hFVIII Gene Research Articles

Intronic regions of the human coagulation factor VIII gene harboring transcription factor binding sites with a strong bias towards the short-interspersed elements

Increasing data show that intronic derived regulatory elements, such as transcription factor binding sites (TFBs), play key roles in gene regulation, and malfunction. Accordingly, characterizing the sequence context of the intronic regions of the human coagulation factor VIII (hFVIII) gene can be important. In this study, the intronic regions of the hFVIII gene were scrutinized based on in-silico methods. The results disclosed that these regions harbor a rich array of functional elements such as repetitive elements (REs), splicing sites, and transcription factor binding sites (TFBs). Among these elements, TFBs and REs showed a significant distribution and correlation to each other. This survey indicated that 31% of TFBs are localized in the intronic regions of the gene. Moreover, TFBs indicate a strong bias in the regions far from splice sites of introns with mapping to different REs. Accordingly, TFBs showed highly bias toward Short Interspersed Elements (SINEs), which in turn they covering about 12% of the total of REs. However, the distribution pattern of TFBs-REs showed different bias in the intronic regions, spatially into the Introns 13 and 25. The rich array of SINE-TFBs and CR1-TFBs were situated within 5′UTR of the gene that may be an important driving force for regulatory innovation of the hFVIII gene. Taken together, these data may lead to revealing intronic regions with the capacity to renewing gene regulatory networks of the hFVIII gene. On the other hand, these correlations might provide the novel idea for a new hypothesis of molecular evolution of the FVIII gene, and treatment of Hemophilia A which should be considered in future studies.

268. Enhanced Gene Expression of Factor VIII by Ultrasound-Mediated Gene Delivery in Dogs

Previously we demonstrated that ultrasound (US)-mediated gene delivery (UMGD) can significantly enhance reporter gene transfer into the mouse and rat livers. This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy. Most significantly, we have achieved therapeutic levels of FVIII following UMGD into hemophilia A mice. Recently we have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine and swine. In order to facilitate the translation of this technology to treat hemophilia, we have recently treated 2 normal dogs with UMGD of FVIII plasmids using an open surgery procedure. Four mg of a high-expressing, liver-specific pBS-HCRHPI-FVIIIA plasmid and 3 ml of Definity® MBs in 8 ml total PBS solution were injected via the segmental portal vein branch with simultaneous exposure of the target liver lobe to therapeutic US (1.1 MHz frequency, 20 cycle pulses, 50 Hz pulse repetition frequency) for 4 minutes using the large diameter transducers. A sham-treated dog received an equivalent pGL4/MB dose, but was not exposed to tUS. We used an apodized dual element unfocused transducer H105 at 2.5Mpa peak negative pressure (PNP) in the first dog experiment and observed low levels of hFVIII gene expression in the treated liver lobes. The second dog experiment was performed using a cylindrically 6.2 MPa at the focal area. One day following treatment, the treated liver lobes were sectioned, and representative sections were fixed and stained for FVIII expression by histochemical staining using a polyclonal anti-FVIII antibody. Untreated normal dog liver and human liver were used as negative and positive controls. Significant hFVIII gene expression was obtained in both treated liver lobes with the expression levels slightly lower than control human liver lobe. Furthermore, fairly homogeneous distribution of FVIII gene expression was observed in hepatocytes. Next, we performed Western blot analysis to confirm if the staining is specific to human FVIII protein. A heavy chain band specific to hFVIII was observed in treated dog plasma and positive hFVIII control, but not in control normal dog plasma and pre-bleed plasma sample from the dog before treatment. Significant enhancement of FVIII-specific clotting activity was also obtained in the second treated dog compared to pre-treatment and the first treated dog. In addition, transaminase levels and histology analysis indicated minimal tissue damage in treated dog livers. Enhancement of FVIII gene expression by UMGD is currently evaluated in hemophilia dogs. Our results sugges that UMGD has great potential for therapeutic treatment of hemophilia A.

