Restoration of Porcine Factor VIII Procoagulant Activity to Hemophilia A Mice with Pre-Existing Immunity to Human Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3284-3284
Author(s):  
Christopher B. Doering ◽  
Bagirath Gangadharan ◽  
Hillary Z. Dukart ◽  
H. Trent Spencer
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Donor Cell ◽  
Activity Levels ◽  
Cell Engraftment ◽  
Fviii Activity ◽  
Human Factor Viii ◽  
Total Body ◽  
High Level

Abstract The development of inhibitory antibodies directed against human factor VIII (hfVIII) remains the most significant clinical complication associated with the treatment of hemophilia A. Recently, we demonstrated that transplantation of genetically-modified hematopoietic stem cells (HSCs) containing a high-expression porcine (HEP) fVIII transgene promoted sustained, high-level fVIII expression in naïve, genetically-immunocompetent hemophilia A mice (Gangadharan et al. 2006. Blood 107, 3859–64). In the current study, HEP-fVIII HSC transplantation (HSCT)-based gene therapy was tested in the setting of hemophilia A complicated by the presence of circulating anti-hfVIII inhibitory antibodies. Following a series of intravenous injections with recombinant hfVIII, hemophilia A mice developed anti-hfVIII antibodies with clinically-significant inhibitory titers. The median ELISA and Bethesda (inhibitory) titers were 2938 and 55 (n = 18), respectively, and a significant correlation between the anti-hfVIII ELISA and Bethesda (inhibitory) titers was observed (P < 0.001). With the exception of two mice, all plasma samples analyzed contained anti-HEP-fVIII cross-reactive antibodies by ELISA, and 5 of 16 displayed detectable anti-HEP-fVIII inhibitory activity. HSCs were transduced ex vivo with recombinant retrovirus carrying a HEP-fVIII transgene and transplanted into lethally-irradiated (11 Gy total body irradiation) hemophilia A mice (n = 11). Twelve wks post-HSCT, all mice displayed high-level donor cell engraftment in peripheral blood mononuclear cells (PBMCs) at 81 ± 22% (mean ± SD) and expressed therapeutic levels of fVIII with a mean activity of 3 ± 1.3 units/ml (Figure 1). A second cohort of mice (n = 7) underwent a similar HSCT gene therapy procedure with the exception that they received a sub-lethal dose of total body irradiation (5.5 Gy). These mice exhibited lower-level donor cell engraftment at 6.5 ± 6% (range 0.7 – 13%) in PBMCs at 12 weeks post-HSCT. At day 9 post-HSCT, the mice in this cohort contained circulating fVIII activity levels at 1 ± 0.6 units/ml (Figure 1). However in 6 of 7 mice, fVIII activity levels returned to baseline (≤0.01 units/ml) by day 28 post-HSCT, and in 4 of 7 mice, fVIII activity remained below the level of detection. The remaining 3 mice displayed 0.22 ± 0.3 (range 0.04 – 0.57) units/ml fVIII activity at 12 weeks post-HSCT and showed a trend towards greater donor PBMC engraftment, 12.8 ± 0.2% versus 1.8 ± 1.3% (P = 0.057). Analysis of the anti-hfVIII ELISA titers post-HSCT in both cohorts of mice revealed that while the titers steadily decreased in the lethally-irradiated mice (initial t1/2 ~ 12 days), the titers remained unchanged in the sub-lethally-irradiated mice possibly explaining the differences observed for donor cell engraftment and fVIII expression between the 2 groups. These data provide proof-of-concept that HSCT-based gene therapy incorporating a HEP-fVIII transgene could be utilized for the treatment of high-risk patients with refractory anti-hfVIII inhibitors. Figure Figure

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.

