation sites has been tested as a therapy for a variety of inflammatory and infectious lung diseases (Chughtai and O’Riordan, 2004). Depending on the size and chemical nature of the inhibitors, they may be administered orally, intravenously, or by an aerosol route. Whatever the mode of administration, the access of therapeutic inhibitors to active proteases is often hampered by physicochemical constraints in the extravascular space and/or by the partitioning of proteases between soluble and solid phases.

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Concluding Remarks

NSPs were first recognized as protein-degrading enzymes but have now proven to be multifunctional components participating in a variety of pathophysiological processes. Thus, they appear as potential therapeutic targets for drugs that inhibit their active site or impair activation from their precursor. Overall, the available preclinical and clinical data suggest that inhibition of NSPs using therapeutic inhibitors would suppress or attenuate deleterious effects of inflammatory diseases, including lung diseases. Depending on the size and chemical nature of inhibitors, those may be administered orally, intravenously, or by aerosolization. But the results obtained until now have not been fully convincing because of the poor knowledge of the biological function of each protease, their spatiotemporal regulation during the course of the disease, the physicochemical constraints associated with inhibitor administration, or the use of animal models in which NSP regulation and specificity differ from those in human. Two different and complementary approaches may help bypass these putative problems. One is to target active proteases by inhibitors at the inflammatory site in animal models in which lung anatomy and physiology are close to those in human to allow in vitro and in vivo assays of human-directed drugs/inhibitors. The other is to prevent neutrophil accumulation at inflammatory sites by impairing production of proteolytically active NSPs using an inhibitor of their maturation protease, DPPI. Preventing neutrophil accumulation at the inflammatory sites by therapeutic inhibition of DPPI represents an original and novel approach, the exploration of which has just started (Méthot et al., 2008). Thus pharmacological inactivation of DPPI in human neutrophils could well reduce membrane binding of PR3 and, as a consequence, neutrophil priming by pathogenic auto-antibodies in WG. In addition, it has been recognized that the intracellular level of NSPs depends on their correct intracellular trafficking. In the future, pharmacological targeting of molecules specifically involved in the correct intracellular trafficking of each NSP could possibly regulate their production and activity, a feature that could be exploited as a therapeutic strategy for inflammatory diseases.

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DNA Replication

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Decades Old DNA Replication Models Called into Question

http://www.genengnews.com/gen-news-highlights/decades-old-dna-replication-models-called-into-question/81251929/

 

Decades Old DNA Replication Models Called into Question

http://www.genengnews.com/media/images/GENHighlight/102252_web8123122217.jpg

A series of electron micrographs show the barrel-shaped helicase, which is the enzyme that separates the two DNA strands, along with other components of the replisome, including polymerase-epsilon (green).[Brookhaven National Laboratory]

  • It may be time to update biology texts to reflect newly published data from a collaborative team of scientists at Rockefeller University, Stony Brook University, and the U.S. Department of Energy’s Brookhaven National Laboratory. Using cutting-edge electron microscopy (EM) techniques, the investigators gathered the first ever images of the fully assembled replisome, providing new insight into the molecular mechanisms of replication.

    “Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

    “Our finding goes against decades of textbook drawings of what people thought the replisome should look like,” remarked co-senior author Michael O’Donnell, Ph.D., professor and head of Rockefeller’s Laboratory of DNA Replication. “However, it’s a recurring theme in science that nature does not always turn out to work the way you thought it did.”

http://www.genengnews.com/Media/images/GENHighlight/102254_web2322915422.jpg

Previously (left), the replisome’s two polymerases (green) were assumed to be below the helicase (tan), the enzyme that splits the DNA strands. The new images reveal one polymerase is located at the front of the helicase, causing one strand to loop backward as it is copied (right). [Brookhaven National Laboratory]

The researcher’s findings focused on the replisome found in eukaryotic organisms, a category that includes a broad swath of living things, including humans and other multicellular organisms. Over the past several decades, there has been an array of data describing the individual components comprising the complex nature of replisome. Yet, until now no pictures existed to show just how everything fit together.

“This work is a continuation of our long-standing research using electron microscopy to understand the mechanism of DNA replication, an essential function for every living cell,” explained co-senior author Huilin Li, Ph.D., biologist with joint appointments at Brookhaven Lab and Stony Brook University. “These new images show the fully assembled and fully activated ‘helicase’ protein complex—which encircles and separates the two strands of the DNA double helix as it passes through a central pore in the structure—and how the helicase coordinates with the two ‘polymerase’ enzymes that duplicate each strand to copy the genome.”

The image and implications from this study were described in a paper entitled “The architecture of a eukaryotic replisome,” published recently through Nature Structural & Molecular Biology.

Traditional models of DNA replication show the helicase enzyme moving along the DNA, separating the two strands of the double helix, with two polymerases located at the back where the DNA strand is split. In this configuration, the polymerases would add nucleotides to the side-by-side split ends as they move out of the helicase to form two new complete double helix DNA strands. However, the images that the researchers collected of intact replisomes revealed that only one of the polymerases is located at the back of the helicase. The other is on the front side of the helicase, where the helicase first encounters the double-stranded helix. This means that while one of the two split DNA strands is acted on by the polymerase at the back end, the other has to thread itself back through or around the helicase to reach the front-side polymerase before having its new complementary strand assembled.