English

High-level expression of recombinant human blood coagulation factor in milk of farm animals at a large scale provides a powerful tool for protein production. However, a bottleneck in recent protein synthesis technologies is the high cost of current transgenic livestock system. Here, we report a simple, rapid, and low-cost protein production method based on a replication-defective adenoviral vector system. The recombinant hfVIII adenoviral vector was generated by using homologous recombination in bacteria and transfected the plasmids into the HEK293 packaging cell line. Goat’s mammary glands at the different physiological stage were infected with the recombinant adenovirus containing a human blood coagulation factor VIII gene. The expression level of bioactive hfVIII from milk sample was confirmed by Western blot analysis and ELISA methods. The hfVIII gene was expressed as a protein of about 26 kDa and no recombinant hfVIII protein was detected in negative control treatments. The hfVIII was expressed with a high activity level up to 1.4 U/ml in the milk of goats. These data suggest that the approach established here could be a low cost and further increase expression efficacy for the recombinant human blood coagulation factor and other biopharmaceutical interest. Key words: Adenovirus vector, human blood coagulation factor VIII, mammary gland, milk, goat, recombinant protein.

Improved Factor VIII Expression with the Introduction of a PACE-Furin Variant into Codon-Optimized Human Factor VIII

Adeno-associated viral (AAV) vector delivery of canine factor VIII (cFVIII) results in long-term dose dependent expression of FVIII in hemophilia A dogs for >8 years (Sabatino 2011). These data demonstrate that AAV-FVIII can significantly improve the disease phenotype. Interestingly, we found that cFVIII is inherently more stable than human FVIII (hFVIII), resulting in increased biological activity (Sabatino 2009). Thus, the AAV dose of cFVIII does not predict the efficacy of hFVIII and preclinical studies with hFVIII will be important for predicting the therapeutic dose of AAV-hFVIII. Furthermore, recent clinical trials for AAV-mediated gene transfer for hemophilia B have demonstrated sustained long-term expression of therapeutic levels of factor IX (FIX) but established that the AAV vector dose may be limiting due to anti-AAV immune responses to the AAV capsid. Therefore, our goal is to generate a novel hFVIII transgene cassette that increases hFVIII expression so that we can achieve therapeutic FVIII levels with a lower AAV dose. In our efforts to develop a hFVIII transgene cassette, we addressed this systematically by comparing different codon-optimized B-domain deleted (BDD) hFVIII genes, alternate promoters as well as hFVIII variants of the PACE-furin cleavage recognition site. We identified a novel codon-optimized hFVIII-BDD transgene (CO3) that expresses 15-fold higher than wild-type hFVIII-BDD. CO3 was introduced into a minimal expression cassette driven by the liver-specific transthyretin (TTR) promoter. At 4 weeks after delivery of AAV8-TTR-hFVIII-BDD and AAV8-TTR-hFVIII-CO3 (1x1011 vector genomes/mouse), the hFVIII expression in the peripheral blood based on ELISA was 35 ± 5 ng/ml and 492 ± 72 ng/ml, respectively. Using a modified transthyretin (TTRm) promoter (222bp), hFVIII expression further increased 2-fold compared to a wild type TTR promoter. In our previous work, we demonstrated that the R1645H substitution in the PACE-furin (P/F) cleavage recognition site (hFVIII-R1645H) confers increased stability and higher biological activity (Siner 2013). More recently, we have characterized a series of deletion variants at this P/F site in hFVIII with similar properties to R1645H. Therefore, in an effort to further enhance the efficacy of our hFVIII gene therapy approach, we introduced the R1645H or a 4 amino acid deletion (residues 1645-1648) of the P/F site (delP/F) into our hFVIII-CO3 transgene. Administration of AAV8-TTR-hFVIII-CO3, AAV8-TTR-hFVIII-CO3-R1645H and AAV8-TTR-hFVIII-CO3-delP/F in hemophilia A mice (1x1011vg/mouse) demonstrated that the hFVIII expression from R1645H (528 ± 30 ng/ml) was higher than CO3 (378 ± 75 ng/ml) at this vector dose. However, introduction of delP/F (TTR-hFVIII-CO3-delP/F) resulted in hFVIII expression that was 1.8-fold higher (685 ± 93 ng/ml)(p = 0.036) than CO3 alone. Importantly, the introduction of the deletion of the PACE-furin recognition site into the codon-optimized hFVIII resulted in an additive effect on the hFVIII expression. Delivery of AAV8-TTRm-hFVIII-CO3-delP/F using the modified TTR promoter resulted in 1018 ± 117 ng/ml hFVIII demonstrating that the TTRm promoter further enhances transgene expression. Notably, the AAV8-TTRm-hFVIII-CO3-delP/F construct results in hFVIII expression that is 30-fold higher than the wild type hFVIII-BDD. Our studies demonstrate that a combination of a liver-specific promoter, codon-optimization and modification of hFVIII at the PACE-furin recognition site (delP/F) significantly increases hFVIII expression in the setting of gene transfer. This work supports the feasibility of lowering the AAV vector dose for a gene-based therapeutic application for hemophilia A. DisclosuresElkouby:Spark Therapeutics: Research Funding. High:Bluebird Bio: Consultancy, Equity Ownership; Alnylam: Consultancy; Spark Therapeutics: Consultancy, Equity Ownership. Sabatino:Spark Therapeutics: Research Funding; Pfizer, Inc.: Research Funding.

