Hemophilia A gene therapy: AAV-mediated delivery of an enhanced F8 cassette to the liver produces supraphysiological levels of human FVIII in vivo

Transcription

1 Hemophilia A gene therapy: AAV-mediated delivery of an enhanced F8 cassette to the liver produces supraphysiological levels of human FVIII in vivo Brigit E. RILEY Director Discovery and Translational Research Sangamo BioSciences USA 1

2 Disclosures for: Brigit E. RILEY In compliance with the PIM* policy, WFH requires the following disclosures be made at each presentation CONFLICT RESEARCH SUPPORT DIRECTOR, OFFICER, EMPLOYEE DISCLOSURE IF CONFLICT OF INTEREST EXISTS Sangamo BioSciences DIRECTOR SHAREHOLDER HONORARIA ADVISORY COMMITTEE CONSULTANT * Postgraduate Institute for Medicine 2

3 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 3

4 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 4

5 H E M O P H I L I A A A N I D E A L D I S E A S E F O R L I V E R - D I R E C T E D G E N E T H E R A P Y Modest increases in Factor VIII levels (>1% of normal) have a positive impact on patient lives Adeno-associated virus (AAV) vectors have shown efficacy in preclinical and clinical studies; stable Factor IX (FIX) expression ~ five years in the clinic Lag in the clinic of AAV human Factor 8 (hf8) gene therapy poor vector yields at clinical scale and dose required to achieve therapeutic FVIII levels We optimized an AAV hf8 cdna vector cassette to improve both vector yields and liver-specific hfviii expression Administration of the enhanced AAV hf8 cdna vector in vivo resulted in 2-8X normal circulating hfviii levels * Higher circulating hfviii levels will enable lower dose in the clinic 5

6 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y 6

7 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y Transgene packaged into AAV vectors Transgene packaged into AAV vectors Therapeutic delivered by a single infusion AAV vectors P TG transgene P } liver- specific promoter TG } therapeutic gene (F8) 7

8 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y Transgene packaged into AAV vectors Transgene packaged into AAV vectors AAV is delivered by a single infusion Therapeutic delivered by a single infusion AAV vectors P TG transgene P } liver- specific promoter TG } therapeutic gene (F8) 8

9 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y Transgene packaged into AAV vectors Transgene packaged into AAV vectors AAV is delivered by a single infusion Therapeutic delivered by a single infusion AAV traffics to liver to deliver transgene into nucleus of liver cells AAV vectors P TG transgene Liver Cell DNA Promoter P } liver- specific promoter TG } therapeutic gene (F8) Therapeutic Gene (hf8) Nucleus Liver Cell Liver produces and secretes therapeutic hfviii protein 9

10 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y Transgene packaged into AAV vectors Transgene packaged into AAV vectors AAV is delivered by a single infusion Therapeutic delivered by a single infusion AAV traffics to liver to deliver transgene into nucleus of liver cells AAV vectors P TG transgene Liver Cell DNA Promoter P } liver- specific promoter TG } therapeutic gene (F8) Therapeutic Gene (hf8) Nucleus Liver Cell Liver produces and secretes therapeutic hfviii protein Transgene is expressed from the liver, but remains separate from the cell s DNA 10

11 H E M O P H I L I A A L I V E R - D I R E C T E D A AV F 8 C D N A G E N E T H E R A P Y Transgene packaged into AAV vectors Transgene packaged into AAV vectors AAV is delivered by a single infusion Therapeutic delivered by a single infusion AAV traffics to liver to deliver transgene into nucleus of liver cells AAV vectors P TG transgene Liver Cell DNA Promoter P } liver- specific promoter TG } therapeutic gene (F8) Therapeutic Gene (hf8) Nucleus Liver Cell Liver produces and secretes therapeutic hfviii protein Transgene is expressed from the liver, but remains separate from the cell s DNA 11

