scholarly journals Adeno-Associated Viral Vector Delivery of Optimized Human Factor VIII Achieves Therapeutic Factor VIII Levels in Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 199-199 ◽  
Author(s):  
Xavier M. Anguela ◽  
Liron Elkouby ◽  
Raffaella Toso ◽  
Marti DiPietro ◽  
Robert J. Davidson ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Ix ◽  
Large Animal ◽  
Wild Type ◽  
Employment Equity ◽  
Human Factor Viii ◽  
Equity Ownership

Abstract Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x1011 vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x1012 vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x1012 vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x1013 vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Elkouby:Spark Therapeutics, Inc.: Employment, Equity Ownership. Toso:Spark Therapeutics, Inc.: Employment, Equity Ownership. DiPietro:Spark Therapeutics, Inc.: Employment, Equity Ownership. Davidson:Spark Therapeutics: Consultancy. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Sabatino:Spark Therapeutics, Inc.: Research Funding.

scholarly journals A Novel Lysine to Arginine Substitution at Position 301 Enhances Activity of Factor IX

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3772-3772
Author(s):  
Daniel Verhoef ◽  
Jonathan H. Foley ◽  
Andrew Goodale ◽  
Emma Macrae ◽  
Jenny McIntosh ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Specific Activity ◽  
Factor Ix ◽  
Wild Type ◽  
Gain Of Function ◽  
Employment Equity ◽  
Knock Out ◽  
Genome Integration ◽  
Equity Ownership ◽  
Knock Out Mice

Abstract Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Neonatal Gene Transfer Induces Tolerance to Human Factor VIII in Mice and Cats.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 215-215
Author(s):  
Lingfei Xu ◽  
Manxue Mei ◽  
Mark E. Haskins ◽  
Karyn Cullen ◽  
Katherine P. Ponder
Keyword(s):  
Gene Therapy ◽  
Immune System ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Large Animal ◽  
Human Factor Viii ◽  
C3h Mice ◽  
Low Levels ◽  
Medium Dose

Abstract Hemophilia A is a bleeding disorder due to Factor VIII (FVIII) deficiency. FVIII is a very immunogenic protein, as approximately 30% of patients develop inhibitory antibodies (inhibitors) after FVIII protein replacement therapy. In addition, inhibitors often develop after gene therapy for hemophilia A. Identification of a treatment that could prevent inhibitor formation would be important. In this study, we explored the possibility of inducing tolerance to FVIII by neonatal gene transfer. An amphotropic gamma retroviral vector (RV) expressing human B domain-deleted FVIII (hFVIII) from the human α1-antitrypsin promoter was used to define the level of hFVIII that is necessary to achieve tolerance. Hemophilia A mice were injected with 1010 (high), 109 (medium) or 108 (low) transducing units (TU)/kg of RV at 2 to 3 days after birth, which resulted in expression of 63%, 7.3% or <2% of normal hFVIII antigen, respectively. Animals that received the high or medium dose of RV achieved hemostasis in vivo, although those that received the low dose did not. None of the mice produced antibodies to hFVIII. Similar results were also achieved in normal C3H mice, although some animals with low expression (2% of normal) developed low levels of anti-hFVIII antibodies. In contrast, hemophilia A and C3H mice that were challenged with hFVIII protein made potent inhibitors. Thus, neonatal gene transfer does not induce antibodies to hFVIII if the level of expression achieved is sufficiently high (>2x10−9 M). Mice that are tolerant to gene transfer are being challenged with recombinant BDD-hFVIII protein to determine if they are truly tolerant. Although induction of tolerance with neonatal gene transfer in mice is encouraging, tolerance has been more difficult to achieve in large outbred animals because their immune systems are more mature. Cats appear to have a much more mature immune system at birth than mice, as they mount a potent cytotoxic T lymphocyte response to canine iduronidase after neonatal gene therapy. Cats were therefore chosen as a large animal model in which to study tolerance induction with neonatal gene transfer. Neonatal cats were injected IV with 8.5x108 TU/kg of RV (medium dose) at day 5 after birth. Four cats achieved 37±3 ng/ml hFVIII (19% of normal) at 2 weeks after RV transduction. Three cats maintained hFVIII expression for 4 months. The two cats with the highest level of expression (~40 ng/ml) never developed antibodies, while the cat with a medium level of expression (30 ng/ml) developed only very low levels of anti-hFVIII antibodies. The cat with the lowest level of expression (20 ng/ml) lost expression at 2 months after RV transduction, and developed high titer inhibitors. We conclude that neonatal gene transfer does not induce antibodies to hFVIII in most cats. However, a high level of hFVIII expression may be necessary to achieve tolerance after neonatal gene transfer in cats. RV-treated cats will be challenged with hFVIII protein to determine if they are truly tolerant. In addition, normal cats will be challenged with hFVIII protein to determine the frequency of inhibitor formation in cats. Since the cat immune system is relatively mature at birth, these data are encouraging that neonatal gene or protein therapy might induce tolerance to hFVIII in humans. (This project is supported by National Hemophilia Foundation and the Bayer Hemophilia Awards Program.)