Quality of rabbit vitrified/thawed transgenic embryos

The aim of our study was to investigate the influence of vitrification on developmental rate and quality (total number of cells, number of blastomeres in inner cell mass (ICM) area, apoptotic index and embryo diameter) of transgenic (carrying an endogenous-hFVIII or exogenous-enhanced green fluorescent protein (EGFP) gene) rabbit embryos. EGFP-positive rabbit embryos were produced under in vitro conditions by the microinjection of foreign genes into the pronucleus of fertilized eggs. The transgenic rabbit embryos with the hFVIII gene were produced by mating homozygous transgenic rabbits and flushing at the single-cell stage. Developmental rate of vitrified/thawed transgenic embryos that reached hatching blastocyst stage (68.00% and 69.00%) and differed significantly (p < 0.001) from those in control embryos (100.00%). Significant difference (p < 0.05) was found in total cell counts between control (117.00 ± 36.00) and vitrified (141.00 ± 34.80) hFVIII-positive embryos. The higher proportion of ICM cells (32.00%) and greatest embryo diameter (130.85 ± 10.90) were found in the control group compared with the transgenic. Ratio of apoptotic cells was significantly higher (p < 0.01) in the control group (2.50%) and vitrified EGFP-positive embryos (2.90%) compared with the vitrified, hFVIII-positive group of embryos (0.70%). Our results demonstrate that neither gene microinjection itself, nor exogenous (EGFP) and endogenous (hFVIII) gene expression interferes with developmental rate and quality of rabbit embryos. However, a combination of microinjection and vitrification significantly decreases (p < 0.001) the survival rate of rabbit embryos.

A Gene Transfer Approach towards Hemophilia A Correction Using the Piggybac Transposon Vector.