Development And Application Of A Minimal-Adenoviral Vector System For Gene Therapy Of Hemophilia A

1999 ◽  
Vol 82 (08) ◽  
pp. 562-571 ◽  
Author(s):  
Steven Josephs ◽  
Jiemin Zhou ◽  
Xiangming Fang ◽  
Ramón Alemany ◽  
Cristina Balagué ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Vector System ◽  
High Level Expression ◽  
Viral Particles ◽  
Human Factor Viii ◽  
High Level

IntroductionHemophilia A and B are the most common bleeding disorders caused by deficiencies of clotting factors VIII and IX, respectively, both of which are X-linked with a recessive heredity.1 Replacement of the deficient factors with frequent intravenous injections of plasma concentrates or recombinant proteins is the standard treatment for these diseases.2 Great efforts have been made for nearly a decade toward developing experimental gene therapy for these diseases and aiming at the development of a medical intervention that is more effective and convenient than the currently available replacement therapies.3 Hemophilia is a suitable clinical model for the development of gene therapy products and has a number of advantages: 1) there is a simple and well defined cause-and-effect relationship between the protein deficiencies and bleeding symptoms; 2) tissue-specific expression and precise regulation of the transgenes are not necessary; 3) well characterized animal models are available for preclinical studies; 4) an unequivocal endpoint for product efficacy can be assessed in clinical trials; and 5) even 1% to 5% of the normal physiological levels of the proteins is therapeutic.For gene therapy of hemophilia, the most challenging hurdle, with respect to the long-term expression of the deficient proteins at adequate levels, is the development of a suitable gene delivery system. Technologies have been evolving from ex vivo to in vivo approaches, from initial use of retroviral vector to recent application of adenviral (Ad) or adeno-associated virus (AAV) vector, demonstrating progress from early results of transient low-level expression to more sustained high-level expression.3 For hemophilia A treatment, Ad vectors are particularly useful, since the liver naturally produces factor VIII, and following intravenous (i.v.) injection, Ad vectors concentrate in the liver. This makes the gene transduction efficiency to liver very high. Adenovirus vectors have been developed for gene therapy due to their high titer, broad infectivity, potential for large payload, and in vivo gene delivery capacity.4 Although the immunogenicity and cytotoxicity associated with the early-generation Ad vectors have been a concern with respect to their clinical application, newly developed vectors, in which the viral coding sequences have been deleted, have significantly reduced the side effects associated with the vectors. The “gutless” Ad vector, or so called helper-dependent, large-capacity, or mini- Ad vectors are the representative examples of these new-generation Ad vectors.5-15 The mini-Ad vector system described in this report was developed based on two major research findings. First, an Ad- SV40 hybrid virus discovered during attempts to grow human Ad in non-permissive monkey COS-7 cells.16 The hybrid virus had a genome structure in which only both ends of the Ad sequences were retained and almost all coding sequences of the Ad genome were replaced by symmetric, tandemly repeated SV40 genomes. The hybrid viruses replicated and were packaged in the presence of a wild-type Ad as a helper. This finding implied that total replacement of the Ad genome was possible to form a mini-Ad vector as long as proper helper function and selective pressure was provided. Secondly, it was discovered that Ad packaging can be attenuated by deleting portions of the packaging signal.17 This finding provided a means to put selective pressure on the helper Ad (referred to as ancillary Ad) by specifically limiting its packaging process and allowing a preferential packaging of the mini-Ad. The system, therefore, is designed to have three main components: the mini-Ad vector, the E1-deleted ancillary Ad, and a production cell line that provides AdE1 complementation.Based on the mini-Ad vector system, MiniAdFVIII was developed. The MiniAdFVIII vector carries a 27 kb expression cassette, in which the full-length human factor VIII cDNA is flanked by a human albumin promoter and cognate genomic sequences. Infection of MiniAdFVIII in vitro showed that the vector mediated expression of functional human factor VIII at levels of 100-200 ng/106 cells per 24 hours in HepG2 and 293 cells. With single-dose intravenous injection of 1011 viral particles in hemophilic mice, MiniAdFVIII produced a sustained high-level expression of human factor VIII (at 100-800 ng/ml for up to 369 days) that corrected the factor VIII-deficient phenotype. Safety studies of MiniAdFVIII showed that there were no significant toxicities in mice and dogs after a single intravenous dose of up to 3×1011 and 6×1012 viral particles, respectively. In this report, other studies for developing the MiniAdFVIII vector with a site-specific integration capability and the development of a human factor VIII-tolerized mouse model for preclinical studies of MiniAdFVIII are described.

