Gene Therapy: Fellow s Conference Fred Hutchinson Cancer Research Center. Zandra K. Klippel

Size: px
Start display at page:

Download "Gene Therapy: Fellow s Conference Fred Hutchinson Cancer Research Center. Zandra K. Klippel"

Transcription

1 Gene Therapy: Did we finally get it right? Fellow s Conference Fred Hutchinson Cancer Research Center University it of Washington Zandra K. Klippel March 2012

2 Introduction >6000 human diseases are monogenic Prevalence at birth: 1/200 Monogenic hematological disorders are the most common among these disorders. Genomics.energy.gov

3 Applications Edelstein, et al. J Gene Med 2004; 6:

4 Gene Therapy 101

5 Definition Procedure that introduces a defined DNA sequence into specific cells either to replace a defective gene, or to impart a new function to the cell in order to induce it to produce a protein that has a putative therapeutic function.

6 Historical Perspective Joshua Lederberg Hemophilia B AAV8 Rosenberg Neomicin Resistance TIL 1989 ADA deficient SCID 1990 X Linked SCID? s OTC patient dies of Adenovirus 5 transfer Leukemia like disease in X linked SCID Edelstein et al. J Gene Med 2004; 6:

7 Historical Perspective Can human cells uptake foreign DNA? SV40 DNA integration studies How do we introduce this DNA? 1973 F.L. Graham Calcium phosphate transfection Retrovirus life cycle and transduction capability 1981 DNA recombination and cloning Friedmann T. Nature Genetics 1992; 2: 93 98

8 Vectors Genetic Material Cell targeted Integration to Human DNA Size of gene Risks Retrovirus RNA Dividing Yes 7 10Kb (8Kb) Insertional mutagenesis Lentivirus RNA Non dividing Yes 9 10Kb (8Kb) Adenovirus dsdna Non dividing No 36Kb (7 8Kb) Insertional mutagenesis HIV Innate immunity Loss of material with divisions Adeno Associated Virus ssdna Non dividing No 4.7 Kb (4.5Kb) Vaccinia, Pox virus, HSV, Naked DNA, lipofection Adaptive Immune response to capside Loss of material with divisions

9 How about Hemophilia B?

10 Hemophilia B X linked bleeding disorder caused by df defect in Factor IX 1/25000 male births. ~3300 males affected in USA.

11 Hemophilia B Treatment 1970 s Lyophilized Factors 1985 Anti HIV Testing 1985 HIV heatinactivated 1964 Cryoprecipitate 1970 s Prophylaxis Home Tx 1982 FIX Gene Cloned 1987 Safer Plasma Products 1997 rfix 2007 Prophylaxis Efficacy 1975 HCV 1982 HIV/AIDS 1994 Immune Tolerance Registry 2002 Tx of HCV Mannucci P M et al. Haemophilia (2008), 14 (Suppl. 3), 10 18

12 Success in Animals Animal Vector Delivery F.IX activity Herzog et al PNAS 1997 Mice raav CMV hfix Intramuscular levels in RAG mice Kay et al Science 1993 PNAS 1994 Herzog et al Nat Med 1999 Mol Ther 2001 Dogs LX cfix Ad/PGK cfix Portal Vein Bleeding time/ptt Dogs raav CMV FIX Intramuscular Bleeding time Snyder et al Nat Med Dogs & raav MFG cfix Portal Vein Bleeding 1999 raav MFG hfix Mice time Mount et al Blood 2002 Dogs *(null mutation) Nathwani et al Blood 2002 Rhesus Macaques raav ApoE4/AAT cfix Portal Vein 5 10% raav 2 CAGG hfix Hepatic A, or Portal V Nathwani et al Blood 2006 Mice scaav2/8 LP1 hfixco Tail vein >1% Nathwani et al Mol Ther 2011 Non Human Primates scaav2/8 LP1 hfixco Peripheral vein 4 8% (2/5) >10%

13 Evidence of gene transfer and expression of FIX after AAV intramuscular injection N=3 N3 Intramuscular injection raav2 CMV hf hf.ix vg/kg Kay MA, et al. Nat Genet. 2000; 24:

14 Other Attempts N=7 N7 Hepatic Artery infusion Vector: raav2 ApoE/Serpina hf.ix Doses: 8x10 10 vg/kg 4x10 11 vg/kg *HCV; AAV NAB 1:2 2x10 12 vg/kg *HCV; AAV NAB 1:17 Manno et al. Nat Med (3); *No HCV; No AAV NAB