12 W I L D T Y P E A AV Single-stranded DNA virus which requires a helper virus for replication No pathology associated with AAV infection Tissue selectivity is determined by capsid composition Tissue Selectivity of AAV Serotypes AAV1 AAV2 AAV3 AAV4 AAV5 AAV6 AAV7 AAV8 AAV9 Liver, Heart, Skeletal muscle Liver, Heart, and muscle Heart, Liver Heart, Lung, Liver Liver Liver, Heart, Skeletal muscle Liver, Skeletal muscle Liver, Heart, Brain, Muscle Liver, Heart, Brain, Lung, Skeletal muscle Vance et al, DOI: /

13 R E C O M B I N A N T A AV Recombinant AAV (raav) has been used extensively for nearly 20 years as a gene therapy vector in preclinical and clinical studies Efficient transduction and long term, stable transgene expression in non-dividing tissues: - Liver, brain and muscle Replication deficient High degree of stability which allows for rigorous methods of vector purification AAV vectors carrying capacity is small (~4.7 kb of DNA) 13

14 G E N E R AT I O N O F R E C O M B I N A N T A AV Wild-Type AAV ITR Recombinant AAV Contains the transgene in place of wild type genes ITR wild type genes Transgene of Interest ITR Replace wild type genes ITR Manufacturing raav Helper plasmid is supplied in trans, together with the transgene to a packaging cell line Helper plasmid ITR ITR Transgene of interest + Highly purified raav cell line Harvest crude raav extract Purify raav - Density gradient - Column purification Within Cell Capsid Assembly Virus Packaging 14

15 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 15

16 W H AT I S O P T I M A L F O R A AV H F 8 C D N A? hf8 is not an ideal gene for AAV: Constrained by hf8 gene size Optimal AAV transgene size is ~4.7 kb; full length hf8 is ~7 kb AAV dose required to achieve therapeutic hfviii levels Low efficiency of transcription/translation Low manufacturing yields of AAV hf8 Clinical scale manufacturing feasibility is limiting Optimal AAV hf8 cdna requires: Shorter coding sequence for hf8 Potential solution is the use of an optimized B-domain deleted sequence (BDD) An optimized robust liver-specific promoter module to drive hf8 expression Improved virus yields 16

17 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 17

18 O P T I M I Z AT I O N O F A AV H F 8 C D N A R E Q U I R E D M U LT I - FA C T O R I A L M O D I F I C AT I O N S Liver Promoter Human Factor 8 B-Domain Deleted (BDD) polya ITR ITR Promoter module modifications Assembled different permutations of liver-specific promoter elements Identified regions of the promoter module that could be improved upon For other elements a systematic mutational design approach was used Transgene modifications Optimized the F8 cassette Other modifications Identified minimal synthetic polya Removed un-necessary nucleic acids Reduced size Optimized sequences outside transgene hfviii protein has the same amino acid sequence as biologics currently in clinic 18

19 I T E R AT I V E P R O C E S S T O I D E N T I F Y A N I M P R O V E D A AV H F 8 C D N A C A S S E T T E Promoter module modifications Virus Yields In Vitro secreted hfviii levels (mrna/protein) and activity Other modifications Transgene modifications In Vivo circulating hfviii levels (mrna/protein) 19

20 Y ie ld (v g ) M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D Stage 1 Stage 2 Stage 3 Stage Iterative Process to Improve Cassette 20

21 Y ie ld (v g ) M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D Stage 1 Stage 2 Stage 3 Stage Iterative Process to Improve Cassette 21

22 Y ie ld (v g ) M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D Stage 1 Stage 2 Stage 3 Stage Iterative Process to Improve Cassette 22

23 Y ie ld (v g ) M O D I F I C AT I O N S I M P R O V E V I R U S Y I E L D Stage 1 Stage 2 Stage 3 Stage fold improvement at the cell factory scale Iterative Process to Improve Cassette At clinical scale; greater than 5-fold improvement in vector yields 23