scholarly journals Hematopoietic stem-cell gene therapy of hemophilia A incorporating a porcine factor VIII transgene and nonmyeloablative conditioning regimens

Blood ◽  
2007 ◽  
Vol 110 (8) ◽  
pp. 2855-2863 ◽  
Author(s):  
Lucienne M. Ide ◽  
Bagirath Gangadharan ◽  
Kuang-Yueh Chiang ◽  
Christopher B. Doering ◽  
H. Trent Spencer
Keyword(s):  
Stem Cell ◽  
Factor Viii ◽  
Hemophilia A ◽  
Immunosuppressive Agents ◽  
Donor Cell ◽  
Hematopoietic Stem ◽  
Cell Engraftment ◽  
Conditioning Regimens ◽  
Low Toxicity ◽  
Total Body

Abstract Insufficient expression of factor VIII (fVIII) is a major hurdle in the development of successful nucleic acid treatments for hemophilia. However, we recently showed that under myeloablative and reduced-intensity total body irradiation (TBI) conditioning, transplantation of hematopoietic stem cells (HSCs) transduced with recombinant retroviruses containing B domain–deleted porcine fVIII (BDDpfVIII) sequences provides curative fVIII levels in a hemophilia A mouse model. In the current study, we tested BDDpfVIII activity after nonmyeloablative conditioning with busulfan, cyclophosphamide, or fludarabine and immunosuppressive agents CTLA4-Ig + anti-CD40L or anti-(murine)thymocyte serum (ATS). ATS is similar in action to anti-(human)thymocyte globulin (ATG), which is used clinically with busulfan in bone marrow transplantations to increase donor cell engraftment. Mice conditioned with busulfan + ATS and that received a transplant of BDDpfVIII-transduced stem-cell antigen 1-positive cells exhibited moderate levels of donor cell chimerism (between 20% and 60%) and achieved sustained fVIII levels more than 1 U/mL. Similar results were observed in mice preimmunized with human fVIII and conditioned with 5 Gy TBI + ATS or busulfan + ATS. These data demonstrate that it is possible to achieve sufficient fVIII expression after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pretransplantation conditioning with targeted immunosuppression, potentially even in the context of preexisting inhibitors.

Direct Comparison of Bioengineered Factor VIII Expression Constructs: Towards an Optimal Transgene for Gene Therapy of Hemophilia A.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5477-5477
Author(s):  
Kerry L. Titus ◽  
Paul Lee ◽  
H. Trent Spencer ◽  
Christopher Doering
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Factor Viii ◽  
Hemophilia A ◽  
Double Mutation ◽  
Specific Amino Acid ◽  
Stable Transfectants ◽  
Glycosylation Sites ◽  
Fviii Activity ◽  
A2 Domain

