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Archive for the ‘Cell Processing System in Cell Therapy Process Development’ Category

Emerging STAR in Molecular Biology, Synthetic Virology and Genomics: Clodagh C. O’Shea: ChromEMT – Visualizing 3D chromatin structure

Article ID #241: Emerging STAR in Molecular and Cell Biology, Synthetic Virology and Genomics: Clodagh C. O’Shea: ChromEMT – Visualizing 3D chromatin structure. Published on 8/31/2017

WordCloud Image Produced by Adam Tubman

Curator: Aviva Lev-Ari, PhD, RN

On 8/28/2017, I attend and covered in REAL TIME the CHI’s 5th Immune Oncology Summit – Oncolytic Virus Immunotherapy, August 28-29, 2017 Sheraton Boston Hotel | Boston, MA

I covered in REAL TIME this event and Clodagh C. O’Shea talk at the conference.

On that evening, I e-mailed my team that

“I believe that Clodagh C. O’Shea will get the Nobel Prizebefore CRISPR

11:00 Synthetic Virology: Modular Assembly of Designer Viruses for Cancer Therapy

Clodagh_OShea

Clodagh O’Shea, Ph.D., Howard Hughes Medical Institute Faculty Scholar; Associate Professor, William Scandling Developmental Chair, Molecular and Cell Biology Laboratory, Salk Institute for Biological Studies

Design is the ultimate test of understanding. For oncolytic therapies to achieve their potential, we need a deep mechanistic understanding of virus and tumor biology together with the ability to confer new properties.

To achieve this, we have developed

  • combinatorial modular genome assembly (ADsembly) platforms,
  • orthogonal capsid functionalization technologies (RapAd) and
  • replication assays that have enabled the rational design, directed evolution, systematic assembly and screening of powerful new vectors and oncolytic viruses.

Clodagh O’Shea’s Talk In Real Time:

  • Future Cancer therapies to be sophisticated as Cancer is
  • Targer suppresor pathways (Rb/p53)
  • OV are safe their efficacy ishas been limited
  • MOA: Specify Oncolytic Viral Replication in Tumor cells Attenuate – lack of potency
  • SOLUTIONS: Assembly: Assmble personalized V Tx fro libraries of functional parts
  • Adenovirus – natural & clinical advantages
  • Strategy: Technology for Assmbling Novel Adenovirus Genomes using Modular Genomic Parts
  • E1 module: Inactives Rb & p53
  • core module:
  • E3 Module Immune Evasion Tissue targeting
  • E4 Module Activates E2F (transcription factor TDP1/2), PI3K
  • Adenovirus promoters for Cellular viral replication — Tumor Selective Replication: Novel Viruses Selective Replicate in RB/p16
  • Engineering Viruses to overcome tumor heterogeneity
  • Target multiple & Specific Tumor Cel Receptors – RapAd Technology allows Re-targeting anti Rapamycin – induced targeting of adenovirus
  • Virus Genome: FKBP-fusion FRB-Fiber
  • Engineer Adenovirus Caspids that prevent Liver uptake and Sequestration – Natural Ad5 Therapies 
  • Solution: AdSyn335 Lead candidat AdSyn335 Viruses targeting multiple cells
  • Engineering Mutations that enhanced potency
  • Novel Vector: Homes and targets
  • Genetically engineered PDX1 – for Pancreatic Cancer Stroma: Early and Late Stage
On Twitter:
 

Engineer Adenovirus Caspids prevent Liver uptake and Sequestration – Natural Ad5 Therapies C. O’Shea, HHDI

 

Scientist’s Profile: Clodagh C. O’Shea

http://www.salk.edu/scientist/clodagh-oshea/

 

EDUCATION

BS, Biochemistry and Microbiology, University College Cork, Ireland
PhD, Imperial College London/Imperial Cancer Research Fund, U.K.
Postdoctoral Fellow, UCSF Comprehensive Cancer Center, San Francisco, U.S.A

VIDEOS

http://www.salk.edu/scientist/clodagh-oshea/videos/

O’Shea Lab @Salk

http://oshea.salk.edu/

AWARDS & HONORS

  • 2016 Howard Hughes Medical Institute Faculty Scholar
  • 2014 W. M. Keck Medical Research Program Award
  • 2014 Rose Hills Fellow
  • 2011Science/NSF International Science & Visualization Challenge, People’s Choice
  • 2011 Anna Fuller Award for Cancer Research
  • 2010, 2011, 2012 Kavli Frontiers Fellow, National Academy of Sciences
  • 2009 Sontag Distinguished Scientist Award
  • 2009 American Cancer Society Research Scholar Award
  • 2008 ACGT Young Investigator Award for Cancer Gene Therapy
  • 2008 Arnold and Mabel Beckman Young Investigator Award
  • 2008 William Scandling Assistant Professor, Developmental Chair
  • 2007 Emerald Foundation Schola

