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Tweets and Re-Tweets of Tweets by @pharma_BI@AVIVA1950 at 2021 Virtual World Medical Innovation Forum, Mass General Brigham, Gene and Cell Therapy, VIRTUAL May 19–21, 2021

REAL TIME EVENT COVERAGE as PRESS by invitation from 2021 Virtual World Medical Innovation Forum at #WMIF2021 @MGBInnovation:

Aviva Lev-Ari, PhD, RN

Tweet Collection Curator:

Aviva Lev-Ari, PhD, RN

UPDATED Twitter Analytics

May 2021  31 days

TWEET HIGHLIGHTS

Top Tweet earned 611 impressions

@MGBInnovation#WMIF Best Global event on Gene Cell Therapy covered in real time @AVIVA1950@pharma_BI Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included pic.twitter.com/9Q2dWikhNd 1  2

View all Tweet activity View Tweet activity

Top Follower followed by 7,598 people

Ryan Gravatt@gravatt FOLLOWS YOU

Christian, father, husband. Owner @RaconteurMC. Strategist for comms, digital. Former award-winning journalist. Proverbs 3:5-6 View profile

Top mention earned 15 engagements

#COVID#vaccines by @Pfizer, @AstraZeneca are probed in @Europe after reports of #heart#inflammation, rare #nerve#disorderpharmaceuticalintelligence.com/2021/05/14/cov… via @pharma_BI@AVIVA1950 1  3View all Tweet activityView Tweet activity

MAY 2021 SUMMARY

Tweets

213

Tweet impressions

17.6K

Profile visits

861

Mentions

211

New followers

2

These are the Tweets and the Re-Tweets

by Day, 5/21, 5/20, 5/19 for

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

Real Time coverage: Aviva Lev-Ari, PhD, RN

LPBI Group’s Logo
Aviva Lev-Ari, PhD, RN, Founder, 1.0 LPBI Group and 2.0 LPBI Group

May 21, 2021

TWEETS AND RE-TWEETS for 2021 World Medical Innovation Forum, Mass General Brigham, Gene and Cell Therapy, VIRTUAL May 19–21, 2021

PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 21, 2021

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 21, 2021

Tweets Originator for Part 1: Aviva Lev-Ari, PhD, RN

From: Mass General Brigham <innovations@partners.org>
Reply-To: <innovations@partners.org>
Date: Monday, May 24, 2021 at 9:31 AM
To: “Aviva Lev-Ari, PhD, RN” <AvivaLev-Ari@alum.berkeley.edu>
Subject: RECAP | World Forum | Day 3 | GCT | CEOs | Harvard | Investors

Notable Tweets
@mandywoodland Fascinating #WMIF2021 panel on mRNA yesterday -“mRNA is the message, and we just have to decide what message we want to deliver to the cell,” said moderator Lindsey Baden, MD. “The promise of this technology could not be more front and center for all of us.”   @LeapsByBayer Congratulations to the 2021 Innovation Discovery Grants winners: @lynchielydia, Peter Sage, @GrishchukL, Benjamin Kleinstiver, Petr Baranov, announced at the #WMIF2021. It’s exciting to see the range of breakthrough research in #geneticdisease at @MassGenBrigham@DrLilitGaribyan Gene and cell therapy have scalability problems that we need to solve. This is what is echoed this week at @MGBInnovation World Medical Innovation Forum. #gct #celltherapy #healthcare #innovation   @MPDexpert “imagine how the future could look if gene therapy cost 1/100th what it does today” @VCAmir @PolarisVC #wmif2021  
@AVIVA1950 #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
Notable Tweets

 

Disruptive Dozen

2021 World Medical Innovation Forum on

YouTube

https://www.youtube.com/results?search_query=Disruptive+Dozen+2021+World+Medical+Innovation+Forum

Example for a TWEET

Aviva Lev-Ari

@AVIVA1950

·

May 21

@MGBInnovation

#WMIF Best Global event on Gene Cell Therapy covered in real time

@AVIVA1950

@pharma_BI

Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

2

2

Example for a RE-TWEET

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

May 21

Thanks

@AVIVA1950

for sharing this screen capture of the impressive lineup of #GCT “Disruptive Dozen” panelists at #WMIF2021

Quote Tweet

Aviva Lev-Ari

@AVIVA1950

 · May 21

@MGBInnovation #WMIF Best Global event on Gene Cell Therapy covered in real time @AVIVA1950 @pharma_BI Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

 PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 21, 2021

Aviva Lev-Ari

@AVIVA1950

·

4h

#WMIF2021

@MGBInnovation

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

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

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

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

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

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

Aviva Lev-Ari

@AVIVA1950

·

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

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3

Aviva Lev-Ari

@AVIVA1950

·

4h

#WMIF2021

@MGBInnovation

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

@pharma_BI

@AVIVA1950

1

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

Aviva Lev-Ari

@AVIVA1950

·

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

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

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

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

Aviva Lev-Ari

@AVIVA1950

·

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

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

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

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

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

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

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

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

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

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

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 21, 2021

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

The # of US patients with Parkinson’s Disease is expected to double over next 30 years. Penelope Hallett PhD, Co-Director of the Neuroregeneration Research Inst

@McLeanHospital

, presents a #regenerativemedicine approach that could alter that trajectory. #WMIF2021

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Our “Capital Formation ’21-30 | Investing Modes Driving GCT Technology and Timing” panelists have taken the stage. #WMIF2021

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

CAR-T therapies have proven remarkably effective. Now,

@MassGenBrigham

researchers including

@MGHCancerCenter

Marcela Maus, MD PhD, are working to expand the reach of this transformative technology. #WMIF2021

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #9. Building the Next Wave of CAR-T-cell Therapies #WMIF2021 #GCT #GeneAndCellTherapy #CellTherapy #CarT #DisruptiveDozen

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #6. Eyes and Ears: Expanding Gene Therapy’s Reach #WMIF2021 #GCT #GeneAndCellTherapy #GeneTherapy #DisruptiveDozen

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

May 20

If you’ve missed some First Look sessions, don’t worry! We’ve got you covered. Our First Look On-Demand videos, featuring 18

@MassGenBrigham

investigators giving previews of their #GCT research, are available to view on the #WMIF2021 conference platform. https://worldmedicalinnovation.org/register/

5

7

You Retweeted

REGENXBIO

@REGENXBIO

·

May 19

This morning at 10:20 a.m. ET, our CEO, Ken Mills, will be participating live on the AAV Success Studies virtual panel at the #WMIF2021, hosted by

@MGBInnovation

. Click here to register: https://bit.ly/33tHTti #Genetherapy

Register | World Medical Innovation Forum – Gene and Cell Therapy

Hear from industry-leading experts discuss the advances and future of GCT in health care. May 19-21, 2021; Mass General Brigham. Register!

worldmedicalinnovation.org

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You Retweeted

Brett P. Monia, Ph.D.

@BPMonia

·

May 20

Looking forward to joining

@MGBInnovation

and global colleagues at #WMIF2021. On Thursday, May 20, my colleagues and I will discuss the advantages of RNA-targeted medicines and how they might shape the future of medicine for patients.

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · May 10

Are you part of the @MassGenBrigham network and interested in #GeneAndCellTherapy? Join us at the World Medical Innovation Forum on 5/19-5/21. Register today! https://worldmedicalinnovation.org/register/ #WMIF2021

1

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You Retweeted

Maria Luiza Gutierrez de Andrade Seixas

@MLGASeixas

·

May 16

Incredible opportunity to get up to speed with the most innovative technologies in medicine ! Gene and cell therapy are revolutionizing healthcare ! #WMIF2021 #MedTwitter

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · May 11

#WMIF2021 is an opportunity for innovators from around the globe to meet, explore, challenge, and reflect on the issues influencing the adoption of novel technologies in #healthcare. Register now to join the conversation: https://worldmedicalinnovation.org/register/

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

Currently, the only cure for some common blood disorders is a bone marrow transplant, which can be risky. Now, gene therapies are also in the works, including a CRISPR-based #genetherapy being tested in clinical trials with encouraging early results. #WMIF2021

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #2. A Genetic Fix for Two Common Blood Disorders #WMIF2021 #GCT #GeneAndCellTherapy #BloodDisorders #DisruptiveDozen

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

Researchers have pinpointed key genes involved in cholesterol and lipid metabolism that represent promising targets for new cholesterol-lowering treatments. #WMIF2021

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #1. A New Generation of Cholesterol-Loweing Therapies #WMIF2021 #GCT #GeneAndCellTherapy #DisruptiveDozen

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1

You Retweeted

Harvard Ophthalmology

@HMSeye

·

May 19

The

@MGBInnovation

#WMIF2021 event kicks of this morning! Congratulations to faculty member and event Co-Chair

@VandenbergheLuk

on putting together such a terrific program. Register: https://bit.ly/3uWYB0E

4

9

You Retweeted

Yulia Grishchuk Lab

@GrishchukL

·

4h

I really enjoyed this remarkable panel #WMIF2021. Thank you Meredith Fisher for moderating and thank you David, Bob and Kush for openly sharing your big picture view

1

4

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

2h

Thank you to our World Medical Innovation Forum Collaborators

@Amplify_Bio

@bostonsci

@CanonUSA

@CatalentPharma

@InterSystems

@nlvcofficial

@onemedical

@ReconStrategy

@SiemensHealth

@thermofisher

@VertexPharma

#WMIF2021

You Retweeted

Tracy Doyle

@doylet

·

5h

Variability, delays, manufacturing as an afterthought make #GCT challenging from an investment POV — need to rethink the ecosystem and drive efficiency, invest in tech innovation says Bob Nelson ARCH Venture Partners

@MGBInnovation

#WMIF2021

1

You Retweeted

Tracy Doyle

@doylet

·

5h

We need to change the scale and scope of how #GCT is advancing from discovery to development — systematization critical. Can’t have thousands of one-off therapies say early-stage investors. Major mis-match between where things are now and what could be.

@MGBInnovation

#WMIF202

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2

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #8. Replacing What’s Lost: Stem Cell Therapies for Diabetes #WMIF2021 #GCT #GeneAndCellTherapy #StemCell #StemCellResearch #Diabetes #DisruptiveDozen

3

2

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

21h

An overview of our CEO Panel featuring Lisa Deschamps of

@NovartisGene

, Kieran Murphy of

@GEHealthcare

and Christian Rommel PhD, of

@Bayer

#WMIF2021

4

7

You Retweeted

Mass General Brigham

@MassGenBrigham

·

4h

Gene and cell therapies could change the future of medicine for patients w chronic disease or rare/ultra-rare disease – hear how

@MassGenBrigham

is working w the GCT ecosystem to drive new discoveries from bench to bedside #GCT #WMIF2021

5

11

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

2h

That’s a wrap! Thank you to everyone who helped make #WMIF2021 such a success, especially our incredible sponsors:

@NovartisGene

@Bayer

@GEHealthcare

@AstellasUS

@biogen

@FujifilmHealth

and more. Full list: https://worldmedicalinnovation.org/sponsors/

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

3h

Disruptive Dozen: 12 Technologies that Will Reinvent GCT #1. A New Generation of Cholesterol-Loweing Therapies #WMIF2021 #GCT #GeneAndCellTherapy #DisruptiveDozen

5

2

You Retweeted

Natalie Artzi

@NatalieArtzi

·

17h

Today I moderated a panel on Gene and Cell Therapy Delivery, Perfecting the Technology. We highlighted non-viral delivery technologies as key enablers of gene therapy and editing. Learn more: https://lnkd.in/d-Xqzqh #WMIF2021

3

12

You Retweeted

Yulia Grishchuk Lab

@GrishchukL

·

5h

Thank you

@MGBInnovation

and

@LeapsByBayer

for this award! Congratulations to

@BKleinstiver

and all other winners!

@MGH_RI

@CGM_MGH

! #WMIF2021

Quote Tweet

Leaps by Bayer

@LeapsByBayer

 · 6h

Congratulations to the 2021 Innovation Discovery Grants winners: @lynchielydia, Peter Sage, @GrishchukL, Benjamin Kleinstiver, Petr Baranov, announced at the #WMIF2021. It’s exciting to see the range of breakthrough research in #geneticdisease at @MassGenBrigham…

Show this thread

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You Retweeted

Natalie Artzi

@NatalieArtzi

·

17h

An artistic description of an exciting panel I led today, at the World Biomedical Innovation Forum, discussing the future of non-viral delivery systems for gene therapy. #MatthewStanton #LauraSeppLorenzino #DouglasWilliams #SonyaMontgomery #WMIF2021

May 20, 2021

TWEETS AND RE-TWEETS for 2021 World Medical Innovation Forum, Mass General Brigham, Gene and Cell Therapy, VIRTUAL May 19–21, 2021

PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 20, 2021

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 20, 2021

Tweets Originator for Part 1: Aviva Lev-Ari, PhD, RN

Example for a TWEET

Aviva Lev-Ari

@AVIVA1950

·

May 21

@MGBInnovation

#WMIF Best Global event on Gene Cell Therapy covered in real time

@AVIVA1950

@pharma_BI

Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

2

2

Example for a RE-TWEET

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

May 21

Thanks

@AVIVA1950

for sharing this screen capture of the impressive lineup of #GCT “Disruptive Dozen” panelists at #WMIF2021

Quote Tweet

Aviva Lev-Ari

@AVIVA1950

 · May 21

@MGBInnovation #WMIF Best Global event on Gene Cell Therapy covered in real time @AVIVA1950 @pharma_BI Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 20, 2021

Aviva Lev-Ari

@AVIVA1950

·

2h

#WMIF2021

@MGBInnovation

Bob Brown, PhD CSO, EVP of R&D, Dicerna small molecule vs capacity of nanoparticles to deliver therapeutics quantity for more molecule is much larger CNS delivery most difficult

@pharma_BI

@AVIVA1950



Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Jeannie Lee, MD, PhD Molecular Biologist, MGH Prof Genetics, HMS 200 disease X chromosome unlock for neurological genetic diseases: Rett Syndrome, autism spectrum disorders female model vs male mice model restore own protein

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Suneet Varma Global President of Rare Disease, Pfizer review of protocols and CGT for Hemophilia Pfizer: You can’t buy Time With MIT Pfizer is developing a model for Hemophilia CGT treatment

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Gallia Levy, MD, PhD CMO, Spark Therapeutics Hemophilia CGT is the highest potential for Global access logistics in underdev countries working with NGOs practicality of the Tx Roche reached 120 Counties great to be part of the Roche

@pharma_BI

@AVIVA1950

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Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

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 

@pharma_BI

@AVIVA1950

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1

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Suneet Varma Global President of Rare Disease, Pfizer Gene therapy at Pfizer small, large molecule and CGT – spectrum of choice allowing Hemophilia patients to marry 1/3 internal 1/3 partnership 1/3 acquisitions  review of protocols

@pharma_BI

@AVIVA1950

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1

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Ron Renaud CEO, Translate Bio What strain of Flu vaccine will come back in the future when people do not use masks. AAV vectors small transcript size fit reach cytoplasm more development coming

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Melissa Moore Chief Scientific Officer, Moderna Flu vaccine knowing the virus variant 45 days for Personalized cancer vaccine one per patient

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Melissa Moore Chief Scientific Officer, Moderna 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

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Melissa Moore Chief Scientific Officer, Moderna How many mRNA can be put in one vaccine: Dose and tolerance to achieve efficacy and the 

@pharma_BI

@AVIVA1950

1

2

Aviva Lev-Ari

@AVIVA1950

·

9h

#WMIF2021

@MGBInnovation

Lindsey Baden, MD Director, Clinical Research, Division of Infectious Diseases, BWH Associate Professor, HMS In vivo delivery process regulatory for new opportunities for same platform new indication using multi valence vaccines

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Ron Renaud CEO, Translate Bio Platform allowing to swap cargo reusing same nanoparticles address disease beyond Big Pharma options for biotech

@pharma_BI

@AVIVA1950

#WMIF2021

@MGBInnovation

Melissa Moore Chief Scientific Officer, Moderna 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

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Ron Renaud CEO, Translate Bio 1.6 Billion doses produced rare disease monogenic correct mRNA like CF multiple mutation infection disease and oncology applications

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

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

@pharma_BI

@AVIVA1950

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Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Melissa Moore CSO, Moderna mRNA vaccine 98% efficacy for Pfizer and Moderna more then 10 years 2015 mRNA was ready (ZIKA, RSV), as the proteine is identify manufacturing temp less of downside in the future ability to store at Ref

@pharma_BI

@AVIVA1950

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1

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Shunfei Yan, PhD Investment Manager, InnoStar Capital Indication driven: Hymophilia,  Allogogenic efficiency therapies Licensing opportunities 

@pharma_BI

@AVIVA1950

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1

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Richard Wang, PhD CEO, Fosun Kite Biotechnology Co. Ltd Possibilities to be creative and capitalize the new technologies for new drug Support of the ecosystem by funding new companies Autologous in patients differences cost challenge

@pharma_BI

@AVIVA1950

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1

Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

Tian Xu, PhD Vice President, Westlake University ICH Chinese FDA -r regulation similar to the US Difference is the population recruitment, in China patients are active participants Dev of transposome non-viral methods, price

@pharma_BI

@AVIVA1950

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Aviva Lev-Ari

@AVIVA1950

·

10h

#WMIF2021

@MGBInnovation

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 delay

@pharma_BI

@AVIVA1950

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 20, 2021

You Retweeted

Vertex Pharmaceuticals

@VertexPharma

·

May 19

We’re excited to attend this week’s #WMIF2021 to talk all things cell and genetic therapies. Join our Chief of VCGT Bastiano Sanna tomorrow at 9:50am EDT for a discussion on the promise of cell therapies for type 1 diabetes. Register now! https://bit.ly/3otngYd

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

8h

John Fish, Board Chair, Brigham Health, Chairman & CEO, Suffolk on the Novartis Main Stage to introduce the “Collaboration is Key: GCT R&D of the Future” fireside chat with Jay Bradner, MD, President, NIBR

@NovartisScience

. #WMIF2021

2

2

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

In our next First Look presentation we’ll hear from Xandra Breakefield PhD & Koen Breyne PhD

@MGHNeurology

@MGHNeurosurg

about their work focused on developing non-viral vectors to enhance #genedelivery. #WMIF2021 #GCT #genetherapy

More Topics

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

May 19

Thomas VanCott, PhD, Chief Technology & Strategy Officer, Catalent Cell & Gene Therapy, says that time, improvements and scaling up in manufacturing will lead to allogeneic cell therapies. He recognizes that upfront costs are high, but will decrease in the long term #WMIF2021

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Ravi Thadhani, CAO

@MassGenBrigham

and Juergen Eckhardt, Head of

@LeapsbyBayer

, are announcing the 2021 Innovation Discovery Grants this afternoon at #WMIF2021.