Abstract A major obstacle for gene therapy of hemophilia A is the achievement of adequate factor VIII (fVIII) expression. Bioengineering strategies have targeted specific sequences within human fVIII that are thought to be responsible for its generally poor expression. Specific amino acid substitutions, L303E/F309S herein referred to as double mutation (DM), function to decrease fVIII binding to BiP, a resident ER chaperone, which results in increased fVIII secretion (Swaroop, Moussalli et al. 1997). Furthermore, addition of 6 N-linked glycosylation sites, designated 226/N6, located within the human B domain also increases human fVIII expression (Miao, Sirachainan et al. 2004). We previously demonstrated that porcine and certain hybrid human/porcine fVIII constructs are expressed at 10 – 14-fold greater levels than human fVIII (Doering, Healey et al. 2002; Doering, Healey et al. 2004). The aim of the current study was to directly compare various fVIII expression constructs in order to determine an optimal transgene for gene therapy applications. The following fVIII constructs were generated: human B-domain-deleted fVIII (HBDD-fVIII), HBDD-fVIII with a 14 amino acid linker between the A2 domain and the activation peptide (HSQ-fVIII), porcine fVIII containing a 24 amino acid linker (HEP-fVIII), hybrid human/porcine-fVIII which has porcine A1 and A3 domains (HP47), and modified HBDD, HSQ and HEP-fVIII constructs containing DM and/or 226/N6. Each construct was transiently transfected into BHK-M cells, and fVIII production between 48 – 72 hrs post-transfection was measured using a one-stage clotting assay. Under these conditions, the addition of the DM and 226/N6 significantly increased fVIII expression for HBDD (P = 0.003), though not for HSQ. Addition of DM or 226/N6 alone did not significantly increase the expression of either human fVIII construct, and furthermore, the addition of DM to HEP-fVIII decreased its expression 98%. HEP-fVIII was expressed at 8-fold or greater levels than any of the other human constructs. Next, ~25 stably transfected BHK-M clones were isolated following transfection with each of the fVIII expression constructs and the rate of fVIII production for each clone was determined. Several clones did not express detectable fVIII activity (<0.01 units/mL) and were excluded from the analysis. Approximately 14% of the total number of clones were excluded, ranging from 0 – 42% for the different constructs. HEP-DM-fVIII was the exception, where 82% of the clones had activity <0.01 units/mL. Mean HEP-fVIII expression was 3.93 ± 3.22 units/mL/24 hr (n = 19) (Figure 1), and HP47 was similarly expressed at 3.21 ± 2.31 units/mL/24 hr (n = 19). All of the HSQ-based constructs and HBDD-DM/226/N6 showed similar mean expression levels (0.28 ± 0.03 units/mL/24 hr) and were significantly higher than HBDD and HBDD-DM, which had a mean of 0.12 ± 0.01 units/mL. In the current study, we provide experimental evidence that the expression of HEP-fVIII and HP47 is superior to other bioengineered fVIII expression constructs, which should eliminate the expression barrier that has hampered the clinical translation of gene therapy for hemophilia A. Figure 1: Stable Transfectants Figure 1:. Stable Transfectants

The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2038-2038
Author(s):  
Qizhao Wang ◽  
Jenni Firrman ◽  
Katie A Pokiniewski ◽  
Wenjing Cao ◽  
Hongying Wei ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Defect ◽  
High Dose ◽  
Coagulation Activity ◽  
Fviii Activity ◽  
Cofactor Activity

Abstract Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies. In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. Disclosures No relevant conflicts of interest to declare.

scholarly journals Transient blockade of the inducible costimulator pathway generates long-term tolerance to factor VIII after nonviral gene transfer into hemophilia A mice

Blood ◽  
2008 ◽  
Vol 112 (5) ◽  
pp. 1662-1672 ◽  
Author(s):  
Baowei Peng ◽  
Peiqing Ye ◽  
Bruce R. Blazar ◽  
Gordon J. Freeman ◽  
David J. Rawlings ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Monoclonal Antibody ◽  
T Cells ◽  
Factor Viii ◽  
Cd4 T Cells ◽  
Hemophilia A ◽  
Term Therapy ◽  
Antibody Treatment ◽  
Inducible Costimulator ◽  
Human Factor Viii