READ 

Clodagh C. O’Shea: ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells | Science

http://science.sciencemag.org/content/357/6349/eaag0025

and 

https://www.readbyqxmd.com/keyword/93030

 

Clodagh C. O’Shea

In Press

Jul 27, 2017 – Salk scientists solve longstanding biological mystery of DNA organization

Sep 22, 2016 – Clodagh O’Shea named HHMI Faculty Scholar for groundbreaking work in designing synthetic viruses to destroy cancer

Oct 05, 2015 – Clodagh O’Shea awarded $3 million to unlock the “black box” of the nucleus

Aug 27, 2015 – The DNA damage response goes viral: a way in for new cancer treatments

Apr 12, 2013 – Salk Institute promotes three top scientists

Oct 16, 2012 – Cold viruses point the way to new cancer therapies

Aug 25, 2010 – Use the common cold virus to target and disrupt cancer cells?

Oct 22, 2009 – Salk scientist receives The Sontag Foundation’s Distinguished Scientist Award

May 15, 2008 – Salk scientist wins 2008 Beckman Young Investigator Award

Mar 24, 2008 – Salk scientist wins 2007 Young Investigator’s Award in Gene Therapy for Cancer

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Ido Sagi – PhD Student @HUJI, 2017 Kaye Innovation Award winner for leading research that yielded the first successful isolation and maintenance of haploid embryonic stem cells in humans.

Reporter: Aviva Lev-Ari, PhD, RN

 

Ido Sagi – PhD Student, Silberman Institute of Life Sciences, HUJI, Israel

  • Ido Sagi’s research focuses on studying genetic and epigenetic phenomena in human pluripotent stem cells, and his work has been published in leading scientific journals, including Nature, Nature Genetics and Cell Stem Cell.
  • Ido Sagi received BSc summa cum laude in Life Sciences from the Hebrew University, and currently pursues a PhD at the laboratory of Prof. Nissim Benvenisty at the university’s Department of Genetics in the Alexander Silberman Institute of Life Sciences.

The Kaye Innovation Awards at the Hebrew University of Jerusalem have been awarded annually since 1994. Isaac Kaye of England, a prominent industrialist in the pharmaceutical industry, established the awards to encourage faculty, staff and students of the Hebrew University to develop innovative methods and inventions with good commercial potential, which will benefit the university and society.

Publications – Ido Sagi

Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors.
Cell Stem Cell 2014 Nov 6;15(5):634-42. Epub 2014 Nov 6.
The New York Stem Cell Foundation Research Institute, New York, NY 10032, USA; Naomi Berrie Diabetes Center & Department of Pediatrics, College of Physicians and Surgeons, Columbia University, New York, NY 10032, USA. Electronic address:

November 2014

 



Stem cells: Aspiring to naivety.
Nature 2016 12 30;540(7632):211-212. Epub 2016 Nov 30.
The Azrieli Center for Stem Cells and Genetic Research, Department of Genetics, Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
November 2016

Download Full Paper

SOURCE

Other related articles on Genetic and Epigenetic phenomena in human pluripotent stem cells published by LPBI Group can be found in the following e-Books on Amazon.com

e-Books in Medicine

https://www.amazon.com/s/ref=dp_byline_sr_ebooks_9?ie=UTF8&text=Aviva+Lev-Ari&search-alias=digital-text&field-author=Aviva+Lev-Ari&sort=relevancerank

9 results for Kindle Store : “Aviva Lev-Ari”

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    Etiologies of Cardiovascular Diseases: Epigenetics, Genetics and Genomics

    Nov 28, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Stephen J. Williams PhD
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    Cancer Therapies: Metabolic, Genomics, Interventional, Immunotherapy and Nanotechnology in Therapy Delivery (Series C Book 2)

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    by Larry H. Bernstein and Demet Sag
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    Metabolic Genomics & Pharmaceutics (BioMedicine – Metabolomics, Immunology, Infectious Diseases Book 1)

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    Regenerative and Translational Medicine: The Therapeutic Promise for Cardiovascular Diseases

    Dec 26, 2015 | Kindle eBook

    by Justin D. Pearlman MD ME PhD MA FACC and Ritu Saxena PhD
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    Cardiovascular Original Research: Cases in Methodology Design for Content Co-Curation: The Art of Scientific & Medical Curation

    Nov 29, 2015 | Kindle eBook

    by Larry H. Bernstein MD FCAP and Aviva Lev-Ari PhD RN
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Reporter and Curator: Dr. Sudipta Saha, Ph.D.

2.1.5.5

2.1.5.5   Promising research for a male birth control pill, Volume 2 (Volume Two: Latest in Genomics Methodologies for Therapeutics: Gene Editing, NGS and BioInformatics, Simulations and the Genome Ontology), Part 2: CRISPR for Gene Editing and DNA Repair

Scientists think excessive population growth is a cause of scarcity and environmental degradation. A male pill could reduce the number of unintended pregnancies, which accounts for 40 percent of all pregnancies worldwide.