1

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You Retweeted

Editas Medicine

@editasmed

·

10h

Today Lisa Michaels, Editas CMO, will participate in the panel “Gene Editing – Achieving Therapeutic Mainstream” at the World Medical Innovation Forum #WMIF2021 in Boston. For those attending, be sure to tune in!

@MassGenBrigham

https://bit.ly/3hx1XTV #geneediting #biotechnology

Gene Editing | Achieving Therapeutic Mainstream – 2021 World Medical Innovation Forum

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…

worldmedicalinnovation.org

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Mass General Brigham Innovation

@MGBInnovation

·

3h

A behind the scenes peek at our “Gene Editing | Achieving Therapeutic Mainstream” moderator & panelists preparing to go live. #WMIF2021

1

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

2h

Thank you to the “Common Blood Disorders | Gene Therapy” moderator David Scadden, MD

@ScaddenLab

@harvardstemcell

and panelists Leslie Kean, MD PhD

@DanaFarberNews

, Samarth Kulkarni, PhD

@CRISPRTX

, Nick Leschly

@bluebirdbio

, Mike McCune, MD PhD

@gatesfoundation

. #WMIF2021

1

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

Kieran Murphy, CEO,

@GEHealthcare

, views GCT as the ultimate precision medicine. AI, machine learning, and data science comprise one of the big disruptive forces that will address misdiagnosis, smooth out workflow, reduce cost and enhance recovery. #WMIF2021

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

2h

Wrapping up Day 2 of #WMIF2021 with the “Gene Expression | Modulating with Oligonucleotide-Based Therapies” panel.

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Juergen Eckhardt, Head of

@LeapsbyBayer

, announces new Bayer mentoring program for Innovation Discovery Grant winners at #WMIF2021.

3

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

2h

In our final First Look session of the day, Pierpaolo Peruzzi, MD PhD,

@BWHNeurosurgery

presents “RNA Therapy for Brain Cancer” #WMIF2021

1

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

Hear from

@intelliatweets

CSO Laura Sepp-Lorenzino, PhD, in our “GCT Delivery | Perfecting the Technology” panel this afternoon! #WMIF2021

Quote Tweet

Intellia Therapeutics

@intelliatweets

 · 6h

Today, Intellia CSO, @LauraSeppLore will be participating in the World Medical Innovation Forum’s panel on Gene and Cell Therapy Delivery, Perfecting the Technology. #WMIF2021 @MGBInnovation. Click here to learn more: https://worldmedicalinnovation.org

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Natalie Artzi, PhD, Assistant Professor

@BrighamWomens

is back with us this afternoon sharing a First Look at “Versatile Polymer-Based Nanocarriers for Targeted Therapy and Immunomodulation.” #WMIF2021 #GCT #geneandcelltherapy

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

8h

We look forward to hearing from

@ViaCyte

VP of Clinical Development, Manasi Jaiman, during the “Diabetes | Grand Challenge” panel today. #WMIF2021

Quote Tweet

ViaCyte

@ViaCyte

 · 8h

Join us at #WMIF2021 today! Our own Manasi Jaiman, VP, Clinical Development, will participate in the Diabetes: Grand Challenge panel to discuss regenerative medicine approaches for T1D utilizing stem-cell derived islet cell replacement therapy.

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

5h

We’ll see you back here after the break for the “GCT Delivery | Perfecting the Technology” panel, featuring moderator Natalie Artzi, PhD,

@BrighamWomens

and panelists from

@EvOx_Ltd

,

@intelliatweets

,

@generationbio

and

@codiakbio

. #WMIF2021

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

Christian Rommel, PhD,EVP, Head, Pharmaceuticals Research & Development,

@Bayer

, discusses how GCT is in the embryonic phase. Bayer is ready to treat its first Parkinson’s patient, and is exploring therapeutic technologies to treat diseases with single gene defects #WMIF2021

1

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

8h

Next up is the #Diabetes | Grand Challenge panel at #WMIF2021 featuring speakers from

@BrighamWomens

@armi_usa

@ViaCyte

@VertexPharma

@Sigilon_Inc

3

5

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

10h

The “Gene Editing | Achieving Therapeutic Mainstream” panel today at 2:55 pm Eastern will discuss the movement of #geneediting technology into the therapeutic mainstream. Join us! #WMIF2021 https://worldmedicalinnovation.org/register/

Quote Tweet

Editas Medicine

@editasmed

 · 10h

Today Lisa Michaels, Editas CMO, will participate in the panel “Gene Editing – Achieving Therapeutic Mainstream” at the World Medical Innovation Forum #WMIF2021 in Boston. For those attending, be sure to tune in! @MassGenBrigham https://bit.ly/3hx1XTV #geneediting #biotechnology

You Retweeted

Atara Bio

@Atarabio

·

2h

Global Head of R&D

@jdupontmd

joined this week’s World Medical Innovation Forum hosted by

@MGBInnovation

to discuss the current state of CAR-T and its future prospects. These conversations are important for the development of potential #CART therapies. #WMIF2021

1

8

You Retweeted

Tracy Doyle

@doylet

·

9h

“We can get to an “n of 1” with mRNA technology says Melissa Moore, PhD, CSO Platform Research,

@moderna_tx

@MGBInnovation

#WMIF2021 #GCT

1

1

You Retweeted

Intellia Therapeutics

@intelliatweets

·

6h

Today, Intellia CSO,

@LauraSeppLore

will be participating in the World Medical Innovation Forum’s panel on Gene and Cell Therapy Delivery, Perfecting the Technology. #WMIF2021

@MGBInnovation

. Click here to learn more: https://worldmedicalinnovation.org

3

4

You Retweeted

TranslateBio

@TranslateBio

·

7h

Graphical representation of this morning’s #mRNA #vaccines panel at

@MGBInnovation

‘s #WMIF2021 — Thanks to the MGB team for facilitating a great discussion!

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 7h

Overview of our #mRNA Vaccines panel today, highlighting improved manufacturing capabilities & potential for #personalizedmedicine. Thank you to Lindsey Baden @bwh_id & panelists Kate Bingham, SV Health Investors, Melissa Moore @moderna_tx and Ron Renaud @TranslateBio #WMIF2021

1

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You Retweeted

Tracy Doyle

@doylet

·

May 19

18

@MassGenBrigham

investigators are ready to give you an early preview of their #GCT research in the First Look sessions at #WMIF2021. Exciting opportunities to dramatically change how disease is treated!

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

7h

Please welcome Marc Casper, CEO

@thermofisher

to the stage for a Fireside Chat moderated by Erin Harris

@ErinHarris_1

, Editor in Chief

@_CellandGene

“Partnering Across the GCT Spectrum” #WMIF2021 #GCT #geneandcelltherapy

4

3

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

The “CEO Panel | Anticipating Disruption | Planning for Widespread GCT” panelists have joined the stage. #WMIF2021

1

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You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

7h

Our “Rare and Ultra Rare Diseases | GCT Breaks Through” panelists on the role of family organizations & patient advocacy groups in moving us forward on the regulatory side – “It’s absolutely essential” #WMIF2021

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Congratulations! Lydia Lynch PhD, Brigham and Women’s Hospital receives an Innovation Discovery Grant for “Generating Superior ‘Killers’ for Adoptive Cell Therapy in Cancer” at #WMIF2021.

@BrighamWomens

@BrighamResearch

2

You Retweeted

Tracy Doyle

@doylet

·

10h

Looking forward to the Diabetes Grand Challenge and how #GCT could help millions of people. Read about what facing this disease and how cell therapies could lessen the burden from Manasi Jaiman, MD, VP, Clinical Development

@ViaCyte

here http://bit.ly/T1Dcelltherapies… #WMIF2021

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 11h

Today is Day 2 of the World Medical Innovation Forum. Which panel you are most excited to see today? Reply and let us know! #WMIF2021 https://worldmedicalinnovation.org/agenda/

2

2

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

Coming up at 12:05 pm Eastern: “CEO Panel | Anticipating Disruption | Planning for Widespread GCT” featuring panelists from

@NovartisGene

@GEHealthcare

@Bayer

and moderated by

@CNBC

Senior Health and Science Reporter

@megtirrell

#WMIF2021

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

10h

Join us at #WMIF2021 to hear from Suneet Varma, Global President of Rare Disease

@Pfizer

, during the “Benign Blood Disorders” today at 9:00 am Eastern. https://worldmedicalinnovation.org/register/

Quote Tweet

Pfizer Inc.

@pfizer

 · May 19

Cell and gene therapies hold promising potential for rare disease, blood cancers, and viral diseases. Register for #WMIF21 to hear about our work to pioneer cutting-edge science across our pipeline to advance breakthroughs that change patients’ lives: https://on.pfizer.com/3f3CGzj

2

1

You Retweeted

Pearl Freier

@PearlF

·

9h

Melissa Moore/Moderna said they are working with Merck on developing personalized cancer vaccines, n of 1 #wmif2021

1

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Congratulations! Peter Sage PhD, Brigham and Women’s Hospital receives an Innovation Discovery Grant for “Novel Strategies to Enhance Tfr Treatment of Autoimmunity” at #WMIF2021

@BrighamWomens

@BrighamResearch

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Congratulations! Yulia Grishchuk PhD, Massachusetts General Hospital, receives an Innovation Discovery Grant for “AAV-Based Gene Replacement Therapy Improves Targeting and Clinical Outcomes in a Childhood CNS Disorder” at #WMIF2021

@MassGeneralNews

@MGH_RI

@CGM_MGH

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Congratulations! Jinjun Shi, PhD, Brigham and Women’s Hospital, receives an Innovation Discovery Grant for “Long-Lasting mRNA Therapy for Genetic Disorders” at #WMIF2021

@BrighamWomens

@BrighamResearch

2

2

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

9h

Final thoughts from “Benign Blood Disorders” panelists on academic/industry collaboration — the pace of #innovation is incredibly exciting, and I think it will be even faster together. #WMIF2021

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

4h

Congratulations! Benjamin Kleinstiver PhD, Massachusetts General Hospital, receives an Innovation Discovery Grant for “Towards a Permanent Genetic Cure for Spinal Muscular Atrophy” at #WMIF2021

@MassGeneralNews

@MGH_RI

@CGM_MGH

2

You Retweeted

Pearl Freier

@PearlF

·

10h

Listening to mRNA vaccines #wmif2021 panel w/ speakers incl SV Health managing partner & ex UK Vaccine Taskforce

@katebingham2

, Moderna CSO Platform Rsrch Melissa Moore,

@TranslateBio

CEO Ron Renaud

@biotech1969

, Brigham/BWH Dir Clinical Research Infectious Disease Lindsey Baden

2

2

You Retweeted

Ned Pagliarulo

@NedPagliarulo

·

May 19

FDA’s Peter Marks, at #WMIF2021, notes # of INDs for gene therapies was flat in 2020 vs. 2019. But the fact IND submissions didn’t decline, he said, is a sign of how strong the gene therapy field is, given pandemic’s disruption.

1

9

21



You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

5h

Coming up this afternoon: the 2021 Innovation Discovery Grants in #geneandcelltherapy. Who will secure additional funding for research to advance #GCT? Join us to watch live. #WMIF2021 https://worldmedicalinnovation.org/register/

2

1

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

1h

Thank you Jeannie Lee, MD PhD

@MGHPathology

, Bob Brown, PhD

@DicernaPharma

, Brett Monia, PhD

@ionispharma

, and Alfred Sandrock, MD PhD

@biogen

for sharing your perspectives on oligonucleotide-based therapies. #WMIF2021

1

2

You Retweeted

Pearl Freier

@PearlF

·

9h

Melissa Moore/Moderna- one advantage of mRNA is ability to do multivalent vaccines she said. She said they are already testing multivalent covid vaccines in clinical trials & testing flu vaccines. #wmif2021

1

3

You Retweeted

Pearl Freier

@PearlF

·

10h

Kate Bingham/SV Health & former head of UK Vaccine Taskforce: they haven’t seen escape variants in UK yet she said. mRNA is quickest platform to address escape variants probably. Needle delivery w/ supply cold chain has been the challenge. Deploying 3 vaccines in UK #WMIF2021

1

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Show this thread

You Retweeted

Tracy Doyle

@doylet

·

6h

Marc Casper

@thermofisher

says gene and cell therapy represents a “phenomenal opportunity to improve patients’ lives” #WMIF2021 #GCT

1

2

You Retweeted

TranslateBio

@TranslateBio

·

7h

Today, our CEO Ron Renaud

@biotech1969

participated in

@MGBInnovation

‘s 2021 World Medical Innovation Forum to discuss the impact of #messengerRNA #vaccines on the industry #WMIF2021 #mRNA

2

10

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

6h

Lisa Deschamps, SVP & Chief Business Officer,

@NovartisGene

, notes that the science behind gene cell therapies is converging with technological development. How therapies are brought to market is still the question, as there is no roadmap when reimagining medicine #WMIF2021

3

4

You Retweeted

Pearl Freier

@PearlF

·

10h

Melissa Moore/Moderna: clear advantage of mRNA vaccine is how quickly we can manufacture the vaccines. Downsides- need 2store at low temperatures & limited shelflife 4storage in refrigerator. I know that both companies [Moderna, Pfizer/BioNTech] r working 2change this #wmif2021

You Retweeted

Novartis Gene Therapies

@NovartisGene

·

6h

We’re committed to addressing the unmet needs of people living with rare genetic diseases. Our SVP, External Innovation and Strategic Alliances, Leah Bloom, discusses the promise #genetherapy holds for communities impacted by rare diseases during #WMIF2021.

2

4

You Retweeted

Tracy Doyle

@doylet

·

6h

Diagnostics and data tools key part of precision medicine complementing gene and cell therapy says

@KieranMurphyCEO

@GEHealthcare

at

@MGBInnovation

#WMIF2021

Meg Tirrell and 2 others

2

2

You Retweeted

Tracy Doyle

@doylet

·

7h

Debating the value of natural history studies in rare/ultra rare disease — panel led by Susan Slaugenhaupt, PhD, scientific director,

@MGH_RI

at #WMIF2021. Challenges include costs, feasibility, timing, comparative data.

1

2

You Retweeted

Tracy Doyle

@doylet

·

8h

Rett’s Syndrome, which primarily affects young girls, has historically been studied in male mice! Jeannie Lee, MD, PhD,

@MassGeneralNews

, and team are exploring how to treat the disease w X chromosome reactivation… and using a female mouse model. Hear more on #GCT at #WMIF2021

2

5

You Retweeted

Tracy Doyle

@doylet

·

10h

Speed of vaccination is critical to prevent escape variants says Kate Bingham, SV Health Investors, UK, at #WMIF2021, exploring what’s next for the technology w panel led by Lindsey Baden MD,

@BrighamWomens

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2

May 19, 2021

TWEETS AND RE-TWEETS for 2021 World Medical Innovation Forum, Mass General Brigham, Gene and Cell Therapy, VIRTUAL May 19–21, 2021

PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 19, 2021

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 19, 2021

Tweets Originator for Part 1: Aviva Lev-Ari, PhD, RN

Example for a TWEET

Aviva Lev-Ari

@AVIVA1950

·

May 21

@MGBInnovation

#WMIF Best Global event on Gene Cell Therapy covered in real time

@AVIVA1950

@pharma_BI

Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

2

2

Example for a RE-TWEET

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

May 21

Thanks

@AVIVA1950

for sharing this screen capture of the impressive lineup of #GCT “Disruptive Dozen” panelists at #WMIF2021

Quote Tweet

Aviva Lev-Ari

@AVIVA1950

 · May 21

@MGBInnovation #WMIF Best Global event on Gene Cell Therapy covered in real time @AVIVA1950 @pharma_BI Disruptive Dozen technologies four are based on Gene Editing, AAV and non viral vector for drug delivery are included

 PART 1: ALL THE TWEETS PRODUCED by @AVIVA1950 on May 19, 2021



Aviva Lev-Ari

@AVIVA1950

·

17h

#WMIF2021

@MGBInnovation

Marcela Maus, MD, PhD Director, Cancer Center, MGH, 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 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

17h

#WMIF2021

@MGBInnovation

Thomas VanCott, PhD Global Head of Product Dev, Gene & Cell Therapy, Catalent 2/3 autologous 1/3 allogeneic  CAR-T high doses scale up is not done today logistics issues centralized vs decentralized allogeneic are health donors

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

17h

#WMIF2021

@MGBInnovation

Ropa Pike, Director,  Enterprise Science & Partnerships, Thermo FIsher Scientific  Centralized biopharma industry is moving  to decentralized models site specific license 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

17h

#WMIF2021

@MGBInnovation

Rahul Singhvi, ScD CEO and Co-Founder, National Resilience, Inc. Investment company in platforms to be shared by start ups in CGT. Production cost of allogeneic: cost of quality 30% reagents 30% cell 30% Test is very expensive 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

Oladapo Yeku, MD, PhD Clinical Assistant in Medicine, MGH Outstanding moderator and most gifted panel on solid tumor success window of opportunities studies 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

Knut Niss, PhD CTO, Mustang Bio tumor hot start in 12 month clinical trial solid tumors Combination therapy will be an experimental treatment long journey checkpoint inhibitors to be used in combination maintenance 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

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

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

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 

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

Jay Short, PhD Chairman, CEO, Cofounder, BioAlta, Inc. Tumor type is not enough for R&D therapeutics other organs are involved in periphery difficult to penetrate solid tumors biologics activated in the tumor only, positive changes

@pharma_BI

@AVIVA1950

1

1

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

Christi Shaw CEO, Kite CAR-T is priority 120 companies in the space Manufacturing consistency  Patients respond with better quality of life

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

18h

#WMIF2021

@MGBInnovation

Stefan Hendriks Global Head, Cell & Gene, Novartis Confirmation the effectiveness of CAR-T therapies, 1 year response to 5 years 26 months Patient not responding a lot to learn Patient after 8 months of chemo can be helped by CAR-T

@pharma_BI

@AVIVA1950



Aviva Lev-Ari

@AVIVA1950

·

19h

#WMIF2021

@MGBInnovation

Jeffrey Infante, MD , Oncology, Janssen R&D 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

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

19h

#WMIF2021

@MGBInnovation

Nino Chiocca, MD, PhD Neurosurgeon-in-Chief BWH, HMS Oncolytic therapy DID NOT WORK Pancreatic Cancer and Glioblastoma Intra-tumoral heterogeniety hinders success Oncolytic VIRUSES – “coldness” GADD-34 20,000 GBM 40,000 pancreatic

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

19h

#WMIF2021

@MGBInnovation

Loic Vincent, PhD Head of Oncology Drug Discovery Unit, Takeda Classification of Patients by prospective response type id UNKNOWN yet, population of patients require stratification

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

Loic 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 IV

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

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?