15 Their Conclusions Evidence of hepatocyte transduction at doses therapeutic in dog studies Neutralizing antibodies define transduction efficiency Patient G ELISPOT Evidence of development of T cell specific immunity it to capside: memory response! Manno et al. Nat Med (3); 342 7

16 Obstacles to Gene Therapy for Hemophilia B Limited transductionefficiency Transient protein expression Immune response High standards for safety

17 ssdna dsdna Transduction Efficiency Genetherapynet.com Nathwani et al. Blood. 2006;107: )

18 Transduction Efficiency AAV2 transduction: 10% of hepatocytes 6 week latency period AAV8: Rapid uncoating of the virus genome x fold transduction in liver Thomas C et al. J. Virol. 2004, 78(6):3110.

19 Immune Response The vector: Adenovirus vs. Adeno Associated Virus Neutralizing antibodies against capside (Manno et al. Nat Med d (3); 342 7) The product: Tissue targeted: muscle vs. liver (Mount et al. Blood 2002)

20 Immune Response Hepatocytes CD8 AAV vectors Modified from High K. Trans Am Clin Climatol Assoc. 2009; 120:

21

22

23 Vector Characteristics Self complementary Codon optimized i Liver specific promoter AAV2/8 capside Geneinfinity.org images.caltech.edu

24 Low Dose: 2x10 11 vg per kg Missense mutation Prophylaxis: accidental injuries Null mutation Prophylaxis: continue through week 23, then less frequent

25 Humoral Immune Response

26 Intermediate Dose: 6x10 11 vg per kg Missense mutation Had higher anti AVV 8 at baseline Missense mutation Prophylaxis: 1 after fall

27 High Dose: 2x10 12 vg per kg Missense mutation Promoter mutation Asymptomatic elevation of ALT Decrease FIXafter intense Prophylaxis: traumatic injuries activity (1/2 marathon!)

28 Conclusions Important progress has been made. Evidence of transduction Evidence of clinical i l benefit Still room to improve and more questions: Dose Need for steroids Immune response Long term effects if any

29 References (I) Herzog, et al. Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection i of ecombinant adeno associated dvirus Proc. Natl. Acad. Sci. SiUSA. 1997; 94: High K. The Jeremiah Metzger Lecture: Gene Therapy for Inherited Disorders: From Christmas Disease to Leber's Amaurosis.Trans Am Clin Climatol Assoc. 2009; 120: Mount JD, et al. Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver directed gene therapy. Blood : Nathwani et al. Self complementary adeno associated virus vectors containing a novel liver specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood : O Connor TO, Crystal RG. Genetic medicines: treatment strategies for hereditary disorders. Nat Rev Genet Apr;7(4): Fi Friedmann T. A bi brief fhistory of gene therapy. Nature Nt Genetics 1992; 2: Edelstein et al. J Gene Med 2004; 6: Nathwani AC et al. Mol Ther. Long term safety and efficacy following systemic administration of a self complementary AAV vector encoding human FIX pseudotyped with serotype 5 and 8 capsid proteins 2011 May;19(5):876 85

30 References (II) Snyder RO et al. Correction of hemophilia B in canine and murine models using recombinant adeno associated dviral vectors. Nat Med Jan;5(1):64 70 Kay MA, et al. In vivo gene therapy of hemophilia B: sustained partial correction in factor IXdeficient dogs. Science Oct 1;262(5130):117 9 Manno CS. Successful transduction of liver in hemophilia by AAV Factor IX and limitations imposed by the host immune response. Nat Med Mar;12(3):342 7 Herzog RW, et al. Long term correction of canine hemophilia B by gene transfer of blood coagulation factor IX mediated by adeno associated viral vector. Nat Med Jan;5(1):56 63 Kay MA, et al. Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector. Nat Genet Mar;24(3): Rapid Uncoating of Vector Genomes Is the Key to Efficient Liver Thomas CE. Transduction with Pseudotyped Adeno Associated Virus Vectors. J. Virol. 2004, 78(6):3110. Mannucci PM. Back to the future: a recent history of haemophilia treatment. Haemophilia (2008), 14 (Suppl. 3), 10 18

31 Gracias.