AbstractAbstract 1477Human Factor VIII (hFVIII) deficiency offers advantages as a disease target for gene therapy as small increases in factor VIII levels will alter the bleeding phenotype. In addition, both mouse and dog models of the disease are available for preclinical studies. Nonviral DNA transposons are genetic elements consisting of inverted terminal DNA repeats which in their naturally occurring configuration flank a transposase coding sequence. The transposase follows a “cut and paste” mechanism to excise the transposon from its original genomic location and insert it into a new locus. The insect derived piggyBac (PB) can be engineered to carry a therapeutic transgene between the inverted terminal repeats. Wu et al and others reported that piggyBac transposase is highly efficient at catalyzing transposition in mammalian cells in vitro (PNAS 103: 15008–15013, 2006). Advantages of this novel nonviral vector system include a large transgene cassette capacity and ease of production and purification. We hypothesize that a PB transposon vector carrying a reporter gene cassette or the human FVIII cDNA along with a codon-optimized (co-) or hyperactive (hyp-) transposase will confer persistent gene expression and correction of the hemophilia A bleeding phenotype with the FVIII cDNA. PB transposons were engineered to carry a puromycin resistance gene (PB puro), a human alpha1 antitrypsin reporter (PB hAAT), or hFVIII gene (B domain deleted or a partial B domain-226 amino acids/N6). We evaluated co- and hyp-transposase-mediated transposition in the Huh-7 human hepatoma cell line to verify function in hepatocytes. Using the PB puro vector, we demonstrated that the hyp-transposase generated a 2 fold higher transposition efficiency than the co-transposase in hepatocytes. We investigated the impact of varying the ratio of transposon to transposase; we screened ratios of 5:1, 2:1, 1:1, 1:2, and 1:5 in the Huh-7 cell line. Overall, the 1:2 and 1:1 ratios gave the greatest transposition efficiency in vitro. We evaluated the in vivo gene transfer efficiency in mice by hydrodynamic tail-vein injection using PB hAAT driven by the murine albumin enhancer/human alpha anti-trypsin promoter. Either a low (5 micrograms transposon) or high (25 micrograms transposon) dose was given with varying amounts of hyp-transposase to generate an in vivo dose response curve. Serum hAAT levels were measured prior to injection and then monthly for 3 months. Results revealed the 1:1 ratio at the high transposon dose generated higher level of expression compared to all other doses with expression stable in all groups for 3 months. PB vectors encoding hFVIII have been prepared, and our studies with these vectors are ongoing. These data show that the PB vector can be used to deliver transgene expression to the liver and achieve long term expression of a secreted protein. Disclosures:Staber:Bayer Healthcare: Research Funding.

Reduction of inhibitor titres by infusion of FVIII gene transduced tolerogenic dendritic cells in haemophilic mice

Background: The development of anti‐factor VIII (FVIII) inhibitory antibodies is a major complication of FVIII replacement therapy in the management of patients with haemophilia A. Evidence is accumulating that tolerogenic dendritic cells (tDCs) generated in vitro can induce regulatory T cells and promote durable antigen‐specific tolerance in vivo.Objective: In this study we used human FVIII transgene expressing tDCs to reduce inhibitor formation in haemophilic mice.Methods and results: The tDCs were generated by cell sorting the CD11clowCD45RBhigh population of cells resulting from culture of lineage negative bone marrow cells in media supplemented with IL‐10 and the neural peptides VIP and PACAP38. Expression of co‐stimulatory molecules CD40, CD80 and CD86 and MHC Class II was negative or low on these cells and they remain unactivated even after stimulation with lipopolysaccharide (LPS) or transduction by foamy virus (FV) vectors. Following transduction by a FV vector expressing a B domain deleted human FVIII transgene, 3–5 × 105 tDCs were infused by tail vein injection into Bal/c haemophilic mice (tDC‐F8) weekly for two doses. Real‐time PCR showed that the transduction efficiency of our FV vector in this population of tDCs was approximately 70%. Subsequently, mice were challenged with four weekly intravenous doses of 0.2 μg rhFVIII. Mice that received no cells (Neg‐Ctrl) and mice that received an equivalent number of non‐transduced tDCs (tDC‐Ctrl) were used as controls. After immunization with rhFVIII, the spleen size, weight and total cell numbers in tDC‐F8 mice were consistently lower than in Neg‐Ctrl and tDC‐Ctrl mice by approximately 50%. Flow cytometry assessment of CD4+ splenocytes showed there were an increased number of cells expressing the regulatory T‐cell markers FOXP3, CD25, CD103, CTLA4 and GITR in tDC‐F8 treated mice. We observed a 60% and 61% reduction in the level of inhibitor titres from tDC‐F8 mice compared with Neg‐Ctrl and tDC‐Ctrl mice. Splenic CD4+ T cell proliferation in response to FVIII stimulation in cells from tDC‐F8 mice was suppressed by approximately 90% compared with proliferation of cells from Neg‐Ctrl and tDC‐Ctrl mice. We also showed that pre‐immunized mice treated with four infusions of hFVIII FV vector transduced tDCs had a reduction in their inhibitor titres by 54% (P &lt; 0.05). No significant change in inhibitor titres were seen in untreated controls or mice given four doses of untransduced tDCs.Summary: These data indicate that hFVIII gene transduced tolerogenic DCs are useful in decreasing inhibitory antibodies in haemophilic mice. More in vivo studies are in progress to confirm the antigen‐specificity and durability of these effects. Our future studies will focus on isolating and characterizing the regulatory T cell populations induced by in vivo administration of transgene modified tDCs.