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

David Reese, MD Executive Vice President, R&D , Amgen Inter lesion injection of agent vs systemic therapeutics cold tumors immune resistant render them immune susptible Oncolytic virus is a Mono therapy addressing the unknown 

@pharma_BI

@AVIVA1950

2

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

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 suseptible Oncolytic virus is a Mono therapy

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

Robert Coffin, PhD Chief R&D 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 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

20h

#WMIF2021

@MGBInnovation

Ann Silk, MD Physician, Dana Farber-Brigham and Women’s Cancer Center, HMS Which person gets oncolytics virus if patient has immune supression due to other indications Safety of oncolytic virus greater than Systemic treatment

@pharma_BI

@AVIVA1950

2

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

@pharma_BI

@AVIVA1950

amazing Conference on the frontier od Science Cell & Gene Therapy

@MGB

top programs for ALS, Brain genetic vasculopathologies and Occular, MEE

@pharma_BI

@AVIVA1950

Quote Tweet

Pearl Freier

@PearlF

 · 21h

Marianne De Backer/Bayer on post M&A & company culture: They acquired AskBio & thought about how to preserve their freedom so they could continue to operate. Bayer decided to keep them independent & so they can operate at arm’s length. #wmif2021



Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

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 @pharma_BI@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

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?

@pharma_BI

@AVIVA1950

2

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

Debby Baron, Worldwide Business Development, Pfizer  Scalability and manufacturing regulatory conversations, clinical programs safety in parallel to planning getting drug to patients

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

Marianne De Backer, PhD Head of Strategy, BD & Licensing, Bayer Absolute Leadership: Gene editing, gene therapy, via acquisition and alliances Operating model of the acquired company discussed acquired continue independence

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

Sean Nolan Board Chairman, Encoded Therapeutics & Affinia Executive Chairman Jaguar Gene Therapy Istari Oncology As acquiree multiple M&A acquirer looks at integration and cultures companies  Traditional integration vs acquisition 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

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 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

21h

#WMIF2021

@MGBInnovation

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 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

22h

2 people unfollowed me // automatically checked by

fllwrs – keep track of who follows and unfollows you on twitter

fllwrs is the easiest way to keep track of your twitter followers

fllwrs.com

Aviva Lev-Ari

@AVIVA1950

·

22h

#WMIF2021

@MGBInnovation

Manny Simons, PhD CEO, Akouos 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

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

22h

#WMIF2021

@MGBInnovation

Mathew Pletcher, PhD SVP, Head of Gene Therapy Research and Technical Operations, Astellas Continue to explore large animal guinea pig not the mice, not primates (ethical issues) for understanding immunogenicity and immune response 

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

22h

#WMIF2021

@MGBInnovation

Mathew 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 

@pharma_BI

@AVIVA1950



Aviva Lev-Ari

@AVIVA1950

·

23h

#WMIF2021

@MGBInnovation

Rick Modi CEO, Affinia Therapeutics Speed R&D Speed better gene construct get to clinic with better design vs ASAP Data sharing clinical experience patients selection, vector selection, mitigation, patient type specific

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

23h

#WMIF2021

@MGBInnovation

Dave Lennon, PhD President, Novartis Gene Therapies big pharma therapeutics not one drug across Tx areas: cell, gene iodine therapy collective learning infrastructure development Acquisitions growth # applications for scaling 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

23h

#WMIF2021

@MGBInnovation

Rick Modi CEO, Affinia Therapeutics Copy, paste EDIT from product A to B novel vectors variant of vector coder optimization choice of indication is critical exploration on larger populations Speed to R&D to better gene construct get

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

23h

#WMIF2021

@MGBInnovation

Louise Rodino-Klapac, PhD EVP, Chief Scientific Officer, Sarepta Therapeutics AV based platform 15 years in development 1 disease indication vs more than one indication stereotype, analytics as hurdle 1st was 10 years 2nd was 3 years

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Katherine High, MD President, Therapeutics, AskBio Three drugs approved in Europe in the CGT Regulatory Infrastructure CGT drug approval – as new class of therapeutics Participants investigators, regulators, patients i.e., MDM 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Peter Marks, MD, PhD Director, Center for Biologics Evaluation and Research, FDA Immune modulators Immunotherapy Genome editing can make use of viral vectors future technologies nanoparticles and liposome encapsulation 50% more staff

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Peter Marks, MD, PhD Director, Center for Biologics Evaluation and Research, FDA Recover Work load for the pandemic Gene Therapies IND application remained flat Rare diseases urgency remains Guidance T-Cell therapy vs Regulation

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Peter Marks, MD, PhD Director, Center for Biologics Evaluation and Research, FDA June 2020 belief that vaccine challenge manufacture scaling up FDA did not predicted the efficacy of mRNA vaccine vs other approaches expected to work

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Jim Holland CEO, http://Backcountry.com Parkinson patient Constraints by regulatory on participation in clinical trial wish to take Information dissemination is critical 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Patricia Musolino, MD, PhD Co-Director Pediatric Stroke and Cerebrovascular Program What is the Power of One – the impact that a patient can have on their own destiny connecting with other participants in same trial can be beneficial

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Barbara Lavery Chief Program Officer, ACGT Foundation Patient has the knowledge of the symptoms and recording all input needed for diagnosis by multiple clinicians Early application for CGT

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Sarah Beth Thomas, RN Professional Development Manager, BWH Outcome is unknown, hope for good, support with resources all advocacy groups, 

@pharma_BI

@AVIVA1950



Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Jack Hogan Patient, MEE Constraints by regulatory on participation in #clinicaltrials advance stage is approved participation Patients to determine the level of #risk they wish to take 

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Barbara Lavery Chief Program Officer, ACGT Foundation Advocacy agency beginning of work Global Genes educational content and out reach to access the information

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Dave Lennon, PhD President, Novartis Gene Therapies Modality one time intervention, long duration of impart, reimbursement, ecosystem FDA works by indications and risks involved, Standards manufacturing payments over time payers

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Dave Lennon, PhD President, Novartis Gene Therapies Promise of CGT realized, what part? #FDA role and interaction in CGT #Manufacturing aspects which is critical

@pharma_BI

@AVIVA1950

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Julian Harris, MD Partner, Deerfield Hope that CGT emerging, how therapies work, #neuro, #muscular, #ocular, #genetic diseases of #liver and of #heart revolution for the industry 900 #IND application 25 approvals #Economic driver 

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Luk 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

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

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 #delivery

@pharma_BI

@AVIVA1950

1

Aviva Lev-Ari

@AVIVA1950

·

May 19

#WMIF2021

@MGBInnovation

Nino Chiocca, MD, PhD Neurosurgeon-in-Chief and Chairman, Neurosurgery, BWH #Oncolytic #Viruses triple threats #Toxic, #braintumors #immunological requires #combination #therapies with #anticancer

@pharma_BI

@AVIVA1950

Part 2: ALL THE RE-TWEETS by @AVIVA1950 on

May 19, 2021

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

17h

Will point of care production become a reality? “Short answer is yes” says Rupa Pike PhD, Director, Enterprise Science & Innovation Partnerships,

@thermofisher

. #WMIF2021 #GCTManufacturing

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Novartis Gene Therapies

@NovartisGene

·

May 18

The field of #genetherapy is growing. New therapies will come to market for rare and chronic diseases, and new therapies will drive scientific innovation and economic growth. #WMIF2021 (2/6)

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Aviva Lev-Ari

@AVIVA1950

·

15h

Very creative two targets

@ScaddenLab

@pharma_BI

@AVIVA1950

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 16h

A behind the scenes look at David Scadden, MD @ScaddenLab presenting his FIRST LOOK: Regenerating T Cell Immunity #WMIF2021 #GCT #Tcells

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Mass General Brigham Innovation

@MGBInnovation

·

16h

In our First Look sessions clinicians/researchers from Harvard-affiliated hospitals highlight the potential of their research & new technologies. Next we’ll hear from Khalid Shah PhD, Vice Chair of Research

@BWHNeurosurgery

#WMIF2021 https://bwhclinicalandresearchnews.org/2021/05/11/look-whos-talking-world-medical-innovation-forum-first-look-speakers/…

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

19h

“Entrepreneurial Growth | Oncolytic Virus” panel, moderated by Reid Huber PhD, Partner

@ThirdRockV

, discusses how small companies can address the challenges of developing #oncolyticvirus therapies. #WMIF2021

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Novartis Gene Therapies

@NovartisGene

·

May 19

The World Medical Innovation Forum is here! During his fireside chat, our President Dave Lennon shares the immense promise ahead for #genetherapy.

@MGBInnovation

#WMIF2021

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Mass General Brigham Innovation

@MGBInnovation

·

May 18

Tomorrow is Day 1 of #WMIF2021! Hear from the world-renowned CEOs, investors, clinicians and scientists bringing game-changing discoveries and insights to #GCT. Register to attend today: https://worldmedicalinnovation.org/register/

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Novartis Gene Therapies

@NovartisGene

·

May 18

We’re at

@MGBInnovation

‘s World Medical Innovation Forum this week, discussing the future of #genetherapy. Here are our five predictions for where the industry is headed. #WMIF2021 (1/6)

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Mass General Brigham Innovation

@MGBInnovation

·

23h

Some incredible #visualnotes from this morning’s co-chair’s panel “The Grand Challenge of Widespread GCT Patient Benefits” #WMIF2021 #GCT #geneandcelltherapy

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Mass General Brigham Innovation

@MGBInnovation

·

May 17

The World Medical Innovation Forum #WMIF2021 is just two days away! Join us to hear the latest in #geneandcelltherapy #healthcare innovation. https://worldmedicalinnovation.org/register/

You Retweeted

BrighamResearch

@BrighamResearch

·

May 16

“We anticipate that our engineered tumor cell platform will have major contributions in finding a cure for #glioblastoma patients,” says

@khalidshahs

 of

@BWHNeurosurgery

. Catch a preview of his #WMIF2021 First Look talk here: https://fal.cn/3fpUL

8

16

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Mass Eye and Ear

@MassEyeAndEar

·

22h

Dr. Eric Pierce

@MassEyeAndEar

@HMSeye

explains at #WMIF2021 why the first FDA-approved gene therapy for inherited disease was for an inherited retinal degeneration, and what lessons have been learned from the success of that treatment.

Mass General Brigham Innovation

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Mass General Brigham Innovation

@MGBInnovation

·

22h

Ravi Thadhani, CAO @MassGeneralBrigham and Juergen Eckhardt, Head of

@LeapsbyBayer

, will be announcing the 2021 Innovation Discovery Grants at #WMIF2021 tomorrow, 5/20 @ 2:00 pm Eastern. https://worldmedicalinnovation.org

Quote Tweet

Leaps by Bayer

@LeapsByBayer

 · 22h

Together with @BayerPharma, we are pleased to be part of #WMIF2021, organized by @MassGenBrigham. This year’s event focuses on the transformative potential of #cellandgene therapy (#GCT).

Show this thread

 

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Mass General Brigham Innovation

@MGBInnovation

·

20h

Welcome back! Our next #WMIF2021 panel, Oncolytic Viruses in #Cancer | Curing #Melanoma and Beyond, features panelists from

@BrighamWomens

@Replimune

@EikonTX

@Amgen

and

@DanaFarber

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Novartis Gene Therapies

@NovartisGene

·

22h

“We are more committed to our mission than ever before – laser-focused on realizing the transformative potential of #genetherapy for patients.” – Dave Lennon, President, during #WMIF2021

Outstanding researcher and speaker

@pharma_BI

@AVIVA1950

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 21h

Patricia Musolino, MD PhD, Co-Director Pediatric Stroke and Cerebrovascular Program at MGH, discusses her work developing #genetherapy treatments for cerebral genetic vasculopathies #GCT #geneandcelltherapy #WMIF2021

1

You Retweeted

Mass Eye and Ear

@MassEyeAndEar

·

23h

Happening now at #WMIF2021.

@MassEyeAndEar

chief and

@HMSeye

chair Dr. Joan Miller moderates a panel on AAV gene therapy featuring director of Inherited Retinal Disorders Service and Ocular Genomics Institute, Dr. Eric Pierce.

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 23h

Our “AAV Success Studies | Retinal Dystrophy | Spinal Muscular Atrophy” panelists have taken the stage. #WMIF2021 @MassEyeAndEar @REGENXBIO @spark_tx @NovartisGene

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CRISPR Therapeutics

@CRISPRTX

·

19h

Attending

@MGBInnovation

World Medical Innovation Forum? Tune in to hear our CEO

@CRISPRSam

speak tomorrow at 3:25pm ET on innovations in cell and gene therapy, followed by a Q&A. Learn more: https://bit.ly/3eWb66R #WMIF2021

4

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Biogen

@biogen

·

15h

We are proud sponsors of the Virtual World Medical Innovation Forum (#WMIF2021). This year’s program will focus on the impact of gene and cell therapy as a way to potentially advance quality patient care, reduce cost and improve outcomes. Learn more:

World Medical Innovation Forum

worldmedicalinnovation.org

You Retweeted

Pearl Freier

@PearlF

·

16h

Jonathan Kraft introducing #wmif2021 session with Pfizer CSO & president of R&D Mikael Dolsten and MGH oncologist & chair of MGH Cancer Center Daniel Haber.

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Aviva Lev-Ari

@AVIVA1950

·

15h

MEE is the leader in cell therapy for retina genetic disease

Quote Tweet

Tracy Doyle

@doylet

 · May 19

Great discussion to open #WMIF2021 on the patient impact of #GCT @MGBInnovation World Medical Innovation Forum twitter.com/AVIVA1950/stat…

You Retweeted

Pearl Freier

@PearlF

·

May 19

Tuning into

@MGBInnovation

#WMIF2021 cell & gene therapy meeting.

@NovartisGene

president Dave Lennon & Deerfield partner Julian Harris having a “fireside chat.” Dave/Novartis: sees gene therapy as driver for economy generating need for highly skilled workers Incl manufacturing

2

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Pearl Freier

@PearlF

·

17h

Kite Pharma CEO (Gilead subsidiary) Christi Shaw said there are 120 biopharma companies working on CAR-T cell therapy & they are continuing to look for new partnerships. She also mentioned logistical challenges currently getting to Israel & helping patients there. #WMIF2021

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Pearl Freier

@PearlF

·

15h

Dolsten/Pfizer discussing their partnership with Ionis.https://ir.ionispharma.com/news-releases/news-release-details/ionis-and-akcea-announce-pfizer-has-initiated-phase-2b-clinical… #wmif2021

Ionis and Akcea announce that Pfizer has initiated a Phase 2b clinical study of vupanorsen (AKCEA…

The Investor Relations website contains information about Ionis Pharmaceuticals, Inc.’s business for stockholders, potential investors, and financial analysts.

ir.ionispharma.com

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Pearl Freier

@PearlF

·

23h

FDA’s Dir of Center for Biologics Evaluation & Research Peter Marks interviewed by Vicki Sato- chairwoman of Vir Biotechnology, ex Vertex president & ex Biogen VP Research. Around June ’20, started 2c progress in covid vaccines w/ enough candidates moving forward #WMIF2021 1/n

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Tracy Doyle

@doylet

·

23h

FDA staffing up on gene therapies personnel by 50% says Peter Marks, MD, PhD, Center for Biologics Evaluation and Research

@US_FDA

at #WMIF2021

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Mass General Brigham Innovation

@MGBInnovation

·

18h

“Once you work on cell and gene therapy, its really hard to go back and work on anything else” says moderator Marcela Maus, MD PhD in our “CAR-T | Lessons Learned | What’s Next” panel #WMIF2021 #GCT #geneandcelltherapy

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Pearl Freier

@PearlF

·

20h

Ex Merck president R&D Roger Perlmutter is now Eikon Therapeutics CEO & is on #WMIF2021 oncolytic virus in cancer panel w/Amgen EVP R&D David Reese, ex BioVex CTO (T-VEC inventor

@robertcoffin3

now

@Replimune

founder/president, Dana-Farber physician Ann Silk, BWH’s Nino Chiocca

You Retweeted

Novartis Gene Therapies

@NovartisGene

·

May 18

During this week’s World Medical Innovation Forum with

@MassGenBrigham

, join our leaders for panels and presentations discussing what’s next for #genetherapy and the key trends shaping the industry as it evolves. #WMIF2021 https://bit.ly/3eYYls4

59 views

0:24 / 0:36

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Pearl Freier

@PearlF

·

16h

Dolsten/Pfizer discussed covid vaccines and real world evidence study in Israel. Was sole provider of vaccines in Israel. 95%-98% efficacy replicated in real world. Well above 90% efficacy in asymptomatic disease. #wmif2021

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Tracy Doyle

@doylet

·

18h

Is CART-T therapy still an industry priority? Panelists say yes! Join us to hear more at the

@MGBInnovation

#WMIF2021

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Pearl Freier

@PearlF

·

18h

CAR-T #WMIF2021 panel w/ MGH’s

@MarcelaMaus

,

@Atarabio

EVP R&D

@jdupontmd

, BMS SVP Hematology/Oncology & Cell Therapy

@KristenHege

,

@KitePharma

CEO Christi Shaw, Novartis Global Head Cell & Gene

@Stefanhendriks5

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Mass General Brigham Innovation

@MGBInnovation

·

16h

ICYMI: An illustration depicting the “AAV Delivery” panel discussion about advances in the area of #AAVGeneTherapy delivery. Thank you to the panelists from

@MGHNeurology

,

@CureFA_org

,

@AstellasUS

and

@AkouosInc

. #geneandcelltherapy #GCT #WMIF2021



Aviva Lev-Ari

@AVIVA1950

·

16h

Like that presentation a lot

@pharma_BI

@AVIVA1950

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 22h

Casey Maguire PhD, Associate Professor of Neurology, at the podium to present his work developing improved #genetherapy vectors. #WMIF2021 “First Look: Enhanced Gene Delivery and Immunoevasion of AAV Vectors without Capsid Modification”

You Retweeted

Mass General Brigham Innovation

@MGBInnovation

·

22h

Casey Maguire PhD, Associate Professor of Neurology, at the podium to present his work developing improved #genetherapy vectors. #WMIF2021 “First Look: Enhanced Gene Delivery and Immunoevasion of AAV Vectors without Capsid Modification”

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Aviva Lev-Ari

@AVIVA1950

·

16h

Best interview of a CSO in the history of Big Pharma

@Pharma_BI

@AVIVA1950

Quote Tweet

Mass General Brigham Innovation

@MGBInnovation

 · 16h

Mikael Dolsten, MD PhD, CSO & President, Worldwide Research, Development and Medical @pfizer takes the stage for a Fireside Chat, moderated by @MGHCancerCenter Daniel Haber, MD, PhD. “Pfizer’s Future in Cell and Gene Therapy” #WMIF2021

You Retweeted

Pearl Freier

@PearlF

·

May 19

Dave Lennon/Novartis: manufacturing has been a roadblock for many cell & gene therapy companies. Expects to see more investments earlier. Engineering advances will unlock scale & address bigger & bigger patient populations. Oppty to ID patients early #WMIF2021

