LIVE 9/20 2PM to 5:30PM New Viruses for Therapeutic Gene Delivery at CHI’s 14th Discovery On Target, 9/19 – 9/22/2016, Westin Boston Waterfront, Boston
http://www.discoveryontarget.com/
http://www.discoveryontarget.com/crispr-therapies/
Leaders in Pharmaceutical Business Intelligence (LPBI) Group is a
Media Partner of CHI for CHI’s 14th Annual Discovery on Target taking place September 19 – 22, 2016 in Boston.
In Attendance, streaming LIVE using Social Media
Aviva Lev-Ari, PhD, RN
Editor-in-Chief
http://pharmaceuticalintelligence.com
#BostonDOT16
@BostonDOT
COMMENTS BY Stephen J Williams, PhD
Gene Therapy Breakthroughs
New Strategies for Better Specificity and Delivery
2:05 Chairman’s Remarks
Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute City of Hope
- CBG (center for biomedicine and Genetics) 20000 sq feet
- CTPC (center therapy production) mainly CART
- CBG 16 years operation do all stem cells and >400 products
- New stem cell Beta cell progenitor
- Do oncolytic VSV
- CTPC is investigator driven CART islet cells,
- Like to do novel work so work with CIRM
- Banking of modified stem cells
- Adherent scale out limitations: cost,inefficient; solution can be suspension
- Establish hESC; plate on CELLstart > Accutase>StemPRO SFM>differentiation process; defined reagents — they use this for cardiomyocyte differentiation: they are functional (inotropy, chronotropy response to isoproterenol) can freeze back cells
- Create a bank of intermediate cells and when you need it for surgery they will put on their matrix, enrich, expand and ship out
- Allogeneic cells: project where take allogeneic neural stem cells to deliver a chemotherapy payload as they like to migrate to brain tumors
- Allogeneic cells: for ALS modified to express GDNF
- HIV resistance with engineered CCR5 negative blood stem cells
- Release assay considerations: viability, sterility, if cryopreserved then can determine identity, viral insertions, VSV-G copy number, endotoxin and potency (FDA is wanting phase I potency assays) for CART potency is % transduced
- Good in vivo activity of the neural stem cells loaded with chemotherapeutic
ALS
- If deliver GDNF to muscle using genetically modified myoblasts
- Best to use fetal stem cells – less issues
Canavan disease: progressive fatal neurologic disorder that begins in infancy and don’t make it past teenage years
- Rossbach is taking autologous cells reprogramming generating iPS cells and then modifying by CRISPR but the CRISPR issues of off target effects persist as well the time required for process and verification; also don’t want to use a selectable marker and put in patients; so you can differentiate the cells and hit them with a lentiviral vector system
They have been named a PACT Center Production Assistance for Cell Therapy where you can apply for a project grant. Applicable for startups up to larger mature companies
They do a standard panel of tests for viral infections.
They work with investigators or companies at all stages of manufacturing processes.
@BeckmanInst
@cityofhope
2:15 Large-Scale Production of Cell Therapies for Regenerative Medicine
Joseph Gold, Ph.D. Director Manufacturing Center for Biomedicine and Genetics, Beckman Research Institute
2:45 Directed Evolution of New Viruses for Therapeutic Gene Delivery
David Schaffer, Ph.D. Professor of Chemical and Biomolecular Engineering, BioEngineering, Molecular and Cell Biology and Neuroscience;
AAV is very safe as many people already infected with it
- Spark (Leber’s cogenital anaurosis
- Hemophilia B
- Lipoprotein lipase deficiency
- Spinal muscular atrophy
- Challenges: are we just getting the ‘low hanging fruit’ eg Spark therapy must be injected after retinal therapy, hemo B needs to be given at high doses
- Their theory was AAV had been evolving for its own purposes so hence the limitations of AAV;
- Utilized 25 different techniques to generate variants of AAV in a library then packaged (each will have its own barcode)
- Broad platform technology: retina, lung, brain and spinal cord
Retinal: AAV may be too large to get through layers of the eye, problems; subretinal injections and damage or retinal detachment. Then they used their whole library in an in-vivo screen (as hard to recapitulate the multi cell layers of the retina).
Cystic Fibrosis
- AAV2H22 variant worked very well to supply the CFTR gene in pig model of cystic fibrosis and increases chloride transport and reduce bacterial load
- Then found pig variant AAV did not work well on human tissue so designed a human variant and worked well in human tissues
- The variant AAV2.5T surrounds sialic acid binding pockets and increases binding and endocytosis
Brain and Spinal Cord: Sanfilippo B trial 8 holes drilled into skull followed by 16 AAV injections
- Injected a generated AAV variant (by evolution process) : engineered AAV2 is 100 fold better getting through blood brain barrier… novel variant undergoes retrograde transport to cortex ; made a cas9 to remove a tdTomato gene overexpressed in mouse and found 90% knockdown
- Also interesting point: the porcine variant did not work in human and the human variant did not work in porcine. Implication for FDA safety and efficacy testing must do in monkeys
They started a spinout 4D Molecular Therapeutics
4:25 Lentiviral Vectors for Gene Therapy
Munapaty Swani, Ph.D. Texas Tech Health Science Center
- Can express multiple shRNA under a separate promoter but toxic so if expressed in miRNA backbone could be safer under a pol II
- How much of flanking sequence is needed?