Immunomodulation of transgene responses following naked DNA transfer of human factor VIII into hemophilia A mice

A robust humoral immune response against human factor VIII (hFVIII) following naked DNA transfer into immunocompetent hemophilia A mice completely inhibits circulating FVIII activity despite initial high-level hFVIII gene expression. To prevent this undesirable response, we compared transient immunomodulation strategies. Eight groups of mice (n = 4-9 per group) were treated with naked DNA transfer of pBS-HCRHPI-hFVIIIA simultaneously with immunosuppressive reagents that included cyclosporine A (CSA), rapamycin (RAP), mycophenylate mofetil (MMF), a combination of CSA and MMF, a combination of RAP and MMF, a monoclonal antibody against murine CD40 ligand (MR1), recombinant murine Ctla4Ig, and a combination of MR1 and Ctla4Ig. All animals except those receiving only CSA exhibited delayed or absent immune responses against hFVIII. The most effective immunosuppressive regimen, the combination of Ctla4Ig and MR1, prevented inhibitor formation in 8 of 9 animals; the ninth had transient low-titer antibodies. All 9 mice of this group produced persistent, therapeutic levels of hFVIII for more than 6 months. When challenged with the T-dependent antigen bacteriophage Phix174, tolerized mice exhibited normal primary and secondary antibody responses, suggesting that transient immunomodulation to disrupt B/T-cell interaction at the time of plasmid injection effectively promoted long-term immune tolerance specific for hFVIII.

Efficient expression of a transgene in platelets using simian immunodeficiency virus‐based vector harboring glycoprotein Ibα promoter:in vivomodel for platelet‐targeting gene therapy

Platelets release several mediators that modify vascular integrity and hemostasis. In the present study, we developed a technique for efficient transgene expression in platelets in vivo and examined whether this targeted-gene-product delivery system using a platelet release reaction could be exploited for clinical applications. Analysis of luciferase reporter gene constructs driven by platelet-specific promoters (the GPIIb, GPIbalpha, and GPVI) revealed that the GPIbalpha promoter was the most potent in the megakaryoblastic cell line UT-7/TPO and human CD34+-derived megakaryocytes. Transduction of UT-7/TPO; CD34+-derived megakaryocytes; and c-Kit+, ScaI+, and Lineage- (KSL) murine hematopoietic stem cells with a simian immunodeficiency virus (SIV)-based lentiviral vector carrying eGFP resulted in efficient, dose-dependent expression of eGFP, and the GPIbalpha promoter seemed to bestow megakaryocytic-specific expression. Transplantation of KSL cells transduced with SIV vector containing eGFP into mice showed that there was preferable expression of eGFP in platelets driven by the GPIbalpha promoter [7-11% for the cytomeglovirus (CMV) promoter, 16-27% for the GPIbalpha promoter]. Furthermore, transplantation of ex vivo-transduced KSL cells by SIV vector carrying human factorVIII (hFVIII) driven by the GPIbalpha promoter induced the production of detectable transcripts of the hFVIII gene and the hFVIII antigen in bone marrow and spleen for at least 90 days and partially corrected the hemophilia A phenotype. Platelet-targeting gene therapy using SIV vectors appears to be promising for gene therapy approaches toward not only inherited platelet diseases but also other hemorrhagic disorders such as hemophilia A.