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Mass General Brigham Innovation

@MGBInnovation

·

19h

Nino Chiocca, MD PhD,

@BWHNeurosurgery

presents FIRST LOOK: Oncolytic Viruses: Turning Pathogens into Anticancer Agents #WMIF2021

You Retweeted

Pearl Freier

@PearlF

·

22h

M&A cell & gene therapy #WMIF2021 panel incl Bain Capital’s Adam Koppel, Bayer’s Head Strategy Business Development & Licensing

@MDDBacker

, Pfizer’s SVP Worldiwde BD Debbie Baron, Eli Lilly VP BD Ken Custer, ex AveXis CEO Sean Nolan now Affinia & Encoded Therapeutics Board Chair

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Pearl Freier

@PearlF

·

21h

Marianne De Backer/Bayer on post M&A & company culture: They acquired AskBio & thought about how to preserve their freedom so they could continue to operate. Bayer decided to keep them independent & so they can operate at arm’s length. #wmif2021

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Resilience

@IncResilience

·

17h

Happening now: our CEO, Rahul Singhvi, speaking at the virtual 2021 World Medical Innovation Forum: http://worldmedicalinnovation.org #WMIF2021 https://pic.twitter.com/Nyc2lXbvUR

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Pearl Freier

@PearlF

·

21h

Ken Custer/Eli Lilly-said they’re relatively new in cell & gene therapy. They invested in 1 of Sean Nolan’s (ex AveXis CEO) new companies,Jaguar Gene Therapy. Lilly’s legacy in neuroscience is noted & bought Prevail last yr. Clinical trial w/ Parkinson’s w/GBA1 mutation #wmif2021

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Mass General Brigham Innovation

@MGBInnovation

·

May 19

Jack Hogan, a patient

@MassEyeAndEar

, was the first in the U.S. to be approved for FDA gene therapy surgery. In 2018 he underwent therapy to treat retinitis pigmentosa by having a synthetic gene inserted into his retina. With improved eyesight he can now play sports #WMIF2021

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Tracy Doyle

@doylet

·

21h

The acquisition market in #GCT: looking for breakthroughs for patients, technologies for intractable diseases, manufacturing expertise, pioneering companies with deep experience — all for “the modality of the future”. M&A panel at #WMIF2021

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Pearl Freier

@PearlF

·

18h

Christi Shaw/Kite Pharma: Only 4 out of 10 patients eligible for CAR-T are being referred for CAR-T cell therapy by oncologists. The other 6 out of 10, referred to palliative care only. Consistency of manufacturing is also very important. #wmif2021 1/n

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presents on the future potential of this revolutionary technology at #WMIF2021

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Marianne De Backer/Bayer on post M&A & company culture: They acquired AskBio & thought about how to preserve their freedom so they could continue to operate. Bayer decided to keep them independent & so they can operate at arm’s length. #wmif2021

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37th Annual J.P. Morgan HEALTHCARE CONFERENCE: #JPM2019 for Jan. 8, 2019; Opening Videos, Novartis expands Cell Therapies, January 7 – 10, 2019, Westin St. Francis Hotel | San Francisco, California, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 1: Next Generation Sequencing (NGS)

37th Annual J.P. Morgan HEALTHCARE CONFERENCE: #JPM2019 for Jan. 8, 2019; Opening Videos, Novartis expands Cell Therapies, January 7 – 10, 2019, Westin St. Francis Hotel | San Francisco, California

Reporter: Stephen J. Williams, PhD

The annual J.P. Morgan Healthcare Conference is the largest and most informative healthcare investment symposium in the industry, bringing together industry leaders, emerging fast-growth companies, innovative technology creators, and members of the investment community.

 

Joe Biden

Joe Biden on the Fight Against Cancer

Former Vice President of the United States joined the J.P. Morgan Healthcare Conference to discuss cancer initiatives.

Watch Video

Bill Gates

Bill Gates on the Current State of Global Health

In his keynote address at the annual J.P. Morgan Healthcare Conference, Bill Gates spoke about the state of healthcare around the world.

Watch Video

CEO Anne

Anne Wojcicki on Disrupting the Healthcare Industry

The CEO of 23andMe discusses at the J.P. Morgan Healthcare Conference how her genomics company is activating the power of the consumer.

Watch Video

  1. Another packed house as panel including Saurabh Saha, & Alexis Borisy discuss the rewiring of R&D for the digital age at Exec Bfast

Novartis Talks Move to Cell and Gene Therapies at JPM

Novartis logo on outdoor wall

Denis Linine / Shutterstock

Following a strong post-hoc analysis of mid-stage data in the fall of 2018, Novartis announced this morning the company’s experimental humanized anti-P-selectin monoclonal antibody was crizanlizumab granted Breakthrough Therapy Status by the U.S.Food and Drug Administration (FDA).

Crizanlizumab received the designation as a treatment for the prevention of vaso-occlusive crises (VOCs) in patients of all genotypes with sickle cell disease (SCD). VOCs, which can be extremely painful for patients, happen when multiple blood cells stick to each other and to blood vessels, causing blockages.

The designation was awarded following results from the Phase II SUSTAIN trial, which showed that crizanlizumab reduced the median annual rate of VOCs leading to health care visits by 45.3 percent compared to placebo. The SUSTAIN study also showed that crizanlizumab significantly increased the percentage of patients who did not experience any VOCs vs placebo, 35.8 percent vs. 16.9 percent.

The FDA designation came one day after the Swiss pharma giant laid out its map for a future of success, sustainability and, if things work out, respect from consumers. In an interview with CNBC Monday, Novartis Chief Executive Officer Vas Narasimhan noted that the company is looking to become an entity that doesn’t draw its profits from treating disease, but will make money by providing cures. He pointed to the moves Novartis has made toward gene and cellular therapies that have the potential to cure patients of various diseases in what many researchers hope could be a “one-and-done” treatment. Narasimhan told CNBC that cures are what society wants and that is something they will value. The challenge will be determining the payment system.

As an example, the company is eying potential approval of a gene therapy for spinal muscular atrophy (SMA), a fatal genetic disease marked by progressive, debilitating muscle weakness in infants and toddlers. Novartis’ gene therapy Zolgensma is expected to be approved by the FDA this year and could have a price tag of between $4 and $5 million. While significantly high, non-profit SMA groups have already suggested that the gene therapy treatment could be more cost-effective than Spinraza, the only approved SMA treatment on the market.

During its presentation at J.P. Morgan, Novartis pointed to the moves it has made as the company pivots to this future of gene and cell therapies. The presentation noted that over the course of 2018, the company made several deals to sell off non-essential businesses, such as the $13 billion sale of its share of a consumer health business to partner GlaxoSmithKline. Not only that, but Novartis also made significant acquisitions to reshape its portfolio, including the $8.7 billion acquisition of AveXis for the SMA gene therapy. The deal for AveXis wasn’t the only gene therapy deal the company struck. Novartis began 2018 with a deal for Spark Therapeutics’ gene therapy Luxturna, a one-time gene therapy to restore functional vision in children and adult patients with biallelic mutations of the RPE65 (retinal pigment epithelial 65 kDa protein) gene.

In his interview with CNBC, Narasimhan said the company is about “platforms,” which also includes radio-ligand therapy. The company forged ahead in that area with two acquisitions, Advanced Accelerator Applications and Endocyte. Radiopharmaceuticals like Endocyte’s Lu-PSMA-617 are innovative medicinal formulations containing radioisotopes used clinically for both diagnosis and therapy. When the Endocyte deal was announced, Novartis noted the field is expected to become an increasingly important treatment option for patients, as well as a key growth driver for the company’s oncology business.

Other posts on the JP Morgan 2019 Healthcare Conference on this Open Access Journal include:

#JPM19 Conference: Lilly Announces Agreement To Acquire Loxo Oncology

36th Annual J.P. Morgan HEALTHCARE CONFERENCE January 8 – 11, 2018

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Stem Cells Used as Delivery Truck for Brain Cancer Drugs

Reporter: Irina Robu, PhD

Medulloblastoma, common brain cancer in children has been very difficult to treat therapeutically with traditional interventions which relies on surgical techniques to remove the bulk of the cancerous tissue. The researchers seen the need for novel treatments of medulloblastomas that have recurred, as well as for treatments that are less toxic overall. For this reason, data from University of North Carolina (UNC) Lineberger Comprehensive Cancer Center and  Eshelman School of Pharmacy published a study in PLOS named “Intra-cavity stem cell therapy inhibits tumor progression in a novel murine model of medulloblastoma surgical resection”, validates how cancer-hunting stem cells can track down and deliver a drug to terminate medulloblastoma cells hiding after surgery.

The technology in the research is an extension of a discovery that won researchers a Nobel Prize in 2012 and showed they could transform skin cells into stem cells. The research team started by reprogramming skin cells into stem cells and genetically engineered them to manufacture a substance that becomes toxic to other cells when exposed to another drug. Inserting the drug carries the stem cells into the brain of laboratory models after surgery decreased the size of tumors by 15 times and extended median survival in mice by 133%.

In this study, the scientists indicated they could shrink tumors in murine models of medulloblastoma, hence extending the rodents life. The approach holds promise for reducing side effects and helping more children with medulloblastoma. Amazingly the researchers also developed a laboratory model of medulloblastoma that allowed them to simulate the way standard care is currently delivered—surgery followed by drug therapy. Using this model, they discovered that after surgically removing a tumor, the cancer cells that remained grew faster.

According to the study investigator, Shawn Hingtgen, PhD, the cells are like a FedEx truck that will deliver cytotoxic agents directly into the tumor to a particular location. In earlier studies, Dr. Hingtgen and his colleagues showed that they could flip skin cells into stem cells that hunt and transport cancer-killing drugs to glioblastoma, the deadliest malignant brain tumor in adults.

Medulloblastoma is cancer that happens mostly in kids between ages of three and eight, and while current therapy has changed survival pretty dramatically, it can still be pretty toxic. The ability to use a patient’s own cells to target the tumor directly would be “the holy grail” of therapy, the investigators trust it could hold capacity for other rare, and sometimes fatal, brain cancer types that occur in children as well.

Source

https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198596

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Skin Regeneration Therapy One of First Tissue Engineering Products Evaluated by FDA

Reporter: Irina Robu, PhD

Under the provisions of 21st Century Cures Act the U.S. Food and Drug Administration approved StrataGraft regenerative skin tissue as the first product designated as a Regenerative Medicine Advanced Therapy (RMAT) produced by Mallinckrodt Pharmaceuticals. StrataGraft is shaped using unmodified NIKS cells grown under standard operating procedures since the continuous NIKS skin cell line has been thoroughly characterized. StrataGraft products are virus-free, non-tumorigenic, and offer batch-to-batch genetic consistency.

Passed in 2016, the 21st Century act allows FDA to grant accelerated review approval to products which meet an RMAT designation. The RMAT designation includes debates of whether priority review and/or accelerated approval would be suitable based on intermediate endpoints that would be reasonably likely to predict long-term clinical benefit.

The designation includes products

  • defined as a cell therapy, therapeutic tissue engineering product, human cell and tissue product, or any combination product using such therapies or products;
  • intended to treat, modify, reverse, or cure a serious or life-threatening disease or condition; and
  • preliminary clinical evidence indicates the drug has the potential to address unmet medical needs for such disease or condition.

According to Steven Romano, M.D., Chief Scientific Officer and Executive Vice President, Mallinckrodt “We are very pleased the FDA has determined StrataGraft meets the criteria for RMAT designation, as this offers the possibility of priority review and/or accelerated approval. The company tissue-based therapy is under evaluation in a Phase 3 trial to assess its efficacy and safety in the advancement of autologous skin regeneration of complex skin defects due to thermal burns that contain intact dermal elements.

SOURCE

https://www.rdmag.com/news/2017/07/skin-regeneration-therapy-one-first-be-evaluated-fda

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Mid Atlantic LRIG 22nd Annual Technology Showcase: Agenda on 3D Bioprinting on Wednesday, May 11, 2016 at Holiday Inn, 195 Davidson Avenue, Somerset, NJ

Reporter: Stephen J. Williams, Ph.D.

 

Symposium Speakers and Topics:

Human Organoids
Hatem E. Sabaawy-Director, Production GMP Facility for Cell and Gene Therapy, RBHS-Robert Wood Johnson Medical School, Rutgers Cancer Institute of New Jersey

Intestinal Organoids for Drug Discovery
Richard Visconti-Associate Principal Scientist, Cellular Pharmacology, Merck Research Laboratories, Kenilworth,  New Jersey

3D Bioprinting
Elizabeth Wu-President, WuZenTech, Edison, New Jersey

Building  Your Brand  Through LinkedIn
Stan Robinson, Jr., LinkedIn Consultant, Helping Professionals with Social Selling, Personal Branding

Register at EventBrite here: https://www.eventbrite.com/e/mid-atlantic-22nd-annual-technology-and-exhibition-tickets-21359945171 

To sign up to be an LRIG member or update your profile, please visit us at http://lrig.org
Hoping to see you on May 11th.
Reserve your spot today!

 

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Transplantation of modified human adipose derived stromal cells expressing VEGF165

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

http://pharmaceuticalintelligence.com/2013-11-03/larryhbern/Transplantation of modified human adipose derived stromal cells expressing VEGF165 

This contribution to the series on stem cells and regenerative medicine deals with transplantation of modified human adipose tissue to repair  ischemic damaged skeletal muscle by apparently increase neovascularization, essentrial for laying down the circulation that feeds the tissue.

Transplantation of modified human adipose derived stromal cells expressing VEGF165 results in more efficient angiogenic response in ischemic skeletal muscle

Evgeny K Shevchenko1*Pavel I Makarevich12Zoya I Tsokolaeva1,Maria A Boldyreva1Veronika Yu Sysoeva2Vsevolod A Tkachuk23 andYelena V Parfyonova12
1Laboratory of angiogenesis, Russian Cardiology Research and Production Complex;  2Lomonosov Moscow State University; 3Laboratory of molecular endocrinology, Russian Cardiology Research and Production Complex, Moscow,  Russia.
Journal of Translational Medicine 2013, 11:138.   http://www.translational-medicine.com/content/11/1/138   http://dx.doi.org/10.1186/1479-5876-11-138
This is an Open Access article distributed under the terms of the Creative Commons Attribution License   http://creativecommons.org/licenses/by/2.0

Abstract

Background

Modified cell-based angiogenic therapy has become a promising novel strategy for ischemic heart and limb diseases. Most studies focused on myoblast, endothelial cell progenitors or bone marrow mesenchymal stromal cells transplantation. Yet adipose-derived stromal cells (in contrast to bone marrow) are abundantly available and can be easily harvested during surgery or liposuction. Due to high paracrine activity and availability ADSCs appear to be a preferable cell type for cardiovascular therapy. Still neither genetic modification of human ADSC nor in vivo therapeutic potential of modified ADSC have been thoroughly studied. Presented work is sought to evaluate angiogenic efficacy of modified ADSCs transplantation to ischemic tissue.

Materials and methods

Human ADSCs were transduced using recombinant adeno-associated virus (rAAV) serotype 2 encoding human VEGF165. The influence of genetic modification on functional properties of ADSCs and their angiogenic potential in animal models were studied.

Results

We obtained AAV-modified ADSC with substantially increased secretion of VEGF (VEGF-ADSCs). Transduced ADSCs retained their adipogenic and osteogenic differentiation capacities and adhesion properties.

  • The level of angiopoetin-1 mRNA was significantly increased in VEGF-ADSC compared to unmodified cells yet
    • expression of FGF-2, HGF and urokinase did not change.

Using matrigel implant model in mice it was shown that

  • VEGF-ADSC substantially stimulated implant vascularization with paralleling increase of capillaries and arterioles.

In murine hind limb ischemia test we found

  • significant reperfusion and revascularization after intramuscular transplantation of VEGF-ADSC compared to controls with no evidence of angioma formation.

Conclusions

Transplantation of AAV-VEGF- gene modified hADSC resulted in stronger therapeutic effects in the ischemic skeletal muscle and may be a promising clinical treatment for therapeutic angiogenesis.

Keywords:

Therapeutic angiogenesis; Cell therapy; Gene modified cells; Adipose stromal cells; Vascular endothelial growth factor; Adeno-associated virus; Ischemia

Background

Despite advances in revascularization techniques, the treatment of ischemic heart and limb diseases remains a worldwide problem. Therapeutic angiogenesis represents alternative new strategy for ischemia resolution that utilizes regenerative capacity of human body and

  • stimulates natural process of
  1. vessel growth,
  2. remodeling and
  3. tissue revascularization [1].

Commonly adopted approaches for therapeutic angiogenesis include

  • direct introduction of recombinant growth factors and gene therapy.

Yet clinical trials have shown several drawbacks of these modalities. Thus low efficacy of recombinant protein administration is explained by

  • dissemination after injection and
  • rapid degradation of therapeutic agent, which
  • requires multiple and long-term infusions thus
    • leading to tremendous expenses [2,3].

Delivery of cDNA coding angiogenic factors via different expression mammalian vectors (plasmids, recombinant viruses) was found more feasible and allowed to achieve great improvement in some cases yet

  • efficacy was still not high enough especially in double blind placebo controlled trials [4].

Many authors discussed possible reasons of gene therapy low efficacy and most of them are univocal to emphasize transfection efficacy and transient transgene expression after plasmid delivery. This can be circumvented by administration of viral vectors but their use is limited due to possible danger of insertional mutagenesis and immune reactions [5,6].

Recently, autologous transplantation of bone marrow stromal cells or endothelial progenitor cells has been shown to enhance angiogenesis and peripheral blood flow [79]. However,

  • the regenerative capacity of these cells decreases with age and
  • in patients with co-morbidities such as diabetes mellitus which reduces efficacy of autologous cell administration, and
  • limited cell viability after transplantation into ischemic tissues also restricts their angiogenic potential [1012].

It was shown in several experimental studies that this problem could be circumvented by gene modified cell therapy strategy utilizing stem or progenitor cells overexpressing angiogenic proteins [13,14]. To develop a feasible and potent gene modified cell therapy for ischemic diseases

  1. the cells should be both effective and accessible in large numbers as well as
  2. the chosen viral vector should be both safe and effective in terms of gene delivery.

The majority of experimental studies have evaluated gene modified bone marrow stromal cells or endothelial progenitor cells for ischemia treatment [1517]. However, cells extracted from bone marrow or peripheral blood after mobilization are available in limited numbers and as for bone marrow cells painful aspiration procedure is required.

In contrast to bone marrow or myoblasts, stromal fraction of adipose tissue contains an abundant population of multipotent stem cells that can be easily harvested in high numbers by minimally invasive surgical techniques [1821]. These adipose –derived stromal cells (ADSCs)

  • share common properties with bone marrow stromal cells and represent a very convenient object for therapeutic use.