24 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 24

25 S E V E R A L M E T H O D S U S E D T O A S S E S S H F V I I I L E V E L S A N D A C T I V I T Y Enzyme-Linked Immunosorbent Assay (ELISA) hfviii magnet Clotting Assay Add Ca 2+ Generation of Factor Xa and thrombin yielding clot formation Activator + phospholipids hfviii FVIII deficient plasma magnet Factor X Chromogenic substrate Chromogenic Assay Factor IXa, Ca 2+, Phospholipid hfviii Factor Xa Factor Xa Chromophore 405 nm Clotting times ACTIVITY Not specific for human FVIII LEVELS Specific for human FVIII ACTIVITY Not specific for human FVIII 25

26 I N V I T R O : H E P G 2 C E L L S E X P E R I M E N TA L D E S I G N AAV2 ITR Liver-specific promoter module hfviii BDD PolyA AAV2 ITR Optimized raav hf8 cdna cassette packaged into AAV2/6 Test article addition Endpoints ELISA for hfviii levels days Clotting assay for hfviii activity Supernatant collection schedule Chromogenic assay for hfviii activity 26

27 h F V III A c tiv ity (U /m L ) h F V III (P e r c e n t N o rm a l) I N V I T R O : H E P G 2 C E L L S G O O D C O R R E L AT I O N B E T W E E N H F V I I I A C T I V I T Y / L E V E L S E L IS A C h r o m o g e n ic A s s a y C lo ttin g A s s a y D o s e Values will be reported as hfviii (Percent Normal) for ELISA, Chromogenic or Clotting where 1 U/mL = 100% Normal 4.8E6 / 2.4E6 / 1.2E6 / 0.6E6 MOI 27

28 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 28

29 I N V I V O : W I L D T Y P E M O U S E D ATA E X P E R I M E N TA L D E S I G N AAV2 ITR Liver-specific promoter module hfviii BDD PolyA AAV2 ITR IV injection of test article Optimized raav hf8 cdna cassette packaged into AAV2/ days Plasma collection schedule Dose 7.2E+12 vg/kg Endpoints ELISA for hfviii levels hf8 mrna tissue biodistribution Immunosuppression regimen of cyclophosphamide (50 mg/kg) 29

30 I N V I V O : W I L D T Y P E M O U S E D ATA S U P R A P H Y S I O L O G I C A L L E V E L S h F V III (P e r c e n t N o rm a l) % hfviii Levels F o rm u la tio n A A V h F 8 c D N A Levels were determined by ELISA 30

31 I N V I V O : W I L D T Y P E M O U S E D ATA H F 8 E X P R E S S I O N I S R E S T R I C T E D T O L I V E R N o rm a liz e d h F V III m R N A V a lu e s 8 hf8 mrna L iv e r B r a in K id n e y T e s te s H e a r t S p le e n L u n g Represents two independent mouse studies 31

32 I N V I V O : H E M O P H I L I A A M O U S E D ATA E X P E R I M E N TA L D E S I G N AAV2 ITR Liver-specific promoter module hfviii BDD PolyA AAV2 ITR Optimized raav hf8 cdna cassette packaged into AAV2/6 IV injection of test article Dose 7.2E+12 vg/kg days 3 months Endpoint Chromogenic assay for hfviii activity Tail vein transection (TVT) for hemostasis Plasma collection schedule 32

33 I N V I V O : H E M O P H I L I A A M O U S E D ATA O V E R V I E W O F H E M O P H I L I A A R C M I C E Hemophilia A R593C mice are tolerized to hfviii because they contain a hf8-r593c transgene under control of a murine albumin promoter hfviii-r593c is frequently observed in Hemophilia A patients, and in mice produces no detectable hfviii protein - Thought to be rapidly degraded in mice, with peptide fragments presented to the immune system Mice also contain a knockout of the mouse F8 gene and are deficient for endogenous mouse FVIII protein Studies were conducted in collaboration with Dr. David Lillicrap at Queen s University 33