859. Cats Produce a CTL Response That Reduces Expression of Human Factor IX and Human Factor VIII after Neonatal Gene Transfer and Represent an Important Model for Translation into Humans

Gene therapy has corrected bleeding manifestations of hemophilia in small and large animals. However, immune responses can abrogate an effect. We previously demonstrated that neonatal gene therapy could induce tolerance to human Factor IX (hFIX) in mice and dogs. However, this approach may not be effective in humans, which probably have a more mature immune system at birth. Immune responses can involve antibody or cytotoxic T lymphocyte (CTL) responses. Antibody formation has been the major problem in animal models that have been evaluated to date. However, CTL responses could also preclude long-term correction of disease. Indeed, a recent study in humans achieved therapeutic expression of hFIX after liver-directed AAV-mediated gene therapy, but levels fell at 1 month due to a CTL response against AAV capsid proteins. We recently demonstrated that cats with mucopolysaccaridosis I mount a potent CTL response to |[alpha]|-L-iduronidase after neonatal gene therapy. We reasoned that cats might be an excellent model to test immune responses to coagulation factors after neonatal gene transfer. These studies used retroviral vectors (RV) expressing hFIX or human Factor VIII (hFVIII), which have differences at |[sim]|69 and |[sim]|212 amino acids, respectively, from the feline proteins. These results might predict responses in animals with null mutations, which represent |[sim]|1% and 20% of patients with hemophilia B and A, respectively. Newborn cats were injected IV with 4x109 transducing units (TU)/kg of hAAT- hFIX-WPRE, which expresses hFIX from the human |[alpha]|1-antitrypsin promoter. This vector does not shut off in vivo, as it directed stable expression of feline 4-sulfatase for over 1 year in cats. Cats achieved 118+/-29 ng/ml (2% of normal) of hFIX in plasma at 1 month. Although expression was stable in 3 of 4 cats, one lost expression at 4.5 months, which was associated with very low levels of RV RNA in the liver, suggesting that a CTL response had occurred. Anti- hFIX antibody levels were very low. Other cats were injected IV with 9x108 or 7x109 TU/kg of hAAT-hFVIII-WPRE at 2 to 3 days after birth, which expresses a B domain-deleted hFVIII. The low dose resulted in 31+/-9 ng/ml of hFVIII in plasma (16% of normal), but expression fell at 4.5 months. Two of 4 cats developed anti- hFVIII antibodies in an ELISA-based assay. All cats had extremely low RV RNA levels in liver, suggesting that a CTL response eradicated expressing cells. Cats that received the high dose RV achieved 125+/-54 ng/ml (63% normal). Although none developed anti-hFVIII antibodies, 2 of 3 lost most activity at 6 months. These data suggest that 25% of cats that received the hFIX gene, and 86% of cats that received the hFVIII gene as newborns developed a CTL response. Future experiments will attempt to block this CTL response with CTLA4-Ig, which was effective in MPS I cats that received neonatal gene therapy. The cat may be an invaluable preclincal model to test gene therapy approaches for hemophilia.

Immunomodulation of Transgene Responses Following Naked DNA Transfer of Factor VIII into Hemophilia A Mice.