Abstract Formation of inhibitory antibodies is a common problem encountered in clinical treatment for hemophilia. Human factor VIII (hFVIII) plasmid gene therapy in hemophilia A mice also leads to strong humoral responses. We demonstrate that short-term therapy with an anti-ICOS monoclonal antibody to transiently block the inducible costimulator/inducible costimulator ligand (ICOS/ICOSL) signaling pathway led to sustained tolerance to hFVIII in hFVIII plasmid–treated hemophilia A mice and allowed persistent, high-level FVIII functional activity (100%-300% of normal). Anti-ICOS treatment resulted in depletion of ICOS+CD4+ T cells and activation of CD25+Foxp3+ Tregs in the peripheral blood, spleen, and lymph nodes. CD4+ T cells from anti-ICOS–treated mice did not proliferate in response to hFVIII stimulation and produced high levels of regulatory cytokines, including interleukin-10 and transforming growth factor-β. Moreover, CD4+CD25+ Tregs from tolerized mice adoptively transferred dominant tolerance in syngeneic hFVIII plasmid-treated hemophilia A mice and reduced the production of antibodies against FVIII. Anti-ICOS–treated mice tolerized to hFVIII generated normal primary and secondary antibody responses after immunization with the T-dependent antigen, bacteriophage Φx 174, indicating maintenance of immune competency. Our data indicate that transient anti-ICOS monoclonal antibody treatment represents a novel single-agent immunomodulatory strategy to overcome the immune responses against transgene product after gene therapy.

scholarly journals Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Liver Perfusion ◽  
Transfer Efficiency ◽  
Clotting Factor ◽  
Gene Transfer Efficiency ◽  
Fviii Activity

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

scholarly journals GO-8: Preliminary Results of a Phase I/II Dose Escalation Trial of Gene Therapy for Haemophilia a Using a Novel Human Factor VIII Variant

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 489-489 ◽  
Author(s):  
Amit C Nathwani ◽  
Edward Tuddenham ◽  
Pratima Chowdary ◽  
Jenny McIntosh ◽  
Doyoung Lee ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Gene Transfer ◽  
Phase I ◽  
Factor Viii ◽  
Board Of Directors ◽  
Research Funding ◽  
Activity Levels ◽  
Advisory Committees ◽  
Preliminary Results ◽  
Fviii Activity