“DNA replication is one of the most fundamental processes of life, so it is every biochemist’s dream to see what a replisome looks like,” stated lead author Jingchuan Sun, EM biologist in Dr. Li’s laboratory. “Our lab has expertise and a decade of experience using electron microscopy to study DNA replication, which has prepared us well to tackle the highly mobile therefore very challenging replisome structure. Working together with the O’Donnell lab, which has done beautiful, functional studies on the yeast replisome, our two groups brought perfectly complementary expertise to this project.”

The positioning of one polymerase at the front of the helicase suggests that it may have an unforeseen function—the possibilities of which the collaborative group of scientists is continuing to study. Whatever the function the offset polymerase ends up having, Drs. Li and O’Donnell hope that it will not only provide them better insight into the replication machinery but that they may uncover useful information that can be exploited for disease intervention.

“Clearly, further studies will be required to understand the functional implications of the unexpected replisome architecture reported here,” the scientists concluded.

 

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Fifth Histone Found to Recruit Proteins for DNA Repair   

http://www.genengnews.com/gen-news-highlights/fifth-histone-found-to-recruit-proteins-for-dna-repair/81251895/

Scientists at the University of Copenhagen say they have located a previously unknown function for histones, which allows for an improved understanding of how cells protect and repair DNA damages. This new discovery may be of great importance to the treatment of diseases caused by cellular changes such as cancer and immune deficiency syndrome.

The study (“Histone H1 couples initiation and amplification of ubiquitin signaling after DNA damage”) is published in Nature.

“I believe that there’s a lot of work ahead. It’s like opening a door onto a previously undiscovered territory filled with lots of exciting knowledge. The histones are incredibly important to many of the cells’ processes as well as their overall wellbeing,” said Niels Mailand, Ph.D., from the Novo Nordisk Foundation Center for Protein Research at the Faculty of Health and Medical Science.

Histones enable the tight packaging of DNA strands within cells. The strands are two meters in length and the cells usually about 100,000 times smaller. Generally speaking, there are five types of histones. Four of them are core histones and they are placed like beads on the DNA strands, which are curled up like a ball of wool within the cells. The role of the histones is already well described in research, and in addition to enabling the packaging of the DNA strands they also play a central part in practically every process related to the DNA-code, including repairing possibly damaged DNA.

The four core histones have tails and, among other things, they signal damage to the DNA and thus attract the proteins that help repair the damage. Between the histone “yarn balls” we find the fifth histone, Histone H1, but up until now its function has not been thoroughly examined.

Using a mass spectrometer, Dr. Mailand and his team have discovered that, surprisingly, the H1 histone also helps summon repair proteins.

“In international research, the primary focus has been on the core histones and their functionality, whereas little attention has been paid to the H1 histone, simply because we weren’t aware that it too influenced the repair process. Having discovered this function in the H1 constitutes an important piece of the puzzle of how cells protect their DNA, and it opens a door onto hitherto unknown and highly interesting territory,” noted Dr. Mailand.

He expects the discovery to lead to increased research into Histone H1 worldwide, which will lead to increased knowledge of cells’ abilities to repair possible damage to their DNA and thus increase our knowledge of the basis for diseases caused by cellular changes. It will also generate more knowledge about the treatment of these diseases.

“By mapping the function of the H1 histone, we will also learn more about the repair of DNA damages on a molecular level. In order to provide the most efficient treatment, we need to know how the cells prevent and repair these damages,” point out Dr. Mailand.

 

Cover All the Bases for Oligonucleotide Analysis

Stephen Luke

Synthetic oligonucleotides have emerged as promising therapeutic agents for the treatment of a variety of diseases, including viral infections and cancer. Researchers are looking at several classes of nucleic acids, such as antisense oligonucleotides, small interfering RNAs (siRNAs), and aptamers, for therapeutic applications.

However, various impurities – product-related, in the starting materials, and arising from incomplete capping of coupling reactions – must be identified and removed and postsynthesis processing must be monitored. Thus, a key challenge in the development and manufacture of oligonucleotide therapeutics is to establish analytical methods that are capable of separating and identifying impurities.

Exploring Better Options for Oligonucleotide LC Separations

 

 

Table 1. Options for oligonucleotide LC separations

Ion-pair, reversed-phase separation of the trityl-on oligos and is relatively simple to perform. This method separates the full-length target oligo, which still has the dMT group attached, from the deprotected failure sequences. The analytical information obtained is limited, so this is generally considered a purification method.

An alternate method, ion-exchange separations of the trityl-off, deprotected oligos uses the negative charge on the backbone of the oligo to facilitate the separation. Resolution is good for the shorter oligos but decreases with increasing chain length. Aqueous eluents are used but oligos are highly charged, and high concentrations of salt are needed to achieve elution from the column, making the technique unsuitable for use with LC/MS.

Finally, ion-pair, reversed-phase separation of the trityl-off, deprotected oligos makes use of organic solvents and mobile phase additives such as TEAA (triethylammonium acetate) or TEA-HFIP (triethylamine and hexafluoroisopropanol) to ion-pair with the negatively charged phosphodiester backbone of the oligonucleotide. High-performance columns deliver excellent resolution. What’s more, methods with volatile mobile phase constituents such as TEA-HFIP are suitable for use with LC/MS, providing useful information to help characterize oligonucleotide structures and sequences.

In Table 1 we summarize some of the options for oligonucleotide analysis by liquid chromatography.

Designed for ion-pair, reversed-phase separation of the trityl-off, deprotected oligos using either TEAA or TEA-HFIP mobile phases –Agilent AdvanceBio Oligonucleotide columns meet these challenges.

more….   http://www.genengnews.com/gen-articles/cover-all-the-bases-for-oligonucleotide-analysis/5594/

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