But, big drug companies long ago dropped out of the search for a male contraceptive pill which is able to chemically intercept millions of sperm before they reach a woman’s egg. Right now the chemical burden for contraception relies solely on the female. There’s not much activity in the male contraception field because an effective solution is available on the female side.

Presently, male contraception means a condom or a vasectomy. But researchers from Center for Drug Discovery at Baylor College of Medicine, USA are renewing the search for a better option—an easy-to-take pill that’s safe, fast-acting, and reversible.

The scientists began with lists of genes active in the testes for sperm production and motility and then created knockout mice that lack those genes. Using the gene-editing technology called CRISPR, in collaboration with Japanese scientists, they have so far made more than 75 of these “knockout” mice.

They allowed these mice to mate with normal (wild type) female mice, and if their female partners don’t get pregnant after three to six months, it means the gene might be a target for a contraceptive. Out of 2300 genes that are particularly active in the testes of mice, the researchers have identified 30 genes whose deletion makes the male infertile. Next the scientists are planning a novel screening approach to test whether any of about two billion chemicals can disable these genes in a test tube. Promising chemicals could then be fed to male mice to see if they cause infertility.

Female birth control pills use hormones to inhibit a woman’s ovaries from releasing eggs. But hormones have side effects like weight gain, mood changes, and headaches. A trial of one male contraceptive hormone was stopped early in 2011 after one participant committed suicide and others reported depression. Moreover, some drug candidates have made animals permanently sterile which is not the goal of the research. The challenge is to prevent sperm being made without permanently sterilizing the individual.

As a better way to test drugs, Scientists at University of Georgia, USA are investigating yet another high-tech approach. They are turning human skin cells into stem cells that look and act like the spermatogonial cells in the testes. Testing drugs on such cells might provide more accurate leads than tests on mice.

The male pill would also have to start working quickly, a lot sooner than the female pill, which takes about a week to function. Scientists from University of Dundee, U.K. admitted that there are lots of challenges. Because, a women’s ovary usually release one mature egg each month, while a man makes millions of sperm every day. So, the male pill has to be made 100 percent effective and act instantaneously.

References:

https://www.technologyreview.com/s/603676/the-search-for-a-perfect-male-birth-control-pill/

https://futurism.com/videos/the-perfect-male-birth-control-pill-is-coming-soon/?utm_source=Digest&utm_campaign=c42fc7b9b6-EMAIL_CAMPAIGN_2017_03_20&utm_medium=email&utm_term=0_03cd0a26cd-c42fc7b9b6-246845533

http://www.telegraph.co.uk/women/sex/the-male-pill-is-coming—and-its-going-to-change-everything/

http://www.mensfitness.com/women/sex-tips/male-birth-control-pill-making

http://health.howstuffworks.com/sexual-health/contraception/male-bc-pill.htm

http://europe.newsweek.com/male-contraception-side-effects-study-pill-injection-518237?rm=eu

http://edition.cnn.com/2016/01/07/health/male-birth-control-pill/index.html

http://www.nhs.uk/Conditions/contraception-guide/Pages/male-pill.aspx

Read Full Post »

LIVE – Afternoon Session added – Gene Therapy Development & & Cell Therapy Bioprocessing – Overcoming Scientific and Development Challenges @Biotech Week Boston, October 5, 2016 Boston Convention and Exhibition Center

#BiotechWeekBoston

 avivalev-ari@alum.berkeley.edu

@pharma_BI

@AVIVA1950

ANNOUNCEMENT

Leaders in Pharmaceutical Business Intelligence (LPBI) Group, Boston

pharma_bi-background0238

will cover in REAL TIME

Biotech Week Boston, October 5, 2016 @ Boston Convention and Exhibition Center

 

In Attendance, streaming LIVE using Social Media

Aviva Lev-Ari, PhD, RN

Editor-in-Chief

http://pharmaceuticalintelligence.com

 

Gene Therapy Development & Production

&

Gene Therapy Development & Production

8:25 am 5 mins

Chairperson’s Opening Remarks

8:30 am 30 mins

Extended Range and Next Generation Chimeric Antigen Receptor T Cells

  • Bruce Levine, Ph.D., Barbara and Edward Netter Associate Professor in Cancer Gene Therapy, University of Pennsylvania

ALL

  1. Lymphoeid Leukemia –  Persisting CTL019 Cells remains – Pediatrics ALL – Relapsed and Refractory B-celll
  2. Bedside back to Bench lessons: Days post infusion
  3. 28 days vs 51 cells
  4. quality of cells: related to Clinical Response
  5. 93% CR rate for r/r ALL after CTL019
  6. Novartis and UPenn Cell Center Building

LIMPHOMA

  1. Diffuse Large B Cell Lymphoma
  2. Engineered T Cells and CHeckpoint ANtibody Therapies: Potential Synergies
  3. Days post-tumor injection: Non-responding patients: infusion of PD1 Inhibitor
  4. After treatment: Durable responses necrosis no tumor left
  5. Single Arm, Open-Label