However the best development of ADSC for angiogenic therapy still needs to be determined.

As for genetic modification of cells the choice of safe and effective gene transfer vector as well as the appropriate transgene determines the quality and safety of the cell product affecting the efficacy of modified cell based therapy. Recombinant adeno-associated viruses (rAAV) are one of the most promising and versatile tools in this field due to

  1. low immunogenicity and
  2. high transduction potency in vitro

in many types of both – dividing and non-dividing mammalian cells. Besides that until now no human disease caused by AAV has been identified [22].

In this study we genetically modified human ADSCs with a key regulator of angiogenesis – VEGF165 [23] via rAAV-transduction and then evaluated effects of rAAV-transduction and VEGF165 overexpression on human ADSC

  1. growth,
  2. differentiation capacity,
  3. adhesion and
  4. angiogenic factor expression as well as
  5. revascularization and
  6. functional improvement

after intramuscular injection in a mice hind limb model.

Methods

Cell culture

(refer to doi:10.1186/1479-5876-11-138)

DNA constructs production of rAAV particles and cell transduction

(refer to doi:10.1186/1479-5876-11-138)

Western blotting and ELISA

(refer to doi:10.1186/1479-5876-11-138)

ADSC proliferation activity assay

To assess population doubling time (PDT) of gene modified (transduced with rAAV at passage 1) or untreated ADSC (passage 2) seeded on 6-well plates (2 × 104 cells/well). After a 9 day incubation average cell numbers for three wells were obtained using a hemocytometer chamber. PDT was calculated as follows:

 PDT=(log2)*t/(log(Nt/N0))

 where t is period of incubation (hours), Nt – endpoint amount of cells, N0 – initial number of cells.

ADSC cell cycle stage analysis by flow cytometry

(refer to doi:10.1186/1479-5876-11-138)

ModFit LT 3.2 software (Verity Software House, USA) was used for analysis of cell distribution over cell cycle stages according to intensity of propidium iodide fluorescence in a wavelength range of 600–625 nm (excitation wavelength – 488 nm). Results are presented as a percentage of cells in S + G2/M stages.

Apoptosis assay

Analysis of spontaneous apoptosis frequency in ADSC culture was performed using Annexin-V FITC Apoptosis Kit (Invitrogen, USA) according to manufacturer’s protocol.

Adipogenic, osteogenic and endothelial differentiation of ADSC

To confirm adipogenesis intracellular lipid droplets were detected using Oil red O staining reagent (Millipore, USA) 2 weeks after induction. To confirm osteogenesis Alizarine Red C staining was used to detect extracellular matrix mineralization 2 and 3 weeks post induction. Endothelial cells were stained for CD31 and VEGFR2 surface antigens and cell counts were obtained using flow cytometry.

Cell attachment assay

(refer to doi:10.1186/1479-5876-11-138)

Flow cytometry

Antigen expression analysis was performed on cell sorter MoFlo (DakoCytomation, Denmark) or flow cytometry scanner BD FACS CantoTM II (BD Pharmingen, USA). 10 000 events were acquired and analyzed for antigen expression.

Quantitative polymerase chain reaction

Quantitative polymerase chain reaction (qPCR) was performed using primers specific for human VEGF165, ANGPT1, HGF, FGF2 and PLAU mRNAs.

Animals

8–10 week-old male BALB/c NUDE mice

Matrigel plug assay

(Refer to doi:10.1186/1479-5876-11-138)

Hind limb ischemia model

Ten week-old male BALB/с NUDE mice were anaesthetized by intraperitoneal injection of 0.3 ml of 2.5% avertin. Femoral artery was separated in its distal part and ligated proximal to its popliteal bifurcation (keeping v. femoralis and n. ischiadicus intact). ADSC, GFP-ADSC or VEGF-ADSC (5×105 cells per animal) were resuspended in 150 μl of PBS, and injected in 3 equally divided doses tom. tibialis anterior, m. gastrocnemius and m. biceps femoris to generate three experimental animal groups: “GFP-ADSC”, “VEGF-ADSC”, “ADSC” (14 animals per group). PBS (150 μl) was injected in negative control “PBS” group. Blood flow was subsequently measured by laser Doppler imaging.

Laser doppler imaging

(Refer to doi:10.1186/1479-5876-11-138)

Muscle explants

M. tibialis anterior explant culture was prepared on matrigel according to Jang et al. [26] protocol and cultured in M199 medium (Gibco, USA), containing 2% FBS. At day 3 and 7 medium was collected for determination of human VEGF165 concentration by ELISA.

Specimen preparation and histological analysis

At designated period (day 20 for muscles, day 14 for matrigel plugs) animals were sacrificed by lethal isoflurane dose followed by cervical dislocation. Afterwards m. tibialis anterior or matrigel implants respectively were harvested,

For muscle necrosis analysis we used routine hematoxylin-eosin staining of formalin-fixed muscle sections. Necrotic tissue was defined by loss of fiber morphology, cytoplasm disruption, inflammatory cells infiltration and fibrosis.

Statistical analysis

Results were analyzed in Statsoft Statistica 6.0 (Statsoft, USA).

Results

Effective transduction of human ADSC by adeno-associated virus serotype 2

Low-passage human ADSC obtained from different donors were transduced using rAAV encoding GFP to assess gene delivery efficacy. Transduced to total cells ratio was counted by flow cytometry. GFP-positive ADSC (GFP-ADSC) were detected as early as day 2 after viral infection. Maximum number of positive cells (65.6±3%) and highest GFP-fluorescence intensity was reached by day 4–5 (Figure 1). GFP signal was detectable for at least 30 days. At day 15 and 30 flow cytometry showed that 45±2% and 25±1.5% of ADSC were GFP-positive respectively.

Figure 1. Human ADSC transduction by recombinant adeno-associated virus

Figure 1. Human ADSC transduction by recombinant adeno-associated virus

A. GFP-positive cell count by FACS in GFP-ADSC culture at day 4 after transduction by rAAV. B.Representative image of GFP-positive human ADSC (green) transduced by rAAV, 100 × magnification.

Increase of VEGF expression and secretion after rAAV transduction of human ADSC

To obtain gene modified ADSC we constructed rAAV vector encoding human VEGF165. In ADSC transduced by rAAV-VEGF (VEGF-ADSC) VEGF165 mRNA level increased 80±15-fold compared to basal expression in unmodified ADSC or GFP-ADSC (Figure 2A). Protein production was analyzed by Western blotting and ELISA. Data presented at Figure 2B, C shows that in VEGF-ADSC secretion of VEGF increased 45-50-fold (4.5±1.8 ng/ml/105 cells) compared to unmodified cells (0.1±0.02 ng/ml/105 cells) or GFP-ADSC (0.09±0.02 ng/ml/105cells). VEGF concentration in conditioned medium decreased over time during VEGF-ADSC cultivation but remained 30-fold higher (2.9±1.1 ng/ml/105 cells) than in controls (0.09 ± 0.02 ng/ml/105 cells) at day 30 post transduction. Material from a total of 10 donors was used to obtain mean values of VEGF expression increase.

 Figure 2. Validation of VEGF165 expression in AAV-modified VEGF-ADSC.
Figure 2. Validation of VEGF165 expression in AAV-modified VEGF-ADSC.

A. VEGFA expression level in human ADSC 10 days after AAV transduction determined by quantitative PCR. B, C. Analysis of VEGF secretion by GFP-ADSC, VEGF-ADSC and unmodified cells using ELISA (B) and immunoblotting (C). In immunosorbent assay protein content was determined in conditioned media samples obtained at days 7 and 30 post genetic modification of ADSC.

rAAV-mediated modification of human ADSC suppresses their proliferation activity yet does not influence apoptosis

We found that proliferation rate of VEGF-ADSC and GFP-ADSC was reduced compared to unmodified cells (Figure 3A). ADSC population doubling time was 61.3±7 h, while for GFP-ADSC and VEGF-ADSC it was 116.9±11 and 145.4±12 h respectively (n=5, p<0.01 vs unmodified cells). At the same time spontaneous apoptosis rate in all three cell cultures was comparable and comprised about 2±0.5% of total cell population.

Figure 3. . Proliferation of gene modified ADSC.

Figure 3. Proliferation of gene modified ADSC.
Population doubling time in GFP-ADSC, VEGF-ADSC and ADSC cultures. Data of five serial runs. B. Cells distribution in S-G2 cell cycle stages according to cytometry analysis of GFP-ADSC, VEGF-ADSC and ADSC. Data of three serial runs.

Analysis of cell cycle stages distribution in ADSC, GFP-ADSC and VEGF-ADSC cultures (Figure 3B) showed that number of cells in S-G2 stages was more than 1.5-fold lower in modified cells: GFP-ADSC (16±4% cells) and VEGF-ADSC (13±6% cells) compared to unmodified ADSC (25±3% cells; n=3; p<0.05 vs unmodified cells).

ADSC adhesion does not change after genetic modification

Interactions with extracellular matrix proteins play important role in incorporation and integration to recipient’s tissue, cell viability and their functional properties upon transplantation. ADSC did adhere on main extracellular protein collagen type 1 as well as vitronectin and fibronectin while almost none of cells attached to laminin-coated plastic. We did not observe statistically significant differences in adhesion properties between ADSC, GFP-ADSC and VEGF-ADSC cultures (Figure 4).

Figure 4. Data from comparative study of ADSC, GFP-ADSC and VEGF-ADSC adhesion on culture plates

 Figure 4. Data from comparative study of ADSC, GFP-ADSC and VEGF-ADSC adhesion on culture plates coated by collagen 1, vitronectin, fibronectin or laminin (n=4).

Modified ADSC retain their adipogenic, osteogenic and endothelial differentiation potential in vitro

To analyze potential influence of viral transduction and transgene overexpression on differentiation capacity of gene modified cells we performed experiments on adipogenic and osteogenic differentiation of ADSC.

Microscopic analysis of gene modified and untreated ADSC stained with Oil Red O reagent after 14 days of incubation in adipogenic media showed >30% of differentiated (visualized by intracellular lipid droplets accumulation) cells (Figure 5). Oil Red O+ cell count did not reveal statistically significant differences in both GFP-ADSC (33.7±8.1%) and VEGF-ADSC (34.1±11.5%) as well as unmodified ADSC (34.3±11.7%). Similar results were obtained in osteogenic differentiation assay of ADSC. It was confirmed by Alizarin Red C staining that detects extracellular matrix mineralization. At 14 days of incubation in osteogenic media we detected dye-positive cells in ADSC, GFP-ADSC, VEGF-ADSC culture. At day 21 it was followed by dramatic increase of extracellular matrix calcification in both – modified and untreated cells without significant differences (Figure 5).

Figure 5. Adipogenic and osteogenic differentiation of gene modified ADSC

Figure 5. Adipogenic and osteogenic differentiation of gene modified ADSC

Representative images of ADSC and VEGF-ADSC cultures stained by Oil Red O (lipid droplets detection, kjadipogenic differentiation, 100 × magnification) and Alizarine Red C (matrix mineralization, osteogenic differentiation, 100 × magnification for “day 14” and 50 × magnification for “day 21”) reagents after incubation in specific differentiation medium, n=3.

Taking into account mitogenic activity of VEGF we analyzed possible effect of genetic modification and VEGF overexpression on endothelial cell fraction in VEGF-ADSC. Using flow cytometry we determined amount of cells that carry CD31 and VEGFR2 endothelial markers in ADSC, GFP-ADSC and VEGF-ADSC (rAAV-modified at passage 1) cultures at passage 2. Less than 1.5% of CD31, VEGFR2-positive cells were detected in all three populations. Subsequently modified and untreated ADSC at passage 2 that reached >90% confluency were subject to incubation in EGM-2 medium to stimulate endothelial differentiation. After 14 days of cultivation in EGM-2 repeated analysis of CD31 and VEGFR2 expression showed that percentage of endothelial marker-positive cells did not change and remained about 1% in all assayed cultures.

Level of angiopoietin-1 mRNA increases in VEGF-ADSC

Using qPCR we studied potential impact of genetic modification and augmented VEGF secretion on expression activity of hepatocyte growth factor (HGF), fibroblast growth factor-2 (FGF2), angiopoietin-1 (ANGPT-1) and urokinase (PLAU) genes in VEGF-ADSC. As shown in Figure 6 we did not find any changes in FGF2 and HGF expression in GFP-ADSC and VEGF-ADSC compared to ADSC. We found a 3-fold increase in urokinase expression in VEGF-ADSC yet it was not statistically significant. At the same time increase of ANGPT-1 expression in VEGF-ADSC was significant and 5.3±0.6-fold higher than in unmodified cells or GFP-ADSC (n=6, p<0.05).

Figure 6. Comparison of ANGPT1, FGF2, PLAU and HGF genes expression

Figure 6. Comparison of ANGPT1, FGF2, PLAU and HGF genes expression by quantitative PCR in GFP-ADSC, VEGF-ADSC and unmodified ADSC. Charts represent relative expression for assayed genes from a total of 6 runs.

Analysis of VEGF and PDGF receptors expression on ADSC surface

Analysis of VEGF receptors expression on human ADSC was carried out to assess possible autocrine action of VEGF on VEGF-ADSC functional properties. Flow cytometry of ADSC and VEGF-ADSC (at passage 1–2) from different donors stained for VEGF receptor 1 and 2 showed <1% of positive cells (Figure 7). Taking into account observation of Ball et al. which indicated platelet-derived growth factor receptors (PDGFRα and PDGFRβ) as facultative receptors for VEGF165 [27] we analyzed the presence of cells which expressed PDGFRβ in human ADSC culture. Using specific monoclonal antibodies and subsequent flow cytometry we found that >90% of human ADSC were positive for PDGFRβ (Figure 7).

Figure 7. Analysis of VEGF and PDGF receptors expression on ADSC surface.

Figure 7. Analysis of VEGF and PDGF receptors expression on ADSC surface. VEGFR1, VEGFR2 or PDGFRβ-positive cell count by flow cytometry in ADSC culture.

Increased vascularisation of matrigel implants after VEGF-ADSC transplantation

We used matrigel plug assay to determine angiogenic properties of gene modified ADSC in vivo. At day 14 matrigel implants were harvested and subject to histological analysis (Figure 8). In negative control group we found only small sporadic capillaries (<1 capillary per FOV) were detected while in “ADSC”, “GFP-ADSC” and “VEGF-ADSC” groups formation of vessel network was more evident. Vessel counts revealed a 2.7-fold increase of CD31-positive vessels in group “VEGF-ADSC” (88.1±10.4 vessels per FOV) compared to “GFP-ADSC” (31.3±6.2 vessels per FOV) and “ADSC” (34.5±11.6 per FOV). Number of smooth muscle actin (SMA)-positive vessels was also 2.5-fold higher in “VEGF-ADSC” (1.7±0.24 vessels per FOV) than in “GFP-ADSC” (0.7±0.3 vessels per FOV) and “ADSC” (0.7±0.2 vessels per FOV). Thus capillaries/SMA+vessels ratio did not vary among experimental groups.

Figure 8. Effect of VEGF-ADSC or ADSC on vascularization of matrigel implants in nude mice.

Figure 8. Effect of VEGF-ADSC or ADSC on vascularization of matrigel implants in nude mice.
A.Representative images of matrigel sections from “VEGF-ADSC” and “ADSC” groups stained by antibodies against murine CD31 and SMA, 100× magnification. B. Capillaries and arterioles count in matrigel implants.

Blood flow recovery after VEGF-ADSC transplantation into ischemic murine limb

Perfusion assessment in hind limb ischemia model showed maximum blood flow recovery in “VEGF-ADSC” group (Figure 9). By day 20 spontaneous reperfusion of ischemic limb in «PBS» group was feeble and did not exceed 30%. In contrast we observed evident augmentation of blood supply in three experimental groups that received cell injections. At the end of experiment perfusion in “ADSC” and “GFP-ADSC” groups reached 50% and 55% respectively. Blood flow recovery after VEGF-ADSC transplantation was much more effective. At day 12 perfusion in group “VEGF-ADSC” significantly exceeded values in “ADSC” and “GFP-ADSC” by 15-20% and towards the end of experiment (day 20) it reached 80-90%. Thus transplantation of ADSC overexpressing VEGF was more effective than of untreated or GFP-ADSC.

Figure 9. Reperfusion of murine ischemic limb after ADSC administration.

Figure 9. Reperfusion of murine ischemic limb after ADSC administration.
A. Representative laser-doppler scans of subcutaneous blood flow in mice from “ADSC” and “VEGF-ADSC” groups obtained at days 4 and 20 after ischemia induction and cell transplantation. B. Dynamics of blood flow recovery in ischemic limbs within 20 days after intramuscular injection of ADSC, GFP-ADSC, VEGF-ADSC or PBS.

Transplantation of VEGF-ADSC reduces necrosis and stimulates stable vessel formation in ischemic muscle

Histological analysis of hematoxylin-eosin stained m. tibialis anterior specimens obtained at day 20 after and cell transplantation showed significant decrease in necrotic tissue span in «VEGF-ADSC» group (31.3±7%) compared to «ADSC» and «GFP-ADSC» groups (54.3±8.4% and 55.63±6.8%). Animals that received PBS injection as a negative control were characterized by the highest muscle necrosis span that reached 84±6.7% (Figure 10).

Figure 10. Morphometric analysis of tissue necrosis in ischemic muscle from study group animals.

Figure 10. Morphometric analysis of tissue necrosis in ischemic muscle from study group animals.
A. Images of hematoxylin-eosin stained m. tibialis anterior sections. Necrotic tissue is marked by black line. (N* – necrotic tissue, B* – border zone, H* – healthy or regenerating tissue). B. Representative images of muscle tissue from different zones of section. Labels: star – vasa in normal muscle tissue with; black dot – inflammatory demarcation zone between anucleic disrupted tissue and regenerating muscle fibers; triangle – regenerating round-shaped muscle fibers with multiple centrally located nuclei. C. Statistical data of necrotic tissue area in “PBS”, “ADSC”, “GFP-ADSC” and “VEGF-ADSC” groups. Measurements made in 4–5 animals per group.

To assess vascular density muscle tissue sections were stained by specific antibodies against mouse CD31 and SMA (Figure 11). Vessel count showed that in “ADSC” and “GFP-ADSC” groups capillary and arteriolar densities were similar reaching 129±11 and 125±14 capillaries/FOV, 1.35±0.12 and 1.37±0.09 arterioles/FOV respectively. In specimens from animals that received VEGF-ADSC capillary density was 189±19 per FOV (p<0.05) with arteriolar density of 3.1±0.2 per FOV (p<0.01). Furthermore, we found that arterioles/CD31+ vessels ratio was similar in all experimental groups and slightly higher in group “VEGF-ADSC” (1% vs 1.6%). In addition morphometric analysis of muscle tissue from group “VEGF-ADSC” did not reveal angioma or abnormal vessel formation.