Naked DNA transfer of liver-specific, high-expressing plasmid pBS-HCRHPI-FVIIIA in Rag2(−/ −) SCID mice produced persistent high-level gene expression of human factor VIII (hFVIII) (Miao, Hum. Gene Ther. 2003). However, in immunocompetent hemophilia A mice, a robust humoral immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity (Ye, Mol. Ther. 2004). Transient immunomodulation strategies were explored to prevent the formation of inhibitory antibody formation. Eight groups of mice (n=8) were treated by naked DNA transfer of plasmid pBS-HCRHPI-FVIIIA. Each group were subjected to treatment with single or combined immunosuppressive regimen: CyclosporineA (CSA) daily for 14 days; Rapamycin daily for 14 days; Mycophenylate mofetil (MMF) daily for 14 days; combination of CSA and MMF; combination of Rapamycin and MMF; a monoclonal antibody (MR1) against murine CD40 ligand on days -1, 1, 2, 7, & 14; recombinant murine Ctla4Ig on days 1 & 2; and combination of MR1 and Ctla4Ig. Combination regimens were given using the same combined schedule and dosages. All animals treated with immunosuppression had delayed or no immune responses against hFVIII except the group treated with CSA only. The most effective treatment was observed in animals treated with the combination of Ctla4Ig and MR1. Seven of 8 animals failed to develop detectable inhibitors. One animal developed transient low-titer antibodies. This group of animals produced persistent, therapeutic levels of hFVIII gene expression for over 6 months. Tolerized animals were subsequently challenged by the T dependent antigen, bacteriophage Φx174, and exhibited a normal primary and secondary response including amplification and isotype switch. These results strongly suggest that transient immunomodulation strategies to disrupt B- and T- cell interactions at the time of plasmid injection is effective to promote long-term immune tolerance that is specific for FVIII without altering subsequent immune responses to other T cell dependent antigens.

Sustained transgene expression by human cord blood derived CD34+ cells transduced with simian immunodeficiency virus agmTYO1-based vectors carrying the human coagulation factor VIII gene in NOD/SCID mice.

Gene therapy is being studied as the next generation therapy for hemophilia and several clinical trials have been carried out, albeit with limited success. To explore the possibility of utilizing autologous bone marrow transplantation of genetically modified hematopoietic stem cells for hemophilia gene therapy, we investigated the efficacy of genetically engineered CD34+ cell transplantation to NOD/SCID mice for expression of human factor VIII (hFVIII). CD34+ cells were transduced with a simian immunodeficiency virus agmTYO1 (SIV)-based lentiviral vector carrying the enhanced green fluorescent protein (eGFP) gene (SIVeGFP) or the hFVIII gene (SIVhFVIII). CD34+ cells transduced with SIV vectors were transplanted to NOD/SCID mice. Engraftment of transduced CD34+ cells and expression of transgenes were studied. We could efficiently transduce CD34+ cells using the SIVeGFP vector in a dose-dependent manner, reaching a maximum (99.6 +/- 0.1%) at MOI of 5 x 10(3) vector genome/cell. After transducing CD34+ cells with SIVhFVIII, hFVIII was produced (274.3 +/- 20.1 ng) from 10(6) CD34+ cells during 24 h in vitro incubation. Transplantation of SIVhFVIII-transduced CD34+ cells (5-10 x 10(5)) at a multiplicity of infection (MOI) of 50 vector genome/cell into NOD/SCID mice resulted in successful engraftment of CD34+ cells and production of hFVIII (minimum 1.2 +/- 0.9 ng/mL, maximum 3.6 +/- 0.8 ng/mL) for at least 60 days in vivo. Transcripts of the hFVIII gene and the hFVIII antigen were also detected in the murine bone marrow cells. Transplantation of ex vivo transduced hematopoietic stem cells by non-pathogenic SIVhFVIII without exposure of subjects to viral vectors is safe and potentially applicable for gene therapy of hemophilia A patients.

