Sustained Factor VIII Expression Research Articles

Inhibiting Activated Protein C Cleavage of Factor VIII for Hemophilia a Gene Therapy

Background Despite progress in adeno-associated virus (AAV) mediated factor VIII (FVIII) gene transfer for hemophilia A (HA), durable and sustained FVIII expression sufficient to ameliorate bleeding in all participants is an unrealized goal. Following the success of using the gain of function FIX-Padua variant in hemophilia B gene therapy, we hypothesized that use of an enhanced hemostatic function FVIII would similarly improve HA gene therapy by: 1) permitting use of lower vector doses, thereby overcoming dose-dependent safety and efficacy limitations and 2) achieving efficacy with lower FVIII antigen. We previously published a FVIII variant resistant to activated protein C (APC) cleavage (FVIII-R336Q/R562Q or FVIII-QQ) with normal specific activity and 4-5 fold improved hemostatic function in recombinant protein experiments (Wilhelm et al. Blood 2021). Aims We evaluated the possible clinical translation of FVIII-QQ for HA AAV-mediated gene therapy by determining the hemostatic benefit and potential prothrombotic and immunological risks in mice. Additionally, FVIII-QQ steady-state expression was 2.5 to 5-fold higher than FVIII-WT. We hypothesized this was due to disrupted cleavage by APC or other serine proteases at R336 and/or R562. Results Survival studies in HA/CD4KO and WT/CD4KO mice treated with AAV vectors to express (h)uman FVIII-WT and hFVIII-QQ in the range of mild HA, normal, and supratherapeutic FVIII levels thus far demonstrate no significant differences in survival at 7-11 month follow up or D-dimer levels after 8 months. Additionally, FVIII-QQ mice were generated using CRISPR/Cas9 to introduce R562Q (WT mice are Q336) for endogenous (m)ouse FVIII-QQ expression. Endogenous mFVIII-QQ mice were viable, fertile, normal weight, and D-dimers did not significantly differ from WT mice, while FVIII activity was ~2-fold higher than WT mice. HA immune competent mice treated with either 6 weekly doses of hFVIII-WT or hFVIII-QQ protein (0.2 μg) or AAV vector to express hFVIII-QQ or hFVIII-WT had no significant differences in inhibitor titers. To evaluate hemostasis, HA/CD4KO mice were treated with AAV vector only differing by 2 amino acids (R336, R362) at 3 different doses (n=6-12 mice/cohort) to express hFVIII-QQ or hFVIII-WT and underwent tail clip challenge. Estimated EC50 and EC80 of vector doses required to normalize blood loss to WT mice were 5 and 10-fold lower, respectively, for hFVIII-QQ versus hFVIII-WT supporting a hemostatic benefit to using the hFVIII-QQ transgene. Interestingly, across 3 different vector constructs differing only by F8 codon optimization, we observed hFVIII-QQ antigen values 2.5 to 5-fold higher than in mice expressing hFVIII-WT. Analysis of liver tissue from HA/CD4KO mice treated with the same vector construct and dose to express hFVIII-WT or hFVIII-QQ demonstrated approximately the same mRNA values, but plasma hFVIII-QQ antigen was 4-fold higher than hFVIII-WT and intracellular hFVIII-QQ antigen was the same or lower than hFVIII-WT antigen. Consistent with our in vivo AAV experiments, preliminary studies of intracellular versus secreted hFVIII antigen in BHK stable cell lines expressing hFVIII-WT or hFVIII-QQ demonstrated similar mRNA values with higher relative secreted hFVIII-QQ protein. To further probe the mechanism of higher hFVIII-QQ steady-state expression, we evaluated a panel of serine proteases (APC, FIXa, FXa, plasmin, PACE/furin and thrombin) for cleavage of FVIII-QQ relative to FVIII-WT protein. As expected, hFVIII-QQ was resistant to APC cleavage. Surprisingly, all other serine proteases tested demonstrated cleavage of hFVIII-WT, but not hFVIII-QQ, at R336 and/or R562 suggesting protease attack at these cleavage sites may occur more than previously appreciated, though the physiologic significance remains to be determined. Conclusion Our studies outline the use of hFVIII-QQ transgene expression thus far does not demonstrate safety concerns and confers hemostatic benefit over hFVIII-WT via: 1) improved hemostatic function and 2) higher steady-state expression. Enhanced steady state hFVIII-QQ expression relative to hFVIII-WT was observed across multiple model systems. Preliminary data demonstrate multiple serine proteases can cleave at the described FVIII APC cleavage sites (R336, R562) suggesting hFVIII-QQ may generally be more proteolytically stable thereby permitting improved steady-state expression.

The Effects of Immunomodulation with Corticosteroids to Manage an AAV Capsid Immune response in the Phase I/II Study of SPK-8011

Background: Hemophilia A (HA) is an inherited blood disorder caused by deficiency or dysfunction of coagulation factor VIII (FVIII). SPK-8011 is an adeno-associated viral (AAV) vector gene therapy targeting hepatocyte expression of FVIII, aiming to provide durable FVIII expression and long-term bleed prevention. A Phase I/II trial of SPK-8011 (NCT03003533/NCT03432520) has demonstrated multiyear sustained FVIII expression and notably reduced bleeding in people with HA, with no major safety concerns. There are challenges around preventing and managing cellular immune responses to transduced hepatocytes. Here, we present the outcomes of participants who received corticosteroids following vector administration to prevent and/or treat a presumed AAV capsid immune response. Methods: In this open-label, multicenter, non-randomized, dose-escalation trial, people with HA were infused with SPK-8011 at one of four doses ranging from 5×1011 to 2×1012 vg/kg (full methods published in George et al. New Engl J Med 2021). Participants had baseline FVIII activity of ≤2% of normal levels and were negative for neutralizing antibodies to SPK200. Participants were monitored for 52 weeks after vector administration and enrolled in a 4-year long-term follow-up trial. Corticosteroids were used reactively at the investigators’ discretion upon a presumed capsid immune response, i.e., decrease in FVIII expression, increase in alanine aminotransferase (ALT) level, and/or positive peripheral blood mononuclear cells using interferon-γ enzyme-linked immunospot (ELISpot) to SPK200 peptides. Following loss of FVIII expression in two participants, a prophylactic regimen was explored. Assessments included duration of corticosteroid use and associated adverse events (AEs). Results: Twenty-three males with HA were dosed with SPK-8011 and 17 received corticosteroids (12 reactively, 5 prophylactically); five did not have evidence of a capsid immune response, and one remains in the window of a capsid immune response in the follow-up period (data cut-off: 30 Jun 2022). The median (range) time to initiation of reactive corticosteroids was 9.3 (4.3-12.1) weeks post vector infusion in the 12 participants, with a median (range) duration of treatment of 8.5 (2-18) weeks. Two of these 12 participants completely lost FVIII expression following a presumed capsid immune response (one initiated emicizumab; one resumed FVIII prophylaxis); FVIII expression was maintained in the remaining 10 participants. Corticosteroids were administered prophylactically (2-4 weeks post vector) in five participants, with transgene expression maintained in all five. In four of these five participants, length of exposure was prolonged (31-49 weeks), with attempted corticosteroid tapering resulting in decreased FVIII expression, increased ALT and/or positive ELISpot assays outside the typical window of capsid immune responses. Four participants had AEs related to corticosteroid therapy (Table), with weight gain (8.7%; n=2) and insomnia (8.7%; n=2) being the most common. The occurrence of AEs attributed to corticosteroids increased with duration of use, particularly in those with exposure of >30 weeks. Prophylactic corticosteroids did