Figure 11. Vascularization of murine ischemic muscles after ADSC administration.

Figure 11. Vascularization of murine ischemic muscles after ADSC administration.
A. Representative images ofm. tibialis anterior sections from “VEGF-ADSC” and “ADSC” groups stained by antibodies against murine CD31 and SMA, 100× magnification. B. Capillaries and arterioles count in m. tibialis anterior sections. Counts made in 5–6 animals per group.

ADSC retain viability and transgene expression after transplantation into ischemic muscle

To evaluate viability of transplanted ADSC after injection into ischemic tissue m. tibialis anterior specimens from “GFP-ADSC” group were harvested at day 7 after induction of ischemia and cell transplantation. Frozen muscle sections were analyzed using fluorescence microscopy that allowed to detect GFP-positive cells distributed throughout muscle (Figure 12A).

1479-5876-11-138-12  Figure 12. Human ADSC viability and VEGF expression

Figure 12. Human ADSC viability and VEGF expression after transplantation to ischemic murine muscle. 

A. Representative image of m. tibialis anterior section from “GFP-ADSC” group obtained at day 7 after ischemia induction and GFP-ADSC injection, 50× magnification. GFP-positive cells are distributed in tissue around injection site.B. Analysis of VEGF165 content by ELISA in explants culture medium from “ADSC”, “GFP-ADSC”, “VEGF-ADSC” groups obtained at days 3 and 20 after cell trasplantation.

Data from experimental studies indicates that prolonged expression of therapeutic transgene is essential for effective stimulation of angiogenesis and ischemic tissue recovery. Muscle explant model was carried out to confirm the presence of viable and functionally active human ADSC overexpressing VEGF in ischemic muscle at hind limb ischemia experiment endpoint. M. tibialis anteriorwere harvested from “ADSC”, “GFP-ADSC” and “VEGF-ADSC” group animals at day 3 and 20 after cell transplantation and cultured as explant in matrigel. In culture medium samples collected after 3 days of “VEGF-ADSC” explant incubation (obtained at day 3 after cell transplantation) human VEGF165 concentration determined by ELISA reached 2.86±0.21 ng/ml (Figure 12B). Protein concentration was expectedly lower (0.145±0.015 ng/ml) in conditioned medium from muscle explants harvested at day 20. In addition comparison of VEGF concentration in culture medium samples collected at day 3 and 7 post incubation of explant culture revealed accumulation of VEGF. It indirectly confirms presence of functionally active human VEGF-ADSC in ischemic muscle up to 20 days post transplantation. In contrast to “VEGF-ASDC” human VEGF165 concentration in explant cultures from “GFP-ADSC” and “ADSC” groups was below limit of detection.

 Discussion

Gene modified cell-based therapy for ischemic disorders: myocardium infarction and limb ischemia is a rapidly evolving trend in experimental and regenerative medicine. Promoting angiogenesis in ischemic tissues via paracrine action of transplanted modified cells is an emerging alternative modality for patients who are unsuitable for surgical and interventional revascularization. Still choice of

  1. appropriate cell type,
  2. angiogenic factor and
  3. gene delivery tool

are crucial issues for efficacy and safety of the method.

Regarding type of cells there are certain issues concerning their derivation and preparation prior to grafting. Thus, embryonic stem cells application is doubtful due to

  1. ethical reasons,
  2. potential risks of teratogenesis and
  3. immune response to their differentiated progenies [28].

Use of endothelial progenitor cells from peripheral blood and bone marrow are limited by

  • expensive procedures of isolation and difficulties in obtaining sufficient amount of cells.

Regarding the latter point it is known that prolonged incubation of cells in vitro prior to transplantation is associated with

  1. potential risks of malignancy,
  2. proliferation decrease and
  3. commitment to terminal differentiation.

Use of skeletal myoblasts or bone marrow derived mesenchymal stromal cells (BMMSC) is associated with painful isolation procedure of muscle biopsy and suprailiac puncture respectively.

ADSC used in our study share a lot of similar properties and characteristics with BMMSC, while they are easier to obtain in sufficient quantity using minimally invasive liposuction procedure. Various data suggests that up to 1.5 × 10adipose stromal cells can be isolated from 1 ml of adipose tissue [29,30]. This allows to reduce the time of cell propagation in vitro prior to transplantation. As for therapeutic angiogenesis,

human ADSC produce a wide spectrum of biologically active molecules – angiogenic growth factors, cytokines, proteases etc. [31,32].

Multiple experimental studies accumulate data on relatively high therapeutic potential of ADSC for tissue regeneration and stimulation of angiogenesis [21,33,34]. However well-known reduction of cell regenerative potential with age and among patients with severe co-morbidities is also relevant for ADSC. Donor age-associated decrease of proliferation activity and differentiation capabilities was shown for human ADSC [35,36]. Angiogenic potential of ADSC also decreases with ageing and is characterized by reduced secretion of

  • VEGF,
  • HGF,
  • angiopoietin-1 and other angiogenic factors [37].

Thus, attempts to improve regenerative potential of ADSC are reasonable.

We have shown high efficacy of rAAV-mediated genetic modification of human ADSC. Using rAAV encoding VEGF165 we obtained human ADSC with increased level of VEGF165 secretion which retained for at least 30 days. VEGF-A and particularly its most abundant 165-amino acid isoform triggers multiple reactions promoting new vessel formation and growth [23] that supported our choice of therapeutic gene in presented study. Observed gradual decrease of transgene expression can be attributed to proliferation activity of ADSC together with known episomal subsistence of rAAV [38]. Moreover cellular mechanism of addressed

  • methylation can be activated after transduction leading to
  • suppression of cytomegalovirus promoter which triggers
  • transgene expression in our vector [39].

Potential influence of genetic modification and transgene expression on cell behavior and functional activity is frequently kept out of consideration while this issue is of great importance, especially for potential clinical application. We examined possible effects of rAAV-transduction and VEGF overexpression on functional properties of ADSC which included

  • proliferation,
  • spontaneous apoptosis,
  • adhesion and
  • differentiation capability.

We observed a decline in ADSC proliferation after modification by rAAV that was evident by

  • increase of population doubling time as well as
  • decrease in number of cells in S–G2 stages of cell cycle.

At the same time spontaneous apoptosis rate did not exceed 2% in modified and unmodified cells. These results contribute to previously published data that showed transient cell cycle arrest after AAV transduction of embryonic fibroblasts and BMMSC [40]. This effect was observed whenever

  • wild-type,
  • recombinant or
  • genome-empty AAV particles were used.

It was suggested that changes in expression profile and decreased proliferation were related to initial stage of virus entry and caused by capsid proteins interaction with cellular signaling pathways [40]. Growth inhibitory effect was transient and

  • proliferation restored to normal level over time of cell passaging [41].

It appears that proliferation decline of rAAV-modified ADSC occurs by a common mechanism.

ADSC are known to be able to differentiate into

  • adipocytes, chondrocytes, osteoblasts, myocytes, neural cells, cardiomyocytes, endothelial and liver cells
  • when cultured in special induction medium [42,43].

Analyzing data from our differentiation experiments we concluded that rAAV-mediated genetic modification of human ADSC and VEGF overexpression did not alter their adipogenic and osteogenic differentiation properties.

There are several observations indicating ability of ADSC for endothelial differentiation [44,45] as well as evidence for presence of small amount of endothelial cells in ADSC population at early passages [18,19]. In our experiments we did not find an increase in amount of cells positive for endothelial markers CD31 and VEGFR2 in VEGF-ADSC compared to unmodified ADSC population. This suggests that VEGF overexpression

  • neither induces endothelial differentiation of modified ADSC
  • or stimulates proliferation of preexisting endothelial cells in ADSC culture.

Adhesion tests conducted in our study were based on a fact that

  • interaction with extracellular matrix proteins is a key factor
  • that contributes to cell viability and integration into host tissue after transplantation [46].

We found that both modified and untreated ADSC showed very common adhesion on collagen type 1, vitronectin and fibronectin. Thus we can suggest that

  • rAAV-mediated genetic modification did not alter expression of adhesion molecules on cell surface of ADSC.

Our results showing low ADSC adhesion on laminin are not surprising taking into account published observations which indicate diminished or lack of α6, α7 and ß1 integrins expression in ADSC-components of α6/ß1 and α7/ß1 receptors for laminin [47,48].

Since VEGF can regulate multiple signaling pathways [23] we next determined whether expression of HGF, FGF2, urokinase and angiopoietin-1 might be altered in VEGF-ADSC. HGF and FGF2 are mitogens and chemoattractants for both endothelial and mural cells and directly participate in angio- and arteriogenesis [4]. Angiopoietin-1 is characterized as a stabilizing factor that provides formation of functionally mature vessel network [49]. Urokinase plasminogen activator is a key regulator of extracellular proteolysis which is

  • responsible for cleavage activation of growth factors and migration of endothelial cells during vessel growth [50,51].

We found almost 3-fold yet not statistically significant increase of urokinase expression while expression of HGF and FGF2 did not change. Another interesting finding is a 5-fold increase of angiopoietin-1 expression in VEGF–ADSC compared to GFP–ADSC or unmodified cells. We assumed that up-regulation of angiopoietin-1 expression occurs due to autocrine action of VEGF165 produced by VEGF-ADSC. However according to our data supported by other studies [30,52] cultured human ADSC population contains <1% of cells that express receptors to VEGF165 – VEGFR1 and VEGFR2. At the same time we found that

  • >90% of ADSC carry receptor to platelet-derived growth factor – PDGFRβ.
There is a published observation that
  • PDGFRα and PDGFRβ can act as a facultative receptor for VEGF [27].
  • it is also known that PDGFR activation leads to increase of angiopoietin-1 expression [53].

Considering that more than 90% of human ADSC are PDGFRβ-positive

  • we can speculate that increased expression of angiopoetin-1 in VEGF-ADSC could be attributed to PDGFRβ-mediated autocrine action of VEGF.
In our study we evaluated therapeutic potential of gene modified human ADSC in terms of their ability to induce angiogenesis in ischemic muscle tissue. It was found that matrigel implants after transplantation of VEGF-ADSC had higher vascular density than after delivery of untreated cells or ADSC transduced by a reporter gene. Along with

  • capillary formation we also found
  • proportional increase in amount of mature blood vessels characterized by smooth-muscle wall.

This can occur due to the fact that cells transplanted in matrigel produce other angiogenic factors besides VEGF that can promote vessel maturation and stabilization.

Key angiogenic property of cell therapies in experimental study is ability to induce reperfusion of ischemic tissue in appropriate animal models. We used hind limb ischemia model to show that

  • VEGF-ADSC transplantation led to significantly higher perfusion restoration than
  • after untreated of GFP-transduced cell administration.

It was also found that intramuscular injection of VEGF-ADSC had a tissue-protective effect and led to vivid decrement of necrosis span. VEGF is known to be significant antiapoptotic factor that can enhance cell survival. We suggest that

  • increased VEGF content during the first days after onset of acute ischemia and cells administration leads to promotion of cell survival and thus to reduction of necrotic disruption in muscle tissue.

We should also point that during the experiment we did not observe any blood flow decrease after cell administration or rapid “plateau” formation like it was previously described for plasmid-mediated gene delivery due to short-term transgene expression [4]. This can be explained by

  • presence of viable and functionally active ADSC that produced VEGF throughout the experiment.

In our muscle explant experiments we showed that VEGF-ADSC retain functional activity even at long terms after injection (up to 27 days) and produce VEGF in detectable quantities. Thus we can confidently attribute

  • tissue protection and restoration of blood flow in mice that received VEGF-ADSC to increased long-term VEGF production by modified cells.

As for decrease of human VEGF content in murine tissue by day 20 we suggest that cells undergo apoptosis over time. Besides that methylation of CMV promoter which drives VEGF expression in our vector could take place. Taking into account that Nude mice were used we find it hard to assume possible rejection of transplanted cells as far as this animal strain lacks T-cells immunity which plays a crucial role in graft rejection. Still, it seems that produced amount of VEGF is sufficient to trigger angiogenesis and relief tissue ischemia via restoration of blood flow.

Histological analysis of ischemic muscle injected with modified VEGF-ADSC revealed that

  • capillary density was significantly higher than in specimens from animals that received untreated cells or GFP-ADSC.

We noticed that this increase was not only due to higher capillary count, but also to SMA-positive blood vessels of arteriolar type. Furthermore arteriole/capillary ratio was constant throughout experimental groups that indicated formation of a stable mature vascular network. Thus, despite high level of VEGF produced by modified ADSC we did not observe any evidence for abnormal tumour-like vascular structures in muscle as it was previously shown e.g. in studies of adenovirus-mediated delivery of VEGF gene [54]. In contrast to matrigel implants experiment in case of skeletal muscle we do not state that increase of vascular density in experimental groups was only due to de novo formed vessels. Besides promoting endothelial cell proliferation VEGF also prevents endothelial apoptosis leading to survival of preexisting vessels. There was surely a vast amount of persisted capillaries in the muscles due to VEGF anti-apoptotic effect of VEGF.

It is often speculated that low efficacy reported in clinical trials using gene delivery of VEGF alone can be explained by its high mitogenic activity which is not supported by vessel stabilizing stimuli and consequently ends up with dissociation of formed capillaries [55]. This led to a concept of combined gene delivery

  • indicating that combinations of angiogenic and vascular stabilizing factors should be used to treat ischemic tissues [5558].

Cell therapy for ischemic disorders has a valuable advantage since transplanted cells produce a whole “cocktail” of biologically active molecules which render combined effect in impaired tissue. We suggest that stable vessel formation observed in our study is

  • mediated by aforementioned ADSC ability to produce a wide spectrum of angiogenic factors
  • including ones responsible for vessel stabilization and maturation: angiopoietin-1, TGF-β, PDGF,
  • which can act synergistically with increased production of VEGF165 by modified cells.

Besides that, genetic modification can alter cell’s expression profile. Observed increase in expression of angiopoietin-1 in VEGF-ADSC can further contribute to

  • formation of mature vascular network that also
  • supports therapeutic effect of transplanted cells.
Increased concentration of VEGF in ischemic tissue plays a substantial role in vessel stabilization and therapeutic effect if maintained over a significant period of time, which was achieved in our study and exceeded a substantial term of 3 weeks.

Conclusions

Thus we can conclude that human ADSC with their accessibility and angiogenic paracrine activity is an appropriate and preferable type of cells for therapeutic angiogenesis. Obtained results indicate that relatively safe rAAV holds great potential for gene transfer into human ADSC. Taken together, we suggest that

  • the use of AAV-modified ADSC overexpressing VEGF165 is a feasible and effective approach for stimulation of stable vascular network formation in ischemic muscle and

can be implied for therapeutic angiogenesis or tissue-engineered transplants. Further study and improvements in vector design, regulated transgene expression, cell preparation and propagation conditions are still to be completed to allow clinical application of modified cell-based therapeuticals.

Abbreviations

ADSC: Adipose derived stromal cells; BMMSC: Bone marrow derived mesenchymal stem cells; CGS: Cell growth supplement; DMEM: Dulbecco’s modified Eagle’s medium; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; FGF2: Fibroblast growth factor 2; FOV: Field of view; GFP: Green fluorescent protein; HEK293T: Human embryonic kidney 293 T; HGF: Hepatocyte growth factor; PDGF: Platelet derived growth factor; PDT: Population doubling time; PBS: Phosphate buffer saline; rAAV: Recombinant adeno-associated virus; SMA: Smooth muscle actin; VEGF: Vascular endothelial growth factor

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The Delicate Connection:  IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

Author and Curator: Demet Sag, PhD, CRA, GCP      

Table of Contents:

  1. Abstract
  2. Dual role for IDO
  3. Immune System and IDO
  4. Autoimmune disorders and IDO
  5. Cancer and Ido
  6. Clinical Interventions
  7. Clinical Trials
  8. Future Actions for Molecular Dx and Targeted Therapies:
  9. Conclusion
  10. References

TABLE 1- IDO Clinical Trials

TABLE 2- Kyn induced Genes

TABLE 3 Possible biomarkers and molecular diagnostics targets

TABLE 4: Current Interventions ______________________________________________________________________________________________________________

ABSTRACT:

Overall purpose is to find a method to manipulate IDO for clinical applications, mainly the focus of this review is is cancer prevention and treatment.  The first study proving the connection between IDO and immune response came from, a very natural event, a protection of pregnancy in human. This led to discover that high IDO expression is a common factor in cancer tumors. Thus, attention promoted investigations on IDO’s role in various disease states, immune disorders, transplantation, inflammation, women health, mood disorders.
Many approaches, vaccines and adjuvants are underway to find new immunotherapies by combining the power of DCs in immune response regulation and specific direction of siRNA.  As a result, with this unique qualities of IDO, DCs and siRNA, we orchestrated a novel intervention for immunomodulation of IDO by inhibiting with small interference RNA, called siRNA-IDO-DCvax.  Proven that our DCvax created a delay and regression of tumor growth without changing the natural structure and characterization of DCs in melanoma and breast cancers in vivo. (** The shRNA IDO- DCvax is developed by Regen BioPhrama, San Diego, CA ,  Thomas Ichim, Ph.D, CSO. and David Koos, CEO)

______________________________________________________________________________________________________________

Double-Edged Sword of IDO: The Good and The Bad for Clinical intervention and Developments

IDO almost has a dual role. There is a positive side of high expression of IDO during pregnancy (29; 28; 114), transplants (115; 116; 117; 118; 119), infectious diseases (96) and but this tolerance is negative during autoimmune-disorders (120; 121; 122), tumors of cancer (123; 124; 117; 121; 125; 126; 127) (127), and mood disorders (46). The increased IDO expression has a double-edged sword in human physiology provides a positive role during protection of fetus and grafts after transplantations but becomes a negative factor during autoimmune disorders, cancer, sepsis and mood disorders.

Prevention of allogeneic fetal rejection is possible by tryptophan metabolism (26) rejecting with lack of IDO but allocating if IDO present (29; 28; 114). These studies lead to find “the natural regulation mechanism” for protecting the transplants from graft versus host disease GVHD (128) and getting rid of tumors.

The plasticity of  mammary and uterus during reproduction may hold some more answers to prevent GVHD and tumors of cancer with good understanding of IDO and tryptophan mechanism (129; 130). After allogeneic bone marrow transplants the risk of solid tumor development increased about 80% among 19,229 patients even with a greater risk among patients under 18 years old (117).  The adaptation of tolerance against host mechanism is connected to the IDO expression (131). During implantation and early pregnancy IDO has a role by making CD4+CD25+Foxp3+ regulatory T cells (Tregs) and expressing in DCs and  MQs  (114; 132; 133).