scholarly journals 680. Non-Viral Transposon Mediated Gene Transfer of Human Factor VIII to Hemophilia A Mice

2006 ◽  
Vol 13 ◽  
pp. S262
Author(s):  
Litao Xie ◽  
Yubin Kang ◽  
Stephen Yant ◽  
Mark Kay ◽  
Paul McCray
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Human Factor Viii

Induction of tolerance to human factor VIII in mice

Blood ◽  
2001 ◽  
Vol 97 (10) ◽  
pp. 3311-3312 ◽  
Author(s):  
Hengjun Chao ◽  
Christopher E. Walsh
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
High Titer ◽  
Inhibitory Antibody ◽  
Adeno Associated Virus ◽  
Immunodeficient Mice ◽  
Human Factor Viii ◽  
Induction Of Tolerance

Abstract This paper reports loss of human factor VIII (hFVIII) inhibitory antibody in immunocompetent C57BL/6 mice. High-titer anti-hFVIII antibody developed in the mice within 7 to 14 days of intraportal administration of adeno-associated virus (AAV) carrying FVIII that coincided with a reduction in plasma hFVIII antigen. Bethesda titers (&gt; 100 units) persisted relatively unchanged for 9 to 10 months. Unexpectedly, at 10 months after injection of the virus, hFVIII protein (up to 59 ng/mL) was detected in 3 mice at the same time as disappearance of hFVIII inhibitor. The level of hFVIII was similar to that found in immunodeficient mice receiving the same dose of recombinant AAV carrying hFVIII without hFVIII inhibitor. These results suggest that tolerance to hFVIII can be induced by sustained expression of hFVIII in a mouse model. Further elucidation of this observation may affect use of FVIII gene transfer in the treatment of inhibitor-positive patients with hemophilia A.

Mechanism of the Immune Response to Human Factor VIII in Murine Hemophilia A

2001 ◽  
Vol 85 (01) ◽  
pp. 125-133 ◽  
Author(s):  
Huiyun Wu ◽  
Mark Reding ◽  
Jiahua Qian ◽  
David Okita ◽  
Ernie Parker ◽  
...  
Keyword(s):  
Immune Response ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Th2 Cells ◽  
Tween 20 ◽  
Sorbitan Monolaurate ◽  
Human Factor Viii ◽  
Ifn Γ ◽  
Th1 And Th2