Abstract Background: Haemophilia A (HA), the most common inherited bleeding disorder, is well suited for gene therapy because a modest increase in the plasma factor VIII (FVIII) levels to ≥1% of normal levels will substantially ameliorate the bleeding diathesis and improve quality of life. Earlier gene transfer strategies for FVIII replacement approaches using plasmid electroporation, retroviral vector, or adenoviral vector failed to achieve persistent phenotypic correction of bleeding. We have recently shown that a single peripheral vein administration of adeno-associated viral (AAV) vectors expressing the FIX transgene results in stable long-term expression of transgenic FIX at therapeutic levels without long term toxicity in patients with severe haemophilia B (ClinicalTrials.gov:NCT00979238). However, the use of AAV vectors for HA gene therapy has been limited by inefficient expression of transgenic FVIII and the large size of the FVIII cDNA. To overcome these obstacles, we developed two AAV-FVIII expression cassettes containing a small synthetic liver specific promoter (HLP) driving the expression of codon optimized FVIII variants. These vectors mediated therapeutic expression of FVIII in murine and non-human primate models (McIntosh et al 2013). The first of these constructs, AAV-HLP-hFVIII-SQ, encoding a B-domain deleted FVIII variant, was recently shown (Rangarajan et al, 2017) to mediate sustained (>1 year) normalisation of factor VIII activity in six of seven participants following a single intravenous infusion of AAV serotype 5 pseudotyped vector. However, high vector doses (6x1013 vector genomes/kg [vg/kg]) were required for efficacy, possibly because this product was manufactured using the insect cell/baculovirus system. In this report we describe the preliminary results of our on-going Phase I/II clinical trial (GO-8) evaluating the second FVIII cassette (AAV-HLP-hFVIII-V3), which contains a 17 amino-acid peptide comprising six N-linked glycosylation motifs from the human FVIII B-domain that are highly conserved through evolution. In murine studies, AAV-HLP-hFVIII-V3 mediated expression of FVIII at 3-fold higher levels when compared to AAV-HLP-hFVIII-SQ. Methods: The safety and efficacy of a single intravenous infusion of AAV8-HLP-hFVIII-V3, pseudotyped with AAV serotype 8 capsid was assessed in three adult men with severe hemophilia A (FVIII activity levels ≤1% of normal) in the context of an Investigator led, Phase I/II, open-label, non-randomized, dose-escalation trial (ClinicalTrials.gov: NCT03001830GO-8). The first subject received a dose of 6x1011vg/kg and the subsequent two patients each received a dose of 2x1012vg/kg. AAV8-HLP-hFVIII-V3 was manufactured in mammalian HEK 293T cells. The subjects have been followed up for 13-47 weeks after vector administration. Results: Peripheral vein administration of AAV8-HLP-hFVIII-V3 was well tolerated in all patients with no infusion-related reactions. Transgenic FVIII was detectable within two weeks and was more than 5 IU/dl by 6 weeks of gene transfer in all three subjects. Factor VIII activity (one stage clotting assay) levels have remained stable at 7±1IU/dl in patient 1 over a period of 47 weeks. The second participant is 20 weeks following administration of 2x1012 vg/kg of AAV8-HLP-hFVIII-V3 and has steady-state FVIII activity of 6±2IU/dl. In the third subject, who was also treated at a dose of 2x1012 vg/kg, the steady state FVIII activity is almost 10 times higher at 69±7 IU/dl. Elevation of serum alanine aminotransferase was observed in patients 1 and 3 at between weeks 4-6 after gene transfer, reaching peak levels that were 1.5 X upper limit of the normal range. Both patients were treated with corticosteroids within 48 hours of the onset of transaminitis with no loss of transgene expression. No participant has developed a FVIII inhibitor. Conclusion: Our preliminary results from the ongoing Phase I/II study demonstrate FVIII activity levels >5% in all three subjects with normalization of FVIII:C levels in one patient. These levels are sufficient to reduce/prevent spontaneous hemorrhage and have been achieved using relatively lower doses of AAV8-HLP-FVIII-V3 than reported previously with a related FVIII expression cassette. No Grade III (CTCAE v4.03) or greater adverse events have been observed over a period of 47 weeks after administration of AAV8-HLP-hFVIII-V3. Disclosures Nathwani: Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Tuddenham:Freeline: Consultancy; BioMarin: Consultancy, Patents & Royalties. Chowdary:Biogen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Baxalta (Shire): Honoraria, Membership on an entity's Board of Directors or advisory committees; Swedish Orphan Biovitrum AB (Sobi): Honoraria; Novo Nordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Freeline: Consultancy. McIntosh:Freeline: Consultancy.

scholarly journals The Factor VIII Variant X5 Enhances Hemophilia a Gene Therapy Efficiency By Its Improved Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3356-3356
Author(s):  
Franziska Horling ◽  
Johannes Lengler ◽  
Wenjing Cao ◽  
Biao Dong ◽  
Bagirath Gangadharan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Liver Cell ◽  
Human Liver ◽  
Hemophilia A ◽  
Employment Equity ◽  
Fviii Activity ◽  
Human Liver Cell ◽  
Equity Ownership

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector efficiency could be improved by employing more potent liver-specific promoters, but this might come at the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause oxidative stress, and if not resolved may even induce cell death. Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5 variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene therapy. Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells and purified to apparent homogeneity using standard procedures. The preparations were assayed for total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice (E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic, humanized HLA-DRB1*1501). Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5 variant revealed similarity of the two proteins and their specific activities in particular, indicating that introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5 transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any increased immunogenic risk compared to BDD-FVIII. Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy. Disclosures Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler:Baxalta Innovations GmbH, a Takeda company: Employment. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. De La Rosa:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Hoellriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Reipert:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

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