MYELOMA

  1. CART19
  2. CART BCMA – cells for Multiple Myeloma – Bone Marrow: Pre-treatment 70% myeloma cells
  3. Ibrutinib enhances chimeric antigen receptor T-cell engraftment and efficacy in Leukemia
  4. Synthetic Biology – Penn Platform Technology: Academic CAR Clinical Trials
  • Academic-Industry COllaboration with Novartis: Human Cell Therapy: 1996-2011
  • FromGene therapy to >>> Immunotherapy, to >>>>>> Gene Editing
  • TMUNITY – new mission with Leukmia and Lymphoma Foundation

9 am30 mins

Novartis Pharma: Stabilizing Lentiviral (LV) vector formulation for CART Application

  1. LV development of Vector Production – for Ex-vivo Therapy
  2. mamamalian cells + plasmid
  3. high titer, sequences liability &FTO
  4. PROCEESS
  • aggregation
  • Trunsduction
  • purification –
  • Phys-chem profile,
  • stable – – Stability in buffers for recovery of infectious particles – Purification
  • low cost
  1. LV: Optimize transient production:
  • EARLY PROCESS-CHECK,
  • FORMULATION SCEEEN – Preformulation of LVs: Drug DIscovery
  • ANALYTICS DEVELOPMENT
  • TECHNICAL-GRADE VECTOR SUPPLY
  1. High throughput screen
  2. stability promoting buffers
  3. stability in process intermediates
  4. Attributes: Preformulation – success criteria – stable in transduction media/conditions’minimal loss of infectious particles
  5. LV with Transgene 1Preformulation: Effect of buffer and salts – 96 different conditions (buffer +salts)
  6. LV Aggregation & Loss of Infectivity – Accelerated Stree Studies
  7. Size Interpretations – Dynamic Light scattering (DLS): Buffer A, E, H
  8. Robustness: Preformulation buffers: Small, Medium, Large Rh:
  • pH 6.0 – 8.5 – X axis and
  • Salt 0 – 200nM – V Axis

9. Freeze-Thaw Studies – Accelerated Stress Studies: w/o Carbohydrate

  •  number of Freeze-thaw cycles X axis vs
  • % Control to standard – V Axis  

10, Infectious Titer: Primary T Cells

Case study: LV with Transgene #2 –

  • Platform formulation for LV is possible
  • Transduction in primary T cells showed high titer and minimal toxicity
  • stability studies of in-process intermediates and LV-DS provided additional evidence
  • 4 promising conditions identified for preformulation of LV vectors from HTS screens

 9:30 am 30 mins

Mesenchymal Stem Cells (MSCs) on Steroids

  • Oren Levy, HMS, Brigham and Women’s Hospital
I. Mesenchymal Stem Cells – readily accessible potent immunomodulatory secretome already used in >600 clinical trials
  1. Uncontrolled cargo’Inefficient targeting to disease sites – A microparticle-based cellular
  2. Particle-in-a cell approach in Prostate Cancer: PSA – Thapsigargin – PLGA – Poly Lactic co Glycol Acid – encapsulated agent’The cellular carriers MSCs
  3. Glioblastoma, Prostate
  4. Drug-loaded MSCs kill prostate cancer cells
  5. Drug release from G114 MP-loaded cells
  6. MDA-MB; LNCaP – Coculture with pretreated MSCs
  7. Drug-loaded MSCs inhibit tumor growth: days post-inoculation vs Probabilirt of Tumor-free SUrvival vs. Tumor Volume
  8. Particle Payloading: Boosting Potency of Administrated Cell Populations – for Drug delivery
  9. Controlled inhibition of the MSC proinflammatory
  10. Donor variability leads to Functional Variability – variability between 7 donors
  11. Budesonide microparticles boosted MSC
  12. Improved imaging of transplanted MSCs
  13. Retention of PLGA
  • MSC: Therapy
  • Betacells – Islet transplantation Macrophages – immunotherpy

II.  Targeting cells to disease site – Surface of MSC – lacks standard “homing”

  • tetharing – rolling – firm adhesion – transmigration- endothelial cells, basement membrane: Healthy vs Inflammed
  • lucocytes
  • Controlling MSC fate via small molecule pretreatment
  • RNA transfection
  • Bone marrow: PSGL-1/SLex MSCs exhibit enhanced homing to healthy and irradiated bone marrow
  1. A multi-step screening platform for identification of small molecules that improve MSC homing: Surface expression, FIrm adhesion, MSC homing
  2. A medium throughput screen identified Ro-31-8425
  3. Upregulated of CD11a, in response to pretreatment  increases MSC firm adhesion
  4. Pretreated MSCs exhibit superior therapeutic impact post systemic adm – suppression of local skin inflammation
  5. Ro-31-8425 Treated MScs va Vehicle treated MScs
  6. boosted clinical impacet in a MS Model
  7. Bradly applicable cell-based therapeutic platform
  8. mRNA transfection
  9. drug microparticles

 

10:30 am30 mins

Genetic Engineering Red Blood Cells for Therapeutic Function

  •  
    Robert J. Deans, Ph.D.,Chief Scientific Officer, Rubius Therapeutics
  • Rubius Therapeutics – Whitehead Institute/DARPA legacy
  • Flagship Venture founded
  • RCTs – development of ALternative Therapeutics
  • Platfoms: Transfusion /donor selection, risk of oncogenicity – uncontrolled division and secretion
  • Allogenic RCTs grown in Culture from Hemopoeitic Stem Cells (HSCs) In vitro and engineered to Purpose along the way
  • Expansion: CD34 + HSCs: Mobilized, cord blood
  • Cytoplasm: Native cytoplasmic, Tethered cytoplasmic , tethered surface native surface
  • Visualizing Red Cell Therapeutics – RCT during differentiation
  • Reliablly use cell surface markers to track the cells as hey differentiate
  • Flow Cytometry
  • Automated platform foe creating Master cell BioBank
  1. Apheresis Transduction
  2. clinical scale
  3. Rapid prototyping capability for candidate Selection, Vector generation, Human CD34+
  4. Prototyping for Pipeline
  5. Exogenous protein is retained: Cultured RBCs circulating in vivo like Human RBCs
  6. Experimental Setup: RCT circulation in similar to HuRBCs
  7. RCTs can possess antibody drugs on their surface for clearance and therapeutic action
  8. Target: scFv antiVirusAg – capture circulating antigen in vitro and in vivo – Virus Antigen – label antibody
  9. Phenylketonuria (PKU) – CNS morbidity from Serum PHE – it is easily measured, gold standard diagnostic and clinical biomarker
  • Novel protein, cell & gene therapy treatment
  • Existing PAL-RBCs have clinical grade potency at currently achievable manufacturing scale: Serum PHE vs Units PAL-RBS IRON
  • dose dependent PHE expression

 

11 am30 mins

Novel Approaches to Gene Editing and Gene Delivery 

In Vivo gene therapy

  •  
    Andrew Scharenberg, M.D.,Professor of Pediatrics, Adjunct Immunology / Co-director, Program in Cell and Gene Therapy, University of Washington / Seattle Children’s Research Institute
  1.  Viral vector tranfer
  2. cell mediated immunity
  3. Humoral immunity
  4. Evasion/tolerance – neonatal adm of vector
  5. Challenges: Unmet need Goal – Primary cell: DNA delivery to nucleus
  • Cells have mupliplr mechanisms to prevent foreign DNA from entering the nucleus
  • Viruses evade these mechanisms

Non-enveloped Virions – classic gene therapy vector AAV

  • Viral capsid serves multiple roles:
  • AAV has no mechanism to neutralize nuclear silencing mechanism

Enveloped Virions: Retro and Lenti-Virus

Re-dosable in vivo gene transfer

LNP’s mimic the endosomal uptake and escape functions of AAV Capsid

  • mRNA delivery to build a capsid “life boat” – reverse transcribe the gemone and send it in
  • RNA packing, minus strand systhesis, plus strand synthesis, budding, ER vesicular transport ENTRY repair

HBV-based Non-Viral RNA Gene Transfer System: Electric Poration (EP) or LNP

Pregenomic (pgRNA) Vector RNA architecture

  1. Viral RNA – Pol transfers to 3′ DR1 for _ strand sysnthesis
  2. Vector RNA – payload cap… pA
  3. pgRNA with GFP cassette flanked by SB Tn sites – Inhibition and Translation areas
  4. BFP vs GFP
  5. Validation of pgRNA reverse transcription: HepG2 cells transfected with indicated RNAs plus POL/CORE/X mRNAs, then cultured for 2 weeks
  6. Plasma DNA vs Tn-pgRNA, Tn-pgRNA + 5B vs COntrol
  7. pgRNA/SB achieves stable luciferase expression
  8. No-Viral RNA Gene Transfer System: Status and Future
  9. Encouraging evidence of functional synthetic recycling RT vector: SERT – Synthetic encoded reverse transcription)
  10. Transposon IR/DR – flaked pgRNA payload

 

11:30 am30 mins

Development of Stem Cell Derived Extracellular Vesicles into a Non-Living Regenerative Therapeutic Drug Candidate

  •  
    Keil  Ph.D., Capricor, Inc.
  1. Cardioshere-Derived Cells – DOnors of cells – Primary cardiac tissue’Key functions – paracrine:
  2. CDC Manufacturing
  3. DOnor Heart from organ procurement —
  4. Cell Therapy: Autologous vs Allogenics
  5. Cell engraftment
  6. Autologous vs Allogenics Clinical Trials:
  7. Caduceus vs Dynamic
  8. Scar tissue

What are exosomes? as a therapeutics

  • rich in RNA
  • CDC EVs: By the numbers
  • Exosomal Markers vs CDC Marker – CD 105