SummaryMice genetically deficient in factor VIII (fVIII) are a model of hemophilia A. As a first step to reproduce in this mouse model what occurs over time in hemophilia A patients treated with human fVIII (hfVIII), we have investigated the time course and the characteristics of their immune response to hfVIII, after multiple intravenous injections. Anti-hfVIII antibodies appeared after four to five injections. They were IgG1 and to a lesser extent IgG2, indicating that they were induced by both Th2 and Th1 cells. Inhibitors appeared after six injections. CD4+ enriched splenocytes from hfVIII-treated mice proliferated in response to fVIII and secreted IL-10: in a few mice they secreted also IFN-γ and in one mouse IL-4, but never IL-2. A hfVIII-specific T cell line derived from hfVIII-treated mice secreted both IL-4 and IFN-γ, suggesting that it included both Th1 and Th2 cells. CD4+ enriched splenocytes of hfVIII-treated mice recognized all hfVIII domains. Thus, hemophilic mice develop an immune response to hfVIII administered intravenously similar to that of hemophilia A patients. Their anti-hfVIII antibodies can be inhibitors and belong to IgG subclasses homologous to those of inhibitors in hemophilic patients; their anti-hfVIII CD4+ cells recognize a complex repertoire and both Th1 and Th2 cytokines, and especially IL-10, may drive the antibody synthesis. Abbreviations used: antibodies, Ab; antigen presenting cells, APC; Arbitrary Units, AU; enzyme-linked immunosorbant assay, ELISA; factor VIII, fVIII; human factor VIII, hf VIII; intravenous, i.v.; optical density, OD; polymerase chain reaction, PCR; phosphate buffered saline solution, PBS; PBS containing 3% bovine serum albumin, PBS/BSA; PBS containing 0.05% polyoxyethylene sorbitan monolaurate, PBS/Tween-20; phytohemoagglutinin, PHA; stimulation index, SI

Identification of a Novel Missense Mutation in Exon 4 of the Human Factor VIII Gene Associated with Sever Hemophilia a Patient

2007 ◽  
Vol 10 (23) ◽  
pp. 4299-4302 ◽  
Author(s):  
Habib Onsori ◽  
Mohammad Ali Hossein . ◽  
Sheideh Montaser-Kou . ◽  
Mohammad Asgharzadeh . ◽  
Abbas Ali Hosseinpou .
Keyword(s):  
Factor Viii ◽  
Missense Mutation ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Viii Gene ◽  
Human Factor Viii ◽  
Exon 4

scholarly journals Sustained expression of therapeutic levels of human factor VIII in mice

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4671-4677 ◽  
Author(s):  
S Connelly ◽  
JM Gardner ◽  
RM Lyons ◽  
A McClelland ◽  
M Kaleko
Keyword(s):  
Factor Viii ◽  
Adenoviral Vector ◽  
Human Factor ◽  
Hemophilia A ◽  
Coagulation Factor ◽  
High Dose ◽  
Specific Enzyme ◽  
Adult Mice ◽  
Human Factor Viii

Deficiency of coagulation factor VIII (FVIII) results in hemophilia A, a common hereditary bleeding disorder. Using a human FVIII-encoding adenoviral vector, Av1ALAPH81, we have demonstrated expression of therapeutic levels of human FVIII in mice sustained for more than 5 months after vector administration. Administration of a high dose (4 x 10(9) plaque-forming units [pfu]) of Av1ALAPH81 to mice resulted in a peak expression of 2,063 ng/mL of human FVIII in the mouse plasma, with levels decreasing to background by weeks 15 to 17. Normal FVIII levels in humans range from 100 to 200 ng/mL and therapeutic levels are as low as 10 ng/mL. Alternatively, administration of 8- to 80-fold lower vector doses (5 x 10(8) pfu to 5 x 10(7) pfu) to normal adult mice resulted in expression of FVIII at therapeutic levels sustained for at least 22 weeks. Detailed analysis of vector toxicity indicated that the high vector dose caused a dramatic elevation of liver-specific enzyme levels, whereas an eight-fold lower vector dose was significantly less hepatotoxic. The data presented here demonstrate that administration of lower, less toxic vector doses allow long-term persistence of FVIII expression.

scholarly journals Successful treatment of canine hemophilia by continuous expression of canine FVIIa

Blood ◽  
2009 ◽  
Vol 113 (16) ◽  
pp. 3682-3689 ◽  
Author(s):  
Paris Margaritis ◽  
Elise Roy ◽  
Majed N. Aljamali ◽  
Harre D. Downey ◽  
Urs Giger ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Hemophilia A ◽  
Viral Vector ◽  
Canine Model ◽  
Factor Vii ◽  
Large Animal Model ◽  
Large Animal ◽  
Spontaneous Bleeding ◽  
Human Factor Viii

Abstract Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor–dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

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