- 30 nt flanking sequence is enough for Drosha processing
- Constructed 1 to 7 shRNA-miR targeting CCR5 and 6 viral genes; all constructs were functional
- Problem with pol ii promoter
- These 7 shRNA miRNA protect against HIV entry if against CCR5 and the 7 viral elements
- Used the non-integrating lentivirus for transient to see if infect T cells or not versus integrating lentivirus ; results non-integrating lentivirus did not infect t cells so safer to use
- CCR5 disruption reduced HIV infection in T cells in vitro;
- ZFN treatment of HIV+ PBMC prevents activation of HIV
- Encapsulted CAS9 within LV; cas9 protein is incorporated within LV and is functional
- First transduce then come in with the Cas9 so made all in one lentivirus with Cas9 and an sgRNA expression vector *******
- This shows that it is possible to put all in a nanoparticle based lentivirus and an all in one may make it easier and safer (supposedly)
4:55 AAV Capsid Design
Miguel Seria Esteves, PhD Associate Professor Neurology, Gene Therapy Center University of Massachusetts Medical School
-AAV replication dependent no known human disease with native AAV
- Multiple barriers to get across blood brain barrier
- AAV9 preferentially target neonatal neurons and adult astrocytes
- Multiple capsids can be used for AAV9 infection in brain but not complete
- Can we design better capsids to give it better tropic properties and better penetration to blood brain barrier
- Using a polyalanine in the 5’ end of the caspid was most efficeint
- Increases gene transfer efficiency especially IN SELECT CELL TYPES; Glial transduction and increased in striatum: increase is structure specific so little in thalamus but good in cerebellum and spinal cord
- AAV9 tranduces also in peripheral tissues with or without modified capsid
Huntington’s Disease
- Polyglutamate disease polyy glu on huntingtin protein
- They get a 40 to 50% reduction of huntingtin but not significant between capsid design
- They did a directed evolution of AAV capsid and generated capsid gene delivery diversity: DNA shuffling and in vivo selection
- AAV-B1 is a new tropic capsid showing transduction of different structures
- Five fold reduction in tropism to the liver but massive increases in muscle and beta exocrine cells and lung
- Presence of neutralizing antibodies is a problem with AAV therapy
- In conclusion unknown mechanisms by whivh a highly hydrophobic string of 19 alanines modifies the CHS tropism of AAV9 kvariants
- Chimeric capsids identified from in vivo screen can reveal interesting patterns of tropism
12:45 PM Screening with shRNA and CRISPR
Ryan Raver, PhD Global Product Manager, Functional Genomics, MilliporeSigma
- KO – Knock Out
- KD – Knock Down
- RNAi -KD
- CRISPR-Cas9 – KO
NEW STRATEGIES FOR BETTER SPECIFICITY AND DELIVERY
2:05 Chairperson’s Remarks
Joseph Gold, Center for Biomedicine and Genetics Beckman Research Institute, City of Hope
2:15 Large Scale Production of cell Therapies for Regenerative Medicine: COmbination Cell and Gene Therapy products
Joseph Gold, Center for Biomedicine and Genetics Beckman Research Institute, City of Hope
- Biological & Cellular GMP manufacturing Core at COH
- Establishing scalable hESC suspension Culture
- Optimized small molecule concentration, induction timing, stirring rates
- Almost Xeno free
- defined
- very good reproducibility
- high purity and yield:
- Immuno-staining,
- FACS – cTnT, sMHC, Alpha-actinin
- Cryopreservation, Multi-electrode Array (MEA)
- hESC-RPE monolayer on synthetic substrate
Combination cell/gene therapy products at COH
- CAR T CCR5-inactivated CD34+ HSPC – Target: AIDS
- adoptive immunotherapy using CAR-Engineering T cells – glioblastoma
2. HIV resistance with engineering CCR5-negative blood stem cells: gene KO by ZFNs
- assay considerations 0 If cryopreservation : Identify, viral insertion, endotoxin, residual beads Potency: CAR T- % transduced cells, CCR5-?-CD34 cells: HIV resistance
3. Glioblastoma
4. In vivo activity of transduced NSCs: Assay consideration – viability, sterility, mycobatom,
5. ALS: degeneration of neurones
- Embryonic stem cells
- Fetal neural stem cells
- Adult stem cells – human Proginetor neural cells
6. Canavan Disease – Y.Shi – ASPA gene mutations cause Canavan disease
Strategy: autologous iPSC-derived, modified neural progenitors: DIferentiate to neural progenitors
Gene therapy: Correction (Off-taregt effects, correct individual cell lines) or Over expression (Copy number, selection, transduce iPSC)
- release assay consideration
- identity – markers and HLA, contaminants, Potency: in vivo efficacy modified autologous
- production Assistance for Cell therapy
- cell therapy manufacturing development
- roles of Biobanks
2:45 Directed Evolution of New Viruses for Therapeutic Gene Delivery
David Schaffer, Ph.D., Professor of Chemical and Biomolecular Engineering, Bioengineering, Molecular and Cell Biology, and Neuroscience; Director, Berkeley Stem Cell Center, University of California, Berkeley
Adeno-associated viral (AAV) vectors have been increasingly successful in clinical trials; however, viruses face many delivery barriers that limit their efficacy for most disease targets. We have developed directed vector evolution – the iterative genetic diversification of a viral genome and functional selection for desired properties – to engineer novel, optimized AAV vectors for efficient, selective delivery for a range of tissue and disease targets.