34 I N V I V O : H E M O P H I L I A A M O U S E D ATA S U P R A P H Y S I O L O G I C A L L E V E L S h F V III (P e r c e n t N o rm a l) h F V III (P e r c e n t N o rm a l) hfviii Activity Day 14 Day % % F o rm u la tio n A A V h F 8 c D N A 0 F o rm u la tio n A A V h F 8 c D N A Activity determined by Chromogenic Activity Assay 34

35 I N V I V O : H E M O P H I L I A A M O U S E D ATA R E D U C E D B L E E D T I M E I N T R E AT E D M I C E T o ta l B le e d in g T im e (m in ) Tail Vein Transection (TVT) p < Normal bleeding time F o rm u la tio n A A V h F 8 c D N A TVT method based on Johansen et al., Haemophilia, 1-7,

36 I N T R O S L I D E / A G E N D A C L I C K T O E D I T M A S T E R T I T L E S T Y L E Background Hemophilia A and AAV Gene Therapy Overview of AAV Factor 8 cdna Approach to Optimizing AAV Factor 8 cdna Evaluating the Optimized AAV Factor 8 cdna in vitro: HepG2 Cells in vivo: Wild Type and Hemophilia A mouse models in vivo: Non-Human Primates (NHPs) Summary and Next Steps 36

37 I N V I V O : N O N - H U M A N P R I M AT E D ATA E X P E R I M E N TA L D E S I G N AAV2 ITR Liver-specific promoter module hfviii BDD PolyA AAV2 ITR Optimized raav hf8 cdna cassette packaged into AAV2/6 IV injection of test article Doses 2.0E+12 vg/kg 6.0E+12 vg/kg days 247 days Plasma collection schedule Endpoints ELISA for hfviii levels Clotting for hfviii activity Bethesda Units (BU) for inhibitory hfviii antibodies Liver enzymes Immunosuppression regimen of Rituxan and Solu-Medrol (10 mg/kg for both) 37

38 I N V I V O : N O N - H U M A N P R I M AT E D ATA S U P R A P H Y S I O L O G I C A L L E V E L S h F V III (P e r c e n t N o rm a l) hfviii Levels % % Dose selection was based on published studies Follow up dose-finding studies are aimed at determining the minimal effective dose for the clinic E E T o ta l D o s e (v g /k g ) Levels were determined by ELISA 38

39 I N V I V O : N O N - H U M A N P R I M AT E D ATA G O O D C O R R E L AT I O N B E T W E E N A C T I V I T Y / L E V E L S h F V III (P e r c e n t N o rm a l) A c tiv ity E L IS A L e v e ls F o rm u la tio n A n im a l ID E v g /k g G ro u p Shown in the Formulation Group: Detection of ~100 % Normal NHP FVIII activity No detection of NHP FVIII levels given the ELISA is specific for human FVIII 39

40 h F V III (P e r c e n t N o rm a l) B U /m L h F V III (P e r c e n t N o rm a l) B U /m L I N V I V O : N O N - H U M A N P R I M AT E D ATA D U R A B I L I T Y A n tig e n L e v e ls A n tig e n L e v e ls B e th e s d a U n its (B U ) S o lu - M e d ro l R e m o v e d B e th e s d a U n its (B U ) S o lu - M e d ro l R e m o v e d D a y s P o s t D o s in g D a y s P o s t D o s in g Stable hfviii levels for over 8-weeks in the absence of all immunosuppression Levels were determined by ELISA 40

41 I N V I V O : N H P I M M U N E T O L E R A N C E C H A L L E N G E D U R A B I L I T Y A simplified view of tolerance is induction of B-cell/T-cell anergy and/or apoptosis in the presence of sustained levels of agent (hfviii) Do the sustained hfviii levels produced from the raav-hf8 prevent re-induction of neutralizing antibodies? raav-hf8 treated NHP were challenged with hfviii biologic hfviii biologic challenge consisted of 4 weekly infusions of 25 U/kg of Xyntha 41