158. Transient Immunosuppresion by CTLA4Ig and Anti-CD40L Promotes Long-Term Tolerance Specifically Against HFVIII in Hemophilia A Mice Following Naked DNA Transfer

Persistent high-level gene expression of human factor VIII (hFVIII) has been achieved by naked DNA transfer of a liver-specific, highexpressing plasmid pBS-HCRHPI-FVIIIA in Rag2(-/-) SCID mice (Miao et al. (2003) Hum. Gene Ther. 14, 1297-1305). However, in immunocompetent hemophilia A mice, a robust immune response against FVIII that followed gene transfer led to complete inhibition of circulating FVIII activity. This immune response was shown to be mediated by antibodies against the transgene product. In this study, transient immunomodulation strategies to disrupt B- and Tcell interactions at the time of plasmid injection were tested to prevent formation of inhibitory antibodies against hFVIII. Three groups of mice (n=8) were treated with plasmid pBS-HCRHPIFVIIIA; group 1 was injected on days -1, 1, 2, 7, and 14 with a monoclonal antibody (MR1) against murine CD40 ligand; group 2 was treated on days 1 and 2 with recombinant murine CTLA4Ig; and group 3 received a combination of MR1 and CTLA4Ig using the same combined schedule and dosages. All animals treated with immunosuppression had delayed immune responses against hFVIII. However, the 8 animals treated with CTLA4Ig alone developed inhibitors 3-6 weeks post-injection. Six of 8 animals treated with MR1 developed inhibitors at 4-10 weeks; the other 2 did not develop inhibitors for up to 3 months. Most notably, 7 of 8 animals treated with the combination regimen of MR1 and CTLA4Ig failed to develop detectable inhibitors for at least 3 months. One animal developed a low titer of inhibitory antibodies that only partially blocked the functional activity of hFVIII in its plasma. All 7 animals that did not develop inhibitory antibodies against hFVIII exhibited persistent, supra-physiologic therapeutic levels of hFVIII gene expression. To evaluate whether immuno-modulation led to long-term defects in host immune function, two animals treated with the combination protocol, and two control animals not receiving immune modulatory therapy were then challenged twice 4 weeks apart with the neoantigen, bacteriophage Φ×174 (2×108 PFU/each challenge). One treated animal exhibited a normal primary and secondary response to bacteriophage. The second animal exhibited a slightly attenuated primary response and a normal response after the 2nd challenge, indicating that this transient immunosuppressive regimen did not induce a lasting immunodeficiency. These results strongly suggest that transient inhibition of co-stimulatory molecules in hFVIII plasmid-treated hemophilia A mice can promote long-term immune tolerance, specific for hFVIII without altering subsequent immune responses to T cell dependent neoantigens.

Sustained high-level expression of full-length human factor VIII and restoration of clotting activity in hemophilic mice using a minimal adenovirus vector

The successful prophylactic treatment of hemophilia A by frequent infusions of plasma concentrates or recombinant factor VIII (hFVIII) indicates that gene therapy may be a potential alternative for the treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all viral genes and carries the full-length hFVIII cDNA under the control of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was propagated with the assistance of an ancillary vector in 293 cells and was purified by CsCl banding. Sustained expression of hFVIII at physiologic levels (100-800 ng/mL) was achieved in mice after a single intravenous injection of MiniAdFVIII. The expressed hFVIII had a structure identical to that of recombinant hFVIII, as determined by Western blot analysis. The functionality of the protein was confirmed by the restoration of blood coagulation capacity in MiniAdFVIII-treated hemophilic mice, as determined by tail clipping observations. Although antivector or antihuman FVIII antibodies at various levels were detected, long-term expression of the transgene was observed in the mice that did not generate antibodies against the transgene product. The vector DNA persisted in the liver tissues of the mice with long-term expression. No significant histopathologic findings or toxicities were observed to be associated with the vector in the MiniAdFVIII-treated C57BL/6 mice. These results support the further development of MiniAdFVIII for clinical trials toward the treatment of hemophilia A.