Gene Therapy- Past, Present and Future. Mark A. Kay MD, PhD Dennis Farrey Family Professor Departments of Pediatrics and Genetics Stanford University

Transcription

1 Gene Therapy- Past, Present and Future Mark A. Kay MD, PhD Dennis Farrey Family Professor Departments of Pediatrics and Genetics Stanford University

2 Definition of gene therapy Gene therapy is the introduction of nucleic acids (e.g. DNA/genes) into somatic cells of the body to correct or prevent a pathological process Think of DNA as a class of pharmaceuticals

3 What Do We Want to Accomplish? Add a gene-restore a missing gene function or supply an RNA or protein that has pharmacological effect Fix a gene change the DNA-mutation repair Silence a gene-from a pathogen, gain of function mutation

4 Vector Categories Viral-Based-Manipulating Natural Existing Viruses to Transfer Therapeutic Genes Non-Viral- DNA Molecules Delivered as Naked DNA, Lipids, or Polymers

5 Which vectors are in use?

6 What diseases are being targeted?

7 Potential factors limiting vector efficacy

8 Adeno-associated viruses Discovered in mid 1960s as a contaminant Family: Parvoviridae Subfamily: Parvovirinae Genus: Dependoparvovirus

9 AAV Vectors AAV ITR Rep Cap ITR DNA AAV Vectors ITR Control element Gene Cassette Therapeutic gene ITR DNA

10 AAV vector production strategies: Helper virus-free system Therapeutic gene ITR pvector ITR Rep phlp Cap CaPO 4 Vector recovery E2A pladeno Transfection in 293 cells VA E4

11 Alternative Production Methods

12 Purification Schemes Various Physical Separation methods Vector purity (e.g. advential agents, DNA contamination) Full to Empty Capsid Ratio Capsid Composition

13 hfix (ng/ml) raav-mediated transgene expression in vivo AAV-EF1a-hFIX to C57BL/6 mice Snyder et al., Nat. Genetics Months Nature Genetics, 1997

14 Dogs are treated Dog colonies are University of North Carolina at Chapel Hill Dogs treated with AAV-FIX vectors Expression for > 7 years First Hemophilia A dog Hemophilia B dog Kenneth M. Brinkhous at the University of North Carolina at Chapel Hill

15 Biotech - Academic Collaboration The ultimate goals are the same The acute similarities & differences Disclosures IP restrictions Survival publish vs perish shareholders

16 First systemic administration of an AAV vector into a human Manno et al., Nature Medicine 2006

17 Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the immune host response hemophilia Therapeutic hfix was demonstrated in a human Unlike all animal models expression in humans was temporary This limitation was likely related to a cell mediated immune directed against hepatocytes containing capsid particles No matter how good the animal models one cannot predict the outcome in humans until you try it in people Manno et al., Nature Medicine 2006

18 Surprises along the way Jessie Gelsinger s Death Seminal Fluid PCR detection of vector DNA genomes--- how to establish significance Unexplained Transaminitis

19 Pseudotyping recombinant vector genomes AAV-2 is prototype vector-isolated from humans Most of the population exposed (e.g. immunity) AAV1 AAV2 AAV3 Cell-mediated immune responses Small number of amino acid changes can have profound effects on the transduction parameters (immunity, efficiency, cell type) AAV4 AAV5 AAV6

20 AAV-2/8 Gene Therapy for Hemophilia B Nathwani et al., New England Journal of Medicine 2011

21 AAV-8 is not an optimal vector for human gene transfer Some humans pre-existing immunity inhibiting any gene transfer Dose response in human is >10x less than expected based on animal studies

22 Various AAV-clinical trials for Hemophilia B ongoing Additional patients with better dosing What about patients with preexisting immunity? What happens if expression wanes over time? How long will it last if treated at birth?

23 How does one predict clinical outcomes from animal studies? Which animals models are most predictive?

24 Reconstitution of mouse liver with human hepatocytes Tyrosine P-hydroxy-phenylpyruvate NTBC Homogentistisic acid (HGA) Maleylacetoacetate (FAA) Succinylacetone HT1 Fumarylacetoacetate (FAA) Hpd-/- Fah-/- Fumarate Acetoacetate Azuma et al., Nature Biotechnology 2007 AV shuffle library screening in FRG mice

25 Molecular shuffling and evolution of new viruses Kay, M.A. Nature Reviews Genetics 2011

26 AAV shuffle library selection in Tissue Culture Cells TC cells AAV library (MOI range) had5 (fixed predetermined MOI) Harvest supernatant on day 3 post infection Human IVIG negative selection Western Blot analysis Vector DNA extraction PCR amplification of CAP gene TOPO subcloning Sequencing Select dilution with the lowest expression of AAV proteins VP1 VP2 VP3 Library MOI

27 Library screening in mice with humanized liver Lisowski et al., Nature 2014

28 Vectorization of AAV capsids obtained from in vivo screen Lisowski et al., Nature 2014

29 Limitations to conventional raav approaches Loss of transgene expression in growing or dividing cells Risk of insertional mutagenesis Problems with re-administration Genome size Pre-existing immunity Cell-mediated immunity

30 Genome editing ithout nucleases

31 Promoterless Gene Targeting Without Nucleases DNA: 5 homology arm to Alb 3 homology arm to Alb ITR 2A hf9 AAV8 capsid Wild-type Alb locus Stop Targeted Alb locus 2A hf9 RNA: Fused mrna Alb 2A hf9 Protein: + Alb 2A P hf9

32 Promoterless Gene Targeting for hfix Barzel et al., Nature 2014

33 Improved Human Capsid + Gene Ride Treat non-quiescent tissues including neonatal liver. No retreatment with raav required Abrogates hepatic genotoxicity (hepatocellular carcinoma) No genotoxicity or immunogenicity associated with nucleases Endogenous gene regulation

34 Where do we go from here?