Clonal deletion mechanism prevents mother to react with paternal products since female mice accepted the paternal MHC antigen-expressing tumor graft during pregnancy and rejected three weeks after delivery (134). CTLA-4Ig gene therapy alleviates abortion through regulation of apoptosis and inhibition of spleen lymphocytes (135).  

 Immune System and IDO DCs are the orchestrator of the immune response (56; 57; 58) with list of functions in uptake, processing, and presentation of antigens; activation of effector cells, such as T-cells and NK-cells; and secretion of cytokines and other immune-modulating molecules to direct the immune response. The differential regulation of IDO in distinct DC subsets is widely studied to delineate and correct immune homeostasis during autoimmunity, infection and cancer and the associated immunological outcomes. Genesis of antigen presenting cells (APCs), eventually the immune system, require migration of monocytes (MOs), which is originated in bone marrow. Then, these MOs move from bloodstream to other tissues to become macrophages and DCs (59; 60).

Initiation of immune response requires APCs to link resting helper T-cell with the matching antigen to protect body. DCs are superior to MQs and MOs in their immune action model. When DCs are first described (61) and classified, their role is determined as a highly potent antigen-presenting cell (APC) subset with 100 to 1000-times more effective than macrophages and B-cells in priming T-cells. Both MQs and monocytes phagocytize the pathogen, and their cell structure contains very large nucleus and many internal vesicles. However, there is a nuance between MQ and DCs, since DCs has a wider capacity of stimulation, because MQs activates only memory T cells, yet DCs can activate both naïve and memory T cells.

DCs are potent activators of T cells and they also have well controlled regulatory roles. DC properties determine the regulation regardless of their origin or the subset of the DCs. DCs reacts after identification of the signals or influencers for their inhibitory, stimulatory or regulatory roles, before they express a complex repertoire of positive and negative cytokines, transmembrane proteins and other molecules. Thus, “two signal theory” gains support with a defined rule.  The combination of two signals, their interaction with types of cells and time are critical.

In short, specificity and time are matter for a proper response. When IDO mRNA expression is activated with CTL40 ligand and IFNgamma, IDO results inhibition of T cell production (4).  However, if DCs are inhibited by 1MT, an inhibitor of IDO, the response stop but IgG has no affect (10).  In addition, if the stimulation is started by a tryptophan metabolite, which is downstream of IDO, such as 3-hydroxyantranilic or quinolinic acids, it only inhibits Th1 but not Th2 subset of T cells (62).

Furthermore, inclusion of signal molecules, such as Fas Ligand, cytochrome c, and pathways also differ in the T cell differentiation mechanisms due to combination, time and specificity of two-signals.  The co-culture experiments are great tool to identify specific stimuli in disease specific microenvironment (63; 12; 64) for discovering the mechanism and interactions between molecules in gene regulation, biochemical mechanism and physiological function during cell differentiation.

As a result, the simplest differential cell development from the early development of DCs impact the outcome of the data. For example, collection of MOs from peripheral blood mononuclear cells (PBMCs) with IL4 and GM-CSF leads to immature DCs (iDCs). On next step, treatment of iDCs with tumor necrosis factor (TNF) or other plausible cytokines (TGFb1, IFNgamma, IFNalpha,  IFNbeta, IL6 etc.) based on the desired outcome differentiate iDCs  into mature DCs (mDCs). DCs live only up to a week but MOs and generated MQs can live up to a month in the given tissue. B cells inhibit T cell dependent immune responses in tumors (65).

AutoImmune Disorders:

The Circadian Clock Circuitry and the AHR

The balance of IDO expression becomes necessary to prevent overactive immune response self-destruction, so modulation in tryptophan and NDA metabolisms maybe essential.  When splenic IDO-expressing CD11b (+) DCs from tolerized animals applied, they suppressed the development of arthritis, increased the Treg/Th17 cell ratio, and decreased the production of inflammatory cytokines in the spleen (136).

The role of Nicotinamide prevention on type 1 diabetes and ameliorates multiple sclerosis in animal model presented with activities of  NDAs stimulating GPCR109a to produce prostaglandins to induce IDO expression, then these PGEs and PGDs converted to the anti-inflammatory prostaglandin, 15d-PGJ(2) (137; 138; 139).  Thus, these events promotes endogenous signaling mechanisms involving the GPCRs EP2, EP4, and DP1 along with PPARgamma. (137).

Modulating the immune response at non-canonical at canonocal pathway while keeping the non-canonical Nf-KB intact may help to mend immune disorders. As a result, the targeted blocking in canonical at associated kinase IKKβ and leaving non-canonocal Nf-kB pathway intact, DCs tips the balance towards immune supression. Hence, noncanonical NF-κB pathway for regulatory functions in DCs required effective IDO induction, directly or indirectly by endogenous ligand Kyn and negative regulation of proinflammatory cytokine production. As a result, this may help to treat autoimmune diseases such as rheumatoid arthritis, type 1 diabetes, inflammatory bowel disease, and multiple sclerosis, or allergy or transplant rejection.

While the opposite action needs to be taken during prevention of tumors, that is inhibition of non-canonical pathway.  Inflammation induces not only relaxation of veins and lowering blood pressure but also stimulate coagulopathies that worsen the microenvironment and decrease survival rate of patients after radio or chemotherapies.Cancer Generating tumor vaccines and using adjuvants underway (140).

Clinical correlation and genetic responses also compared in several studies to diagnose and target the system for cancer therapies (127; 141; 131).  The recent surveys on IDO expression and human cancers showed that IDO targeting is a candidate for cancer therapy since IDO expression recruiting Tregs, downregulates MHC class I and creating negative immune microenvironment for protection of development of tumors (125; 27; 142).  Inhibition of IDO expression can make advances in immunotherapy and chemotherapy fields (143; 125; 131; 144).

IDO has a great importance on prevention of cancer development (126). There are many approaches to create the homeostasis of immune response by Immunotherapy.  However, given the complexity of immune regulations, immunomodulation is a better approach to correct and relieve the system from the disease.  Some of the current IDO targeted immunotherapy or immmunomodulations with RNA technology for cancer prevention (145; 146; 147; 148; 149; 150) or applied on human or animals  (75; 151; 12; 115; 152; 9; 125) or chemical, (153; 154) or  radiological (155).  The targeted cell type in immune system generally DCs, monocytes (94)T cells (110; 156)and neutrophils (146; 157). On this paper, we will concentrate on DCvax on cancer treatments.

 T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

T-reg, regulatory T cells; Th, T helper; CTLA-4, cytotoxic T lymphocyte-associated antigen 4; TCR, T cell receptor; IDO, indoleamine 2,3-dioxygenase. (refernece: http://www.pnas.org/content/101/28/10398/suppl/DC)

IDO and the downstream enzymes in tryptophan pathway produce a series of immunosuppressive tryptophan metabolites that may lead into Tregs proliferation or increase in T cell apoptosis (62; 16; 27; 158), and some can affect NK cell function (159).

The interesting part of the mechanism is even without presence of IDO itself, downstream enzymes of IDO in the kynurenine tryptophan degradation still show immunosuppressive outcome (160; 73) due to not only Kyn but also TGFbeta stimulated long term responses. DC vaccination with IDO plausible (161) due to its power in immune response changes and longevity in the bloodstream for reversing the system for Th17 production (162).

Clinical Interventions are taking advantage of the DC’s central role and combining with enhancing molecules for induction of immunity may overcome tolerogenic DCs in tumors of cancers (163; 164).

The first successful application of DC vaccine used against advanced melanoma after loading DCs with tumor peptides or autologous cell lysate in presence of adjuvants keyhole limpet hematocyanin (KLH) (165).  Previous animal and clinical studies show use of DCs against tumors created success (165; 166; 167) as well as some problems due to heterogeneity of DC populations in one study supporting tumor growth rather than diminishing (168).

DC vaccination applied onto over four thousand clinical trial but none of them used siRNA-IDO DC vaccination method. Clinical trials evaluating DCs loaded ex vivo with purified TAAs as an anticancer immunotherapeutic interventions also did not include IDO (Table from (169). This table presented the data from 30 clinical trials, 3 of which discontinued, evaluating DCs loaded ex vivo with TAAs as an anticancer immunotherapy for 12 types of cancer [(AML(1), Breast cancer (4), glioblastoma (1), glioma (2), hepatocellular carcinoma (1), hematological malignancies (1), melanoma (6), neuroblastoma sarcoma (2), NSCLC (1), ovarian cancer (3), pancreatic cancer (3), prostate cancer (10)] at phase I, II or I/II.

Tipping the balance between Treg and Th17 ratio has a therapeutic advantage for restoring the health that is also shown in ovarian cancer by DC vaccination with adjuvants (161).  This rebalancing of the immune system towards immunogenicity may restore Treg/Th17 ratio (162; 170) but it is complicated. The stimulation of IL10 and IL12 induce Treg produce less Th17 and inhibiting CTL activation and its function (76; 171; 172) while animals treated with anti-TGFb before vaccination increase the plasma levels of IL-15 for tumor specific T cell survival in vivo (173; 174) ovarian cancer studies after human papilloma virus infection present an increase of IL12 (175).

Opposing signal mechanism downregulates the TGFb to activate CTL and Th1 population with IL12 and IL15 expression (162; 173).  The effects of IL17 on antitumor properties observed by unique subset of CD4+ T cells (176) called also CD8+ T cells secrete even more IL17 (177).

Using cytokines as adjuvants during vaccination may improve the efficacy of vaccination since cancer vaccines unlike infections vaccines applied after the infection or disease started against the established adoptive immune response.  Adjuvants are used to improve the responses of the given therapies commonly in immunotherapy applications as a combination therapy (178).

Enhancing cancer vaccine efficacy via modulation of the microenvironment is a plausible solution if only know who are the players.  Several molecules can be used to initiate and lengthen the activity of intervention to stimulate IDO expression without compromising the mechanism (179).  The system is complicated so generally induction is completed ex-vivo stimulation of DCs in cell lysates, whole tumor lysates, to create the microenvironment and natural stimulatory agents. Introduction of molecules as an adjuvants on genetic regulation on modulation of DCs are critical, because order and time of the signals, specific location/ tissue, and heterogeneity of personal needs (174; 138; 180). These studies demonstrated that IL15 with low TGFb stimulates CTL and Th1, whereas elevated TGFb with IL10 increases Th17 and Tregs in cancer microenvironments.

IDO and signaling gene regulation

For example Ret-peptide antitumor vaccine contains an extracellular fragment of Ret protein and Th1 polarized immunoregulator CpG oligonucleotide (1826), with 1MT, a potent inhibitor of IDO, brought a powerful as well as specific cellular and humoral immune responses in mice (152).

The main idea of choosing Ret to produce vaccine in ret related carcinomas fall in two criterion, first choosing patients self-antigens for cancer therapy with a non-mutated gene, second, there is no evidence of genetic mutations in Ret amino acids 64-269. Demonstration of proliferating hemangiomas, benign endothelial tumors and often referred as hemangiomas of infancy appearing at head or neck, express IDO and slowly regressed as a result of immune mediated process.

After large scale of genomic analysis show insulin like growth factor 2 as the key regulator of hematoma growth (Ritter et al. 2003). We set out to develop new technology with our previous expertise in immunotherapy and immunomodulation (181; 182; 183; 184), correcting Th17/Th1 ratio (185), and siRNA technology (186; 187).  We developed siRNA-IDO-DCvax. Patented two technologies “Immunomodulation using Altered DCs (Patent No: US2006/0165665 A1) and Method of Cancer Treatments using siRNA Silencing (Patent No: US2009/0220582 A1).

In melanoma cancer DCs were preconditioned with whole tumor lysate but in breast cancer model pretreatment completed with tumor cell lysate before siRNA-IDO-DCvax applied. Both of these studies was a success without modifying the autanticity of DCs but decreasing the IDO expression to restore immunegenity by delaying tumor growth in breast cancer (147) and in melanoma (188).  Thus, our DCvax specifically interfere with Ido without disturbing natural structure and content of the DCs in vivo showed that it is possible to carry on this technology to clinical applications.

Furthermore, our method of intervention is more sophisticated since it has a direct interaction mechanism with ex-vivo DC modulation without creating long term metabolism imbalance in Trp/Kyn metabolite mechanisms since the action is corrective and non-invasive.

There were several reasons.

First, prevention of tumor development studies targeting non-enzymatic pathway initiated by pDCs conditioned with TGFbeta is specific to IDO1 (189).

Second, IDO upregulation in antigen presenting cells allowing metastasis show that most human tumors express IDO at high levels (123; 124).

Third, tolerogenic DCs secretes several molecules some of them are transforming growth factor beta (TGFb), interleukin IL10), human leukocyte antigen G (HLA-G), and leukemia inhibitory factor (LIF), and non-secreted program cell death ligand 1 (PD-1 L) and IDO, indolamine 2.3-dioxygenase, which promote tumor tolerance. Thus, we took advantage of DCs properties and Ido specificity to prevent the tolerogenicity with siRNA-IDO DC vaccine in both melanoma and breast cancer.

Fourth, IDO expression in DCs make them even more potent against tumor antigens and create more T cells against tumors. IDOs are expressed at different levels by both in broad range of tumor cells and many subtypes of DCs including monocyte-derived DCs (10), plasmacytoid DCs (142), CD8a+ DCs (190), IDO compotent DCs (17), IFNgamma-activated DCs used in DC vaccination.  These DCs suppress immune responses through several mechanisms for induction of apoptosis towards activated T cells (156) to mediate antigen-specific T cell anergy in vivo (142) and for enhancement of Treg cells production at sites of vaccination with IDO-positive DCs+ in human patients (142; 191; 192; 168; 193; 194). If DCs are preconditioned with tumor lysate with 1MT vaccination they increase DCvax effectiveness unlike DCs originated from “normal”, healthy lysate with 1MT in pancreatic cancer (195).  As a result, we concluded that the immunesupressive effect of IDO can be reversed by siRNA because Treg cells enhances DC vaccine-mediated anti-tumor-immunity in cancer patients.

Gene silencing is a promising technology regardless of advantages simplicity for finding gene interaction mechanisms in vitro and disadvantages of the technology is utilizing the system with specificity in vivo (186; 196).  siRNA technology is one of the newest solution for the treatment of diseases as human genomics is only producing about 25,000 genes by representing 1% of its genome. Thus, utilizing the RNA open the doors for more comprehensive and less invasive effects on interventions. Thus this technology is still improving and using adjuvants. Silencing of K-Ras inhibit the growth of tumors in human pancreatic cancers (197), silencing of beta-catenin in colon cancers causes tumor regression in mouse models (198), silencing of vascular endothelial growth factor (VGEF) decreased angiogenesis and inhibit tumor growth (199).

Combining siRNA IDO and DCvax from adult stem cell is a novel technology for regression of tumors in melanoma and breast cancers in vivo. Our data showed that IDO-siRNA reduced tumor derived T cell apoptosis and tumor derived inhibition of T cell proliferation.  In addition, silencing IDO made DCs more potent against tumors since treated or pretreated animals showed a delay or decreased the tumor growth (188; 147)

 

Clinical Trials:

First FDA approved DC-based cancer therapies for treatment of hormone-refractory prostate cancer as autologous cellular immunotherapy (163; 164).  However, there are many probabilities to iron out for a predictive outcome in patients.

Table 2 demonstrates the current summary of clinical trials report.  This table shows 38 total studies specifically Ido related function on cancer (16), eye (3), surgery (2), women health (4), obesity (1), Cardiovascular (2), brain (1), kidney (1), bladder (1), sepsis shock (1), transplant (1),  nervous system and behavioral studies (4), HIV (1) (Table 4).  Among these only 22 of which active, recruiting or not yet started to recruit, and 17 completed and one terminated.

Most of these studies concentrated on cancer by the industry, Teva GTC ( Phase I traumatic brain injury) Astra Zeneca (Phase IV on efficacy of CRESTOR 5mg for cardiovascular health concern), Incyte corporation (Phase II ovarian cancer) NewLink Genetics Corporation Phase I breast/lung/melanoma/pancreatic solid tumors that is terminated; Phase II malignant melanoma recruiting, Phase II active, not recruiting metastatic breast cancer, Phase I/II metastatic melanoma, Phase I advanced malignancies) , HIV (Phase IV enrolling by invitation supported by Salix Corp-UC, San Francisco and HIV/AIDS Research Programs).

Many studies based on chemotherapy but there are few that use biological methods completed study with  IDO vaccine peptide vaccination for Stage III-IV non-small-cell lung cancer patients (NCT01219348), observational study on effect of biological therapy on biomarkers in patients with untreated hepatitis C, metastasis melanoma, or Crohn disease by IFNalpha and chemical (ribavirin, ticilimumab (NCT00897312), polymorphisms of patients after 1MT drug application in treating patients with metastatic or unmovable refractory solid tumors by surgery (NCT00758537), IDO expression analysis on MSCs (NCT01668576), and not yet recruiting intervention with adenovirus-p53 transduced dendric cell vaccine , 1MT , radiation, Carbon C 11 aplha-methyltryptophan- (NCT01302821).

Among the registered clinical trials some of them are not interventional but  observational and evaluation studies on Trp/Kyn ratio (NCT01042847), Kyn/Trp ratio (NCT01219348), Kyn levels (NCT00897312, NCT00573300),  RT-PCR analysis for Kyn metabolism (NCT00573300, NCT00684736, NCT00758537), and intrinsic IDO expression of mesenchymal stem cells in lung transplant with percent inhibition of CD4+ and CD8+ T cell proliferation toward donor cells (NCT01668576), determining polymorphisms (NCT00426894). These clinical trials/studies are immensely valuable to understand the mechanism and route of intervention development with the data collected from human populations   

Future Actions for Molecular Dx and Targeted Therapies:

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors.  (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Viable tumor environment. Tumor survival is dependent upon an exquisite interplay between the critical functions of stromal development and angiogenesis, local immune suppression and tumor tolerance, and paradoxical inflammation. TEMs: TIE-2 expressing monocytes; “M2” TAMs: tolerogenic tumor-associated macrophages; MDSCs: myeloid-derived suppressor cells; pDCs: plasmacytoid dendritic cells; co-stim.: co-stimulation; IDO: indoleamine 2,3-dioxygenase; VEGF: vascular endothelial growth factor; EGF: epidermal growth factor; MMP: matrix metaloprotease; IL: interleukin; TGF-β: transforming growth factor-beta; TLRs: toll-like receptors. (reference: http://www.hindawi.com/journals/cdi/2012/937253/fig1/)

Current survival or response rate is around 40 to 50 % range.  By using specific cell type, selected inhibition/activation sequence based on patient’s genomic profile may improve the efficacy of clinical interventions on cancer treatments. Targeted therapies for specific gene regulation through signal transduction is necessary but there are few studies with genomics based approach.

On the other hand, there are surveys, observational or evaluations (listed in clinical trials section) registered with www.clinicaltrials.gov that will provide a valuable short-list of molecules.  Preventing stimulation of Ido1 as well as Tgfb-1gene expression by modulating receptor mediated phosphorylation between TGFb/SMAD either at Mad-Homology 1 (MH1) or Mad-Homology 1 (MH2) domains maybe possible (79; 82; 80). Within Smads are the conserved Mad-Homology 1 (MH1) domain, which is a DNA binding module contains tightly bound Zinc atom.

Smad MH2 domain is well conserved and one the most diverse protein-signal interacting molecule during signal transduction due to two important Serine residues located extreme distal C-termini at Ser-Val-Ser in Smad 2 or at pSer-X-PSer in RSmads (80). Kyn activated orphan G protein–coupled receptor, GPR35 with unknown function with a distinct expression pattern that collides with IDO sites since its expression at high levels of the immune system and the gut (63) (200; 63).  

The first study to connect IDO with cancer shows that group (75).  The directly targeting to regulate IDO expression is another method through modulating ISREs in its promoter with RNA-peptide combination technology. Indirectly, IDO can be regulated through Bin1 gene expression control over IDO since Bin1 is a negative regulator of IDO and prevents IDO expression.  IDO is under negative genetic control of Bin1, BAR adapter–encoding gene Bin1 (also known as Amphiphysin2). Bin1 functions in cancer suppression since attenuation of Bin1 observed in many human malignancies (141; 201; 202; 203; 204; 205; 206) .  Null Bin-/- mice showed that when there is lack of Bin1, upregulation of IDO through STAT1- and NF-kB-dependent expression of IDO makes tumor cells to escape from T cell–dependent antitumor immunity.

This pathway lies in non-enzymatic signal transducer function of IDO after stimulation of DCs by TGFb1.  The detail study on Bin1 gene by alternative spicing also provided that Bin1 is a tumor suppressor.  Its activities also depends on these spliced outcome, such as  Exon 10, in muscle, in turn Exon 13 in mice has importance in role for regulating growth when Bin1 is deleted or mutated C2C12 myoblasts interrupted due to its missing Myc, cyclinD1, or growth factor inhibiting genes like p21WAF1 (207; 208).

On the other hand alternative spliced Exon12A contributing brain cell differentiation (209; 210). Myc as a target at the junction between IDO gene interaction and Trp metabolism.  Bin1 interacts with Myc either early-dependent on Myc or late-independent on Myc, when Myc is not present. This gene regulation also interfered by the long term signaling mechanism related to Kynurenine (Kyn) acting as an endogenous ligand to AHR in Trp metabolite and TGFb1 and/or IFNalpha and IFNbeta up regulation of DCs to induce IDO in noncanonical pathway for NF-kB and myc gene activations (73; 74).  Hence, Trp/Kyn, Kyn/Trp, Th1/Th17 ratios are important to be observed in patients peripheral blood. These direct and indirect gene interactions place Bin1 to function in cell differentiation (211; 212; 205).

Regulatory T-cel generation via reverse and non-canonical signaliing to pDCs

Table 3 contains the microarray analysis for Kyn affect showed that there are 25 genes affected by Kyn, two of which are upregulated and 23 of them downregulated (100). This list of genes and additional knowledge based on studies creating the diagnostics panel with these genes as a biomarker may help to analyze the outcomes of given interventions and therapies. Some of these molecules are great candidate to seek as an adjuvant or co-stimulation agents.  These are myc, NfKB at IKKA, C2CD2, CREB3L2, GPR115, IL2, IL8, IL6, and IL1B, mir-376 RNA, NFKB3, TGFb, RelA, and SH3RF1. In addition, Lip, Fox3P, CTLA-4, Bin1, and IMPACT should be monitored.

In addition, Table 4 presents the other possible mechanisms. The highlights of possible target/biomarkers are specific TLRs, conserved sequences of IDO across its homologous structures, CCR6, CCR5, RORgammat, ISREs of IDO, Jak, STAT, IRFs, MH1 and MH2 domains of Smads. Endothelial cell coagulation activation mechanism and pDC maturation or immigration from lymph nodes to bloodstream should marry to control not only IDO expression but also genesis of preferred DC subsets. Stromal mesenchymal cells are also activated by these modulation at vascular system and interferes with metastasis of cancer. First, thrombin (human factor II) is a well regulated protein in coagulation hemostasis has a role in cell differentiation and angiogenesis.

Protein kinase activated receptors (PARs), type of GPCRs, moderate the actions. Second, during hematopoietic response endothelial cells produce hematopoietic growth factors (213; 214). Third, components of bone marrow stroma cells include monocytes, adipocytes, and mesenchymal stem cells (215). As a result, addressing this issue will prevent occurrence of coagulapathologies, namely DIC, bleeding, thrombosis, so that patients may also improve response rate towards therapies. Personal genomic profiles are powerful tool to improve efficacy in immunotherapies since there is an influence of age (young vs. adult), state of immune system (innate vs. adopted or acquired immunity). Table 5 includes some of the current studies directly with IDO and indirectly effecting its mechanisms via gene therapy, DNA vaccine, gene silencing and adjuvant applications as an intervention method to prevent various cancer types.

CONCLUSION

IDO has a confined function in immune system through complex interactions to maintain hemostasis of immune responses. The genesis of IDO stem from duplication of bacterial IDO-like genes.  Inhibition of microbial infection and invasion by depleting tryptophan limits and kills the invader but during starvation of trp the host may pass the twilight zone since trp required by host’s T cells.  Thus, the host cells in these small pockets adopt to new microenvironment with depleted trp and oxygen poor conditions. Hence, the cell metabolism differentiate to generate new cellular structure like nodules and tumors under the protection of constitutively expressed IDO in tumors, DCs and inhibited T cell proliferation.

On the other hand, having a dichotomy in IDO function can be a potential limiting factor that means is that IDOs impact on biological system could be variable based on several issues such as target cells, IDO’s capacity, pathologic state of the disease and conditions of the microenvironment. Thus, close monitoring is necessary to analyze the outcome to prevent conspiracies since previous studies generated paradoxical results.

Current therapies through chemotherapies, radiotherapies are costly and effectiveness shown that the clinical interventions require immunotherapies as well as coagulation and vascular biology manipulations for a higher efficacy and survival rate in cancer patients. Our siRNA and DC technologies based on stem cell modulation will provide at least prevention of cancer development and hopefully prevention in cancer.

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Abstract:

The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review, the  properties of IDO such as basic molecular genetics, biochemistry and genesis will be discussed.

IDO belongs to globin gene family to carry oxygen and heme.  The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn.

The well understood functional genomics and mechanisms is important to translate basic science for clinical interventions of human health needs. In conclusion, overall purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

The first part of the review concerns the basic science information gained overall several years that lay the foundation where translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

Table of Contents:

  • Abstract

1         Introduction: IDO gene encodes a heme enzyme

2        Location, location, location

3        Molecular genetics

4        Types of IDO:

4.1       IDO1,

4.2       IDO2,

4.3       IDO-like proteins

5        Working mechanisms of IDO

6        Infection Diseases and IDO

7. Conclusion

  1. 1.     Indoleamine 2, 3-dioxygenase (IDO) gene encodes a heme enzyme

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC=1.13.11.52) that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin.

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.

After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

  1. 2.     Location, Location and Location

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).

IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).

 

 3.     Molecular Genetics of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.
(reference: http://atlasgeneticsoncology.org/Genes/IDO2ID44387ch8p11.html)

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).  Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).

Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).

The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

4.     There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).

There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

4.1.  IDO1:

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).  The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms. Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (http://www.pnas.org/content/103/8/2611/F3.expansion.html)

4.2. IDO2:

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).   Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46).

Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

4.3. IDO-like proteins: The Origin of IDO:

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.

Bacterial IDO has two types of IDOs that are group I and group II IDO (48).  These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).   Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.

Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins are destined to evolve from a common ancestor, which  is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52) IDO like globin widely distributed among gastropodic mollusks (53; 51).  The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).

The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53) Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.

No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

Mechanisms of IDO:

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

 IDO mechanism for immune response

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63).

Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

 Indoleamine-Pyrrole 2,3,-Dioxygenase; IDO dioxygenase; Indeolamine-2,3

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response. When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).

Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68). SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).

This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91).

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103).

Utilizing specific TLR stimulation to link between innate and acquired responses can be possible through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105).  Double stranded (ds) RNAs through TLR3 (106; 107), Lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

IDO action

Then, the specificity is established by correct pairing of a TLR with its proinflammatory cytokines, so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, if the DCs are stimulated with IL-6, DCs relieve the suppression of effector T cells by regulatory T cells (113).

The modification of IDO+ monocytes manage towards specific subset of T cell activation with specific TLRs are significantly important (94).

The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) and CMV was possible, but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys since single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These data show that TLRs has ligand specificity, signal transduction pathways, expression profiles and cellular localization so design of experiments should follow these rules.

Conclusion:

Overall our purpose of this information is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.  This first part of the review concerns the basic science information gained overall several years that lay the foundation that translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

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Reporter Aviral Vatsa, PhD MBBS

Annual treatment costs for musculoskeletal diseases in the US are roughly 7.7% (~ $849 billion) of total gross domestic product. Such disorders are the main cause of physical disability in US. Almost half of all chronic conditions in people can be attributed to bone and joint disorders. In addition there is increasing ageing population and associated increases in osteoporosis and other diseases, rising incidences of degenerative intervertebral disk diseases and numbers of revision orthopedic arthroplasty surgeries, and increases in spinal fusions. All these factors contribute towards the increasing requirement of bone regeneration and reconstruction methods and products. Delivery of therapeutic grade products to bone has various challenges. Parenteral administration limits the efficient delivery of drugs to the required site of injury and local delivery methods are often expensive and invasive. The theme issue of Advance Drug Delivery reviews focuses on the current status of drug delivery to bone and the issues facing this field. Here is the first part of these reviews and research articles.

1. Demineralized bone matrix in bone repair: History and use

Abstract

Demineralized bone matrix (DBM) is an osteoconductive and osteoinductive commercial biomaterial and approved medical device used in bone defects with a long track record of clinical use in diverse forms. True to its name and as an acid-extracted organic matrix from human bone sources, DBM retains much of the proteinaceous components native to bone, with small amounts of calcium-based solids, inorganic phosphates and some trace cell debris. Many of DBM’s proteinaceous components (e.g., growth factors) are known to be potent osteogenic agents. Commercially sourced as putty, paste, sheets and flexible pieces, DBM provides a degradable matrix facilitating endogenous release of these compounds to the bone wound sites where it is surgically placed to fill bone defects, inducing new bone formation and accelerating healing. Given DBM’s long clinical track record and commercial accessibility in standard forms and sources, opportunities to further develop and validate DBM as a versatile bone biomaterial in orthopedic repair and regenerative medicine contexts are attractive.

2. Biomimetic hydrogels for controlled biomolecule delivery to augment bone regeneration

Abstract

The regeneration of large bone defects caused by trauma or disease remains a significant clinical problem. Although osteoinductive growth factors such as bone morphogenetic proteins have entered clinics, transplantation of autologous bone remains the gold standard to treat bone defects. The effective treatment of bone defects by protein therapeutics in humans requires quantities that exceed the physiological doses by several orders of magnitude. This not only results in very high treatment costs but also bears considerable risks for adverse side effects. These issues have motivated the development of biomaterials technologies allowing to better control biomolecule delivery from the solid phase. Here we review recent approaches to immobilize biomolecules by affinity binding or by covalent grafting to biomaterial matrices. We focus on biomaterials concepts that are inspired by extracellular matrix (ECM) biology and in particular the dynamic interaction of growth factors with the ECM. We highlight the value of synthetic, ECM-mimicking matrices for future technologies to study bone biology and develop the next generation of ‘smart’ implants.

 

3. Calcium phosphate cements as drug delivery materials

Abstract

Calcium phosphate cements are used as synthetic bone grafts, with several advantages, such as their osteoconductivity and injectability. Moreover, their low-temperature setting reaction and intrinsic porosity allow for the incorporation of drugs and active principles in the material. It is the aim of the present work to: a) provide an overview of the different approaches taken in the application of calcium phosphate cements for drug delivery in the skeletal system, and b) identify the most significant achievements. The drugs or active principles associated to calcium phosphate cements are classified in three groups, i) low molecular weight drugs; ii) high molecular weight biomolecules; and iii) ions.

4. Silk constructs for delivery of musculoskeletal therapeutics

Abstract

Silk fibroin (SF) is a biopolymer with distinguishing features from many other bio- as well as synthetic polymers. From a biomechanical and drug delivery perspective, SF combines remarkable versatility for scaffolding (solid implants, hydrogels, threads, solutions), with advanced mechanical properties and good stabilization and controlled delivery of entrapped protein and small molecule drugs, respectively. It is this combination of mechanical and pharmaceutical features which renders SF so exciting for biomedical applications. This pattern along with the versatility of this biopolymer has been translated into progress for musculoskeletal applications. We review the use and potential of silk fibroin for systemic and localized delivery of therapeutics in diseases affecting the musculoskeletal system. We also present future directions for this biopolymer as well as the necessary research and development steps for their achievement.

5. Demineralized bone matrix as a vehicle for delivering endogenous and exogenous therapeutics in bone repair

Abstract

As a unique human bone extract approved for implant use, demineralized bone matrix (DBM) retains substantial amounts of endogenous osteoconductive and osteoinductive proteins. Commercial preparations of DBM represent a clinically accessible, familiar, widely used and degradable bone-filling device, available in composite solid, strip/piece, and semi-solid paste forms. Surgically placed and/or injected, DBM releases its constituent compounds to bone sites with some evidence for inducing new bone formation and accelerating healing. Significantly, DBM also has preclinical history as a drug carrier by direct loading and delivery of several important classes of therapeutics. Exogenous bioactive agents, including small molecule drugs, protein and peptide drugs, nucleic acid drugs and transgenes and therapeutic cells have been formulated within DBM and released to bone sites to enhance DBM’s intrinsic biological activity. Local release of these agents from DBM directly to surgical sites in bone provides improved control of dosing and targeting of both endogenous and exogenous bioactivity in the context of bone healing using a clinically familiar product. Given DBM’s long clinical track record and commercial accessibility in standard forms and sources, opportunities to formulate DBM as a versatile matrix to deliver therapeutic agents locally to bone sites in orthopedic repair and regenerative medicine contexts are attractive.

6. Nanofiber-based delivery of bioactive agents and stem cells to bone sites

Abstract

Biodegradable nanofibers are important scaffolding materials for bone regeneration. In this paper, the basic concepts and recent advances of self-assembly, electrospinning and thermally induced phase separation techniques that are widely used to generate nanofibrous scaffolds are reviewed. In addition, surface functionalization and bioactive factor delivery within these nanofibrous scaffolds to enhance bone regeneration are also discussed. Moreover, recent progresses in applying these nanofiber-based scaffolds to deliver stem cells for bone regeneration are presented. Along with the significant advances, challenges and obstacles in the field as well as the future perspective are discussed.

 
7. Intra-operatively customized implant coating strategies for local and controlled drug delivery to bone

Abstract

Bone is one of the few tissues in the human body with high endogenous healing capacity. However, failure of the healing process presents a significant clinical challenge; it is a tremendous burden for the individual and has related health and economic consequences. To overcome such healing deficits, various concepts for a local drug delivery to bone have been developed during the last decades. However, in many cases these concepts do not meet the specific requirements of either surgeons who must use these strategies or individual patients who might benefit from them. We describe currently available methods for local drug delivery and their limitations in therapy. Various solutions for drug delivery to bone focusing on clinical applications and intra-operative constraints are discussed and drug delivery by implant coating is highlighted. Finally, a new set of design and performance requirements for intra-operatively customized implant coatings for controlled drug delivery is proposed. In the future, these requirements may improve approaches for local and intra-operative treatment of patients.


8. Local delivery of small and large biomolecules in craniomaxillofacial bone

Abstract

Current state of the art reconstruction of bony defects in the craniomaxillofacial (CMF) area involves transplantation of autogenous or allogenous bone grafts. However, the inherent drawbacks of this approach strongly urge clinicians and researchers to explore alternative treatment options. Currently, a wide interest exists in local delivery of biomolecules from synthetic biomaterials for CMF bone regeneration, in which small biomolecules are rapidly emerging in recent years as an interesting adjunct for upgrading the clinical treatment of CMF bone regeneration under compromised healing conditions. This review highlights recent advances in the local delivery small and large biomolecules for the clinical treatment of CMF bone defects. Further, it provides a perspective on the efficacy of biomolecule delivery in CMF bone regeneration by reviewing presently available reports of pre-clinical studies using various animal models.

9. Immobilized antibiotics to prevent orthopaedic implant infections

Abstract

Many surgical procedures require the placement of an inert or tissue-derived implant deep within the body cavity. While the majority of these implants do not become colonized by bacteria, a small percentage develops a biofilm layer that harbors invasive microorganisms. In orthopaedic surgery, unresolved periprosthetic infections can lead to implant loosening, arthrodeses, amputations and sometimes death. The focus of this review is to describe development of an implant in which an antibiotic tethered to the metal surface is used to prevent bacterial colonization and biofilm formation. Building on well-established chemical syntheses, studies show that antibiotics can be linked to titanium through a self-assembled monolayer of siloxy amines. The stable metal–antibiotic construct resists bacterial colonization and biofilm formation while remaining amenable to osteoblastic cell adhesion and maturation. In an animal model, the antibiotic modified implant resists challenges by bacteria that are commonly present in periprosthetic infections. While the long-term efficacy and stability is still to be established, ongoing studies support the view that this novel type of bioactive surface has a real potential to mitigate or prevent the devastating consequences of orthopaedic infection.

10. Local delivery of nitric oxide: Targeted delivery of therapeutics to bone and connective tissues

Abstract

Non-invasive treatment of injuries and disorders affecting bone and connective tissue remains a significant challenge facing the medical community. A treatment route that has recently been proposed is nitric oxide (NO) therapy. Nitric oxide plays several important roles in physiology with many conditions lacking adequate levels of NO. As NO is a radical, localized delivery via NO donors is essential to promoting biological activity. Herein, we review current literature related to therapeutic NO delivery in the treatment of bone, skin and tendon repair.

Bibliography

  1. Demineralized bone matrix in bone repair: History and use
  2. Biomimetic hydrogels for controlled biomolecule delivery to augment bone regeneration
  3. Calcium phosphate cements as drug delivery materials
  4. Silk constructs for delivery of musculoskeletal therapeutics
  5. Demineralized bone matrix as a vehicle for delivering endogenous and exogenous therapeutics in bone repair
  6. Nanofiber-based delivery of bioactive agents and stem cells to bone sites
  7. Intra-operatively customized implant coating strategies for local and controlled drug delivery to bone
  8. Immobilized antibiotics to prevent orthopaedic implant infections
  9. Local delivery of nitric oxide: Targeted delivery of therapeutics to bone and connective tissues

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