42 I N V I V O : N H P I M M U N E T O L E R A N C E C H A L L E N G E N O A P P E A R A N C E O F I N H I B I T O R Y A N T I B O D I E S h F V III (P e r c e n t N o rm a l) B U /m L h F V III (P e r c e n t N o rm a l) B U /m L X y n t h a C h a l e n g e h F V III (P e r c e n t N o rm a l) P o s itiv e fo r In h ib ito ry A n tib o d ie s B U /m L Control Group AAV hf8 cdna Dose Groups A n tig e n L e v e ls B e th e s d a U n its (B U ) D a y s P o s t D o s in g X y n t h a C h a l e n g e X y n t h a C h a l l e n g e N o E v id e n c e o f In h ib ito ry A n tib o d ie s N o E v id e n c e o f In h ib ito ry A n tib o d ie s Regions shaded gray are above the BU cutpoint thus positive for inhibitory antibodies A n tig e n L e v e ls A n tig e n L e v e ls B e th e s d a U n its (B U ) D a y s P o s t D o s in g B e th e s d a U n its (B U ) D a y s P o s t D o s in g hfviii levels ~150% hfviii levels ~10% 42

43 L iv e r E n z y m e (U /L ) h F V III (P e r c e n t N o rm a l) L iv e r E n z y m e s (U /L ) h F V III (P e r c e n t N o rm a l) I N V I V O : N O N - H U M A N P R I M AT E D ATA W E L L TO L E R AT E D Control Group High Dose Group A S T A S T A L T A L T * upper limit of normal * upper limit of normal D a y s P o s t D o s in g D a y s P o s t D o s in g Elevated levels observed post-liver biopsies (day 41) * ALT = Alanine Aminotransferase, upper limit of normal,126 U/L AST = Aspartate Aminotransferase, upper limit of normal,120 U/L 43

44 L iv e r E n z y m e (U /L ) h F V III (P e r c e n t N o rm a l) L iv e r E n z y m e s (U /L ) h F V III (P e r c e n t N o rm a l) I N V I V O : N O N - H U M A N P R I M AT E D ATA W E L L TO L E R AT E D Control Group High Dose Group A S T A S T A L T h F V III L e v e ls A L T h F V III L e v e ls * upper limit of normal * upper limit of normal D a y s P o s t D o s in g D a y s P o s t D o s in g Elevated levels observed post-liver biopsies (day 41) * ALT = Alanine Aminotransferase, upper limit of normal,126 U/L AST = Aspartate Aminotransferase, upper limit of normal,120 U/L 44

45 I N V I V O : N O N - H U M A N P R I M AT E D ATA P R E L I M I N A R Y R E S U LT S D O S E - F I N D I N G S T U D Y h F V III (P e r c e n t N o rm a l) hfviii Levels Preliminary results. Dose-finding study demonstrates high hfviii production from GMP-clinical scale manufacturing process 1 6 E E E E T o ta l D o s e (v g /k g ) Levels were determined by ELISA 45

46 S U M M A RY A N D F U T U R E P L A N S Administration of AAV hf8 cdna, engineered to improve vector yields and liver-specific hfviii expression, resulted in supraphysiological levels in vivo - Wild type mice - Hemophilia A R593C mice - NHPs Good correlation between assays used to measure circulating hfviii protein - Levels by ELISA and activity by Chromogenic or Clotting assays Sustained hfviii levels from the raav-hf8 prevented re-induction of neutralizing antibodies with biologic challenge suggestive of induced tolerance (even in the context of a xenogeneic setting) Ongoing studies are aimed at determining the minimal effective dose Goal of filling the IND, second half of

47 A C K N O W L E D G E M E N T S Judy Greengard Lisa King Eudean Garces Stephen Ballaron Daniel Richards Melanie Butler Carolyn Gasper Kathy Meyer Dale Ando Richard Surosky Alicia Goodwin Andrea Kang Tim Gabriele Hung Tran Jennifer Huang David Lillicrap Christine Hough Dominique Cartier Kate Nesbitt Courtney Dwyer Kassandra Herbert Mike Holmes Jeff Boonsripisal Derek Liu Rainier Amora Lei Zhang 47

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