Acute MI Model

  • Viable mass increased, Myocyte size increased, EF increased,
  • Identified 146a as a major player in cardio-protection
  • CAP2003 manufacturing Process
  • CDC-EV Manufsacturing Overview vs CDC Product
  • Conditioned Medium collection
  • day 1 vs 15 days
  • Drug Product Formulation
  • Choosing a Formulation Buffer: pH
  • 30 days stability
  • Process Robustness: Particle Size and pH: 4 diffrent donors (variability is related to culture and to patients) – 10 formulation runs: EV size vs diafiltrate pH (average =7)
  • Process Robustness: Concentration and Cargo
  • Human mir panel v3.0
  • nCounter miRNA analysis
  • Particle foes not increase linearly with protein
  • Process Robustness: miRNA 146 & 210
  • Process Impurities & Residual: Residual  Fibronectin in CM vs BSA wash-out
  • CDC-EVs have anti-apoptotic, anti fibrotic, immunomodulatory and pro-regenerative activities similar to CDCs
  • New indications
  • Pathway to iND: Pre-Clinical Efficacy
  • Average Total clinical Score
  1. conjunctival injection
  2. corneal opacification
  3. corneal area affected
  4. corneal neovascularization
  5. aqueous flare
  6. Ongoing Pre-Clinical: biodistribution,
  7. Upscaling Exosomes: Bioreactor CDCs
  8. Current Process vs Target Process

Next Gen Storage: Lyophilization (Astraunatu EV

State of Capricor’s Art

 

Cell Therapy Bioprocessing – Innovations in Cell Processing – Part 2

 

2:10 pm 5 mins

Chairperson’s Remarks

 

2:15 pm30 mins

CAR T production in G-Rex: Less is More

  •  
    Juan Vera, M.D., Associate professor Center for Cell and Gene Therapy, Baylor College of Medicine., Baylor College of Medicine
  • Adoptive T-cells Transfer
  • Donor Lymphocytes blood draw — antigen specificities == cell expansion == infusion back to aptient – antigen specific T-cells to iincrease immunogenecity
  • Low seeding density results in greater fold expansion
  • Low cell density + Greater cell expansion: Optimal time for cell harvest vs maximum cell density
  • Volume of Media vs Cells density
  • Upfront media addition resulted in shortest culture period
  • Conventional cultureware  – risk for contamination
  • G-Rex – Plate vs G-Rex – G-REX 5; G-Rex 100; G-REX -500 (flacks)
  • superior cellnumber not due to increase cell number – improved cell output
  1. phase 1 – what is optimal seeding density – cells expansion in 12 days
  2. phase 2 – optimal volume in media – I=1L
  3. phase 3 –
  • are observations reproducible?
  • Cell harvest with The GatheRex – in 5 minutes you collect one billion cells (density
  • CAR-T cells: Preparation of genetically-modified T cells
  • PSCA – solid Tumor
  • NT T cells – suppress tumor cells by immunoresponse to antigen specific to CAR-T
  • CAR-T cell expansion in G-REX – day in culture vs Glucose concentration – inverse correlation
  • Cell performance of production of CAR=t in G-REX
  • Phynotype of CAR-T cells: 24 wells vs G-REX
  • CCR7 vs CD62L: Conventional vs G-REX  – superior Phynotype of CAR-T Cells expanded inside G-REX
  • stimulation beads = not used i=with G-REX
  • media vs cell ratio – serology
  • Persistance of CAR-T cells 6 month post transfer – rechallenge the cells ramping up withstand multiple re-challenge
  • Post transfection – transduction 3 days after

2:45 pm40 mins

Approaches for Determining Technologies to Pursue for Cell Therapy Bioprocessing

  • Brian Murphy, Ph.D., Director of Development, Celgene Cellular Therapeutics

Celgene cellular therapy

  • PDA-002 -MSC
  • PNK-007 – NK cells
  • TST-001
  • GM-
Technologies – CMC Feasibility: make enough cells, the right phynotypes make them GMP – secure cell supply
  • preclinical
  • Phase 1
  • phase 2
  • phase 3 – commercial: Controllable, scaleable, compliant, cost-effective

Scale up: Phase appropriate under scale or over scale vs utilization

  • TIPS: Cell experience need be controlled
  • Static culture technology vs suspension culture
  • Stability in media
  • metabolite
  • Biology Informs Technology: Control critical parameters vs Failure mode
  • After Biology – other factors:
  1. cost
  2. capacity

PDA-002 – Two tier banking system for Cell therapies – adherent cells grown in suspension or microcarriers

Growth on microcarriers in Bioreactor

  • innoculation efficacy
  • harvest formulation
  • fill
  • cryopresevation drug substance

Phase 2 – Process technology

Phace 3 – Process technology for Dose increase — Drug Product — Shipping Product

Biological needs of the cels: Day 11 Media x vs Media Y

Impact of a 35 day Culture — long process: Process duration, Trouble shooting Characterization:

  • 7 days,
  • 21 days
  • 35 days

 

PNK-007: Process Overview – sensitive at few time points in while grown in suspension

Supply Projection & Inventory: 1,2,3 years

Phase 1

phase 2

Phase 3

Conclusion Multiple Groups involved in Production

  • Bioprocess development
  • Stem cell research
  • Analytical development
  • Manufacturing Operations
  • Quality Assurance

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Cell Therapy Bioprocessing – Innovations in Process Technologies, Friday, October 7, 2016, Boston Convention and Exhibition Center

Reporter: Aviva Lev-Ari, PhD, RN

Informa Life Sciences lead the market in providing quality, expert-led conferences; delivering the expert knowledge our clients need to excel in their professional roles and guaranteeing a competitive advantage for their organisations.

Our diverse portfolio covers the Pharmaceutical (Drug Discovery, Clinical Development, Regulatory Affairs, Biopharmaceutical, Generics and Business Strategy), Medical Devices and Diagnostics, Fine Chemicals and Agrochemicals and Veterinary Medicine arenas

http://www.informa-ls.com/

8 am 5 mins

Chairperson’s Remarks

  • Jon A. Rowley, Ph.D., RoosterBio Inc.

10 am 30 mins

Applications for Use of the Lovo Cell Processing System in Cell Therapy Process Development and Manufacturing

  • Alaina C. Schlinker, Ph.D., Fresenius Kabi USA, LLC

10:30 am 30 mins

TBA

  • Philip G. Vanek, Ph.D., GE Healthcare

11 am 30 mins

Platform Processes for PSC Derived Product Manufacture

  • Nick Timmins

11:30 am 30 mins

Building the 3rd Pillar of Medicine: Bioprocessing for Cell Therapies

  • Steve K.W. Oh, Bioprocessing Technology Institute

12 pm 45 mins

Enabling Technologies for Efficient Downstream Processing of AAV Viral Vectors

  • Orjana Terova, Purification, Thermo Fisher Scientific

1:55 pm 5 mins

Chairperson’s Remarks

  • Dominic Clarke, Charter Medical

2 pm 30 mins

Innovative Device Development for Cell Therapies

  • Jamie Piret, Sc.D., The University of British Columbia

2:30 pm 30 mins

Applying Single-Use Technologies

  • Paula Alves, Ph.D., iBET, Portugal

3 pm 30 mins

Manufacturing Multiple Cell Based Products Simulaneously

  • Gail K. Naughton, Ph.D., Histogen Inc.

3:30 pm 30 mins

Scale Out iPSC Derivation and Differentiation Processes for Cell Therapy

  • Wen Bo Wang, Ph.D., Cellular Dynamics International, a FujiFilm company

SOURCE

https://lifesciences.knect365.com/cell-therapy-bioprocessing/agenda/3?stream=1

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Chromatin Remodeling Enzymes: The Human Protein Methyltransferases

Reporter: Aviva Lev-Ari, PhD, RN

 

 

 

 

 

Methyltransferases are enzymes that facilitate the transfer of a methyl (-CH3) group to specific nucleophilic sites on proteins, nucleic acids or other biomolecules. They share a reaction mechanism in which the nucleophilic acceptor site attacks the electrophilic carbon of S-adenosyl-L-methionine (SAM) in an SN2 displacement reaction that produces a methylated biomolecule and S-adenosyl-L-homocysteine (SAH) as a byproduct. Methylation reactions are essential transformations in small-molecule metabolism, and methylation is a common modification of DNA and RNA. The recent discovery of dynamic and reversible methylation of amino acid side chains of chromatin proteins, particularly within the N-terminal tail of histone proteins, has revealed the importance of methyl ‘marks’ as regulators of gene expression. Human protein methyltransferases (PMTs) fall into two major families – protein lysine methyltransferases (PKMTs) and protein arginine methyltransferases (PRMTs) – that are distinguishable by the amino acid that accepts the methyl group and by the conserved sequences of their respective catalytic domains. Given their involvement in many cellular processes, PMTs have attracted attention as potential drug targets, spurring the search for small-molecule PMT inhibitors. Several classes of inhibitors have been identified, but new specific chemical probes that are active in cells will be required to elucidate the biological roles of PMTs and serve as potent leads for PMT-focused drug development.

 

Protein lysine methyltransferases (PKMTs)

The phylogenetic tree shows 51 genes predicted to encode PKMTs, which are positioned in the tree on the basis of the similarities of their amino acid sequences. This tree excludes one validated PKMT, DOT1L, which lacks a SET domain – the catalytic domain conserved in this family – and clusters more closely with the PRMTs. The tree has four major branches, and each branch contains enzymes with validated methyltransferase activity (highlighted in red). Some PKMTs add a single methyl group, resulting in a mono-methylated product (Kme), whereas others produce di-(Kme2) or tri-methylated (Kme3) lysine modifications. Many of the validated PKMTs methylate lysines on histones, though nonhistone substrates have also been identified.

 

Protein arginine methyltransferases (PRMTs)

The human PRMT phylogenetic tree comprises 45 predicted enzymes including the PKMT DOT1L. There are two major types of PRMTs; both catalyze the formation of mono-methylarginine (Rme1) but distinct reaction mechanisms yield symmetric (Rme2s) or asymmetric (Rme2a) dimethylarginine. A small number of predicted PRMTs have validated activity (highlighted in blue). In addition to PRMTs, this tree includes validated RNA methyltransferases (highlighted in green) and biosynthetic enzymes (highlighted in violet). It remains uncertain whether these latter enzymes have PRMT activity, despite their shared structural features. Substrates for the enzymes shown include RNA, metabolites, histones and RNA-binding and spiceosomal proteins.

 

More info: http://www.epizyme.com/epigenetics/about-epigenetics/chromatin-modifying-enzymes/

Sourced through Scoop.it from: www.genautica.com

See on Scoop.it – Cardiovascular and vascular imaging

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Researchers unlock the mysteries of how cells rush to a wound and heal it

Reporter: Aviva Lev-Ari, PhD, RN

 

 

A multidisciplinary research team has discovered how cells know to rush to a wound and heal it — opening the door to new treatments for diabetes, heart disease and cancer. The findings shed light on the mechanisms of cell migration, particularly in the wound-healing process. The results represent a major advancement for regenerative medicine, in which biomedical engineers and other researchers manipulate cells’ form and function to create new tissues, and even organs, to repair, restore or replace those damaged by injury or disease.

 

The answer, it turns out, involves delicate interactions between biomechanical stress, or force, which living cells exert on one another, and biochemical signaling. The University of Arizona researchers discovered that when mechanical force disappears — for example at a wound site where cells have been destroyed, leaving empty, cell-free space — a protein molecule, known as DII4, coordinates nearby cells to migrate to a wound site and collectively cover it with new tissue. What’s more, they found, this process causes identical cells to specialize into leader and follower cells. Researchers had previously assumed leader cells formed randomly. “The results significantly increase our understanding of how tissue regeneration is regulated and advance our ability to guide these processes,” said Pak Kin Wong, UA associate professor of mechanical and aerospace engineering and lead investigator of the research.

 

Wong’s team observed that when cells collectively migrate toward a wound, leader cells expressing a form of messenger RNA, or mRNA, genetic code specific to the DII4 protein emerge at the front of the pack, or migrating tip. The leader cells, in turn, send signals to follower cells, which do not express the genetic messenger. This elaborate autoregulatory system remains activated until new tissue has covered a wound.

 

The same migration processes for wound healing and tissue development also apply to cancer spreading, the researchers noted. The combination of mechanical force and genetic signaling stimulates cancer cells to collectively migrate and invade healthy tissue.

 

Biologists have known of the existence of leader cells and the DII4 protein for some years and have suspected they might be important in collective cell migration. But precisely how leader cells formed, what controlled their behavior, and their genetic makeup were all mysteries — until now. “Knowing the genetic makeup of leader cells and understanding their formation and behavior gives us the ability to alter cell migration,” Wong said.

 

With this new knowledge, researchers can re-create, at the cellular and molecular levels, the chain of events that brings about the formation of human tissue. Bioengineers now have the information they need to direct normal cells to heal damaged tissue, or prevent cancer cells from invading healthy tissue.

Source: www.eurekalert.org

See on Scoop.it – Cardiovascular and vascular imaging

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Using RNA-seq and targeted nucleases to identify mechanisms of drug resistance in acute myeloid leukemia

Reporter: Aviva Lev-Ari, PhD, RN

 

 

The evolution from microarrays to transcriptome deep-sequencing (RNA-seq) and from RNA interference to gene knockouts using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and Transcription Activator-Like Effector Nucleases (TALENs) has provided a new experimental partnership for identifying and quantifying the effects of gene changes on drug resistance. Here we describe the results from deep-sequencing of RNA derived from two cytarabine (Ara-C) resistance acute myeloid leukemia (AML) cell lines, and present CRISPR and TALEN based methods for accomplishing complete gene knockout (KO) in AML cells. We found protein modifying loss-of-function mutations in deoxycytidine kinase (dck) in both Ara-C resistant cell lines. The first kinase in the dNTP salvage pathway is  DCK, the rate limiting enzyme in the metabolic activation of Ara-C. Numerous studies have shown DCK expression is frequently downregulated in cells that are unresponsive to Ara-C2, 3, 4, 5, 6, 7. CRISPR and TALEN-based KO of Dckdramatically increased the IC50 of Ara-C and introduction of a DCK overexpression vector into Dck KO clones resulted in a significant increase in Ara-C sensitivity. This effort demonstrates the power of using transcriptome analysis and CRISPR/TALEN-based KOs to identify and verify genes associated with drug resistance.

Source: www.nature.com

See on Scoop.it – Cardiovascular Disease: PHARMACO-THERAPY

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