- DNA (gene therapy and editing) >> RNAi (antisense) >> Protein: Small molecules and monoclonal antibodies
- Dlivery
- Adeno Associated Viral Vectors Adenoviral helper genes
- AAV2, efficacy in Leber’s Congenital samaurosis (AAV)
- Spinal muscular, lipolrotein lipase deficiency (AAV)
Gene Delivery
- neutralization of pre-esisting antibodies
- target tissue – deep penetration
- Inefficient transduction to target cells
- target specific cells
Fitness as Therapy – virus evolutionary: Tropism and immunity
- AAV directed Vector evolution : Input /sequence >> Diversity/generation >> Packaging
- Retinoschisis Model – Eye therapeutic gene to protect vision – AAV – transduction of retinal cell, vitreas in Human different scale — eventually Macualr degeneration Therapy for: Photo receptor is the target for therapy
- Dog: Fundus Imaging of Engineered AAV: Variant Expression GFP Pool Carrying
Lung- Cystic Fibrosis (mucus production amplifies (enhanced transduction) due to MAC3 translation error) – Gene Therapy – cilia function to restore ability to clear mucos: Variant evolved on Human airway epithelia – AA particles
- AAV2>>> AAV%>> T mutation
- efficacy must be improved
Brain and Spinal Cord
- Scull – 8 drills followed by 16 AAV injections
- Spinal cord: injuction in muscle
Synthetic version of AAV — Engineered AAV for enhanced Retrograde Transport
- AAV2-retrograded transport – Noval variant Undergoes Transport along multiple Projections
- Cas9 – Retrograde Delivery of Cas9 to cortex of mouse – KD –
Summary
- Virus as gene delivery mechanism
- designer AAV variants
3:15 Sponsored Presentation (Opportunity Available)
3:45 Refreshment Break in the Exhibit Hall with Poster Viewing and Poster Competition Winner Announced
4:25 Lentiviral Vectors for Gene Therapy
Manjunath N. Swamy, MD, Professor of Biomedical Sciences and Co-DIrector of the Center of Emphasis in Infectious Diseases. Paul L Foster School of Medicine, Texas Tech University Health Science Center
- RNAi targets for HIV
- Expression of multiple shRNAs
- COnstruction od 7 shRNA-miR targeting CCR5 and 6 viral genes – protect against both 5 ans x4 tropic HIV-1
- shRNA expression does not decrease with distance from promoter
- Lentiviral vector to express ZFNs: HIV envelop – ZFN-mediated CCR5 gene editing in Primary T cells
- ZFN treatment of HIV+PBMC prevents activation of HIV
- Strategy to encapsulate Cas9 Protein wihtin LV : AIm to deliver Cas9 protein deliver sgRNA expression vector. A Lentiviral
- Gene editing by all-in-one Lentivirus = to prepack
4:55 AAV Caspid Engineering
Miguel Sena Esteves, Ph.D., Associate Professor, Department of Neurology, Gene Therapy Center, University of Massachusetts Medical School
Adeno-associated virus vectors have become the leading platform for development of in vivo gene therapies for neurological diseases. We have developed new AAV vectors for widespread gene delivery to the CNS through vascular infusion in adult animals through peptide grafting and in vivo library selection. These new neurotropic AAVs have achieved CNS-wide silencing of gene expression using gene-specific microRNAs.
- Adeno-associated virus – Paravovirus family
- Recombinant AAV vectors carry gene expression cassette of choice flanked by two
- CNS – route of gene delivery
- Crossing BBB
- Systemic delivery of AAV9 vectors: IV
- Peptide grafting – in vivo selection of novel CNS: DIstribution of GFP transduced cells – robust neuronal transducture with transduction AAV-AS
- motor cortex, Straiatum, thalamus, motor cortex, ventral horn of spinal cord, cerebelum, liver, muscle, oculomotor nerve, nucleus of oculomotor nerve
Huntington’s Disease > 40 CAG vs <26 CAG in normal – Peptide grafting of AAV vectors for CNS
- DNA shuffling and in vivo selection: Brain vs Liver
Next generation of AAV vectors for CNS
- Liver, pancreas, lung — same pattern
neural transduction after vascular delivery
- AAV-B1 caspid vs AAV8 – 19 amino acids
Conclusion
New capsids with improved CNS tropism
19 alanines modifies the CNS tropism of AAV9 variants
Chimeric caspids identified from in vivo screens
5:25 Welcome Reception in the Exhibit Hall with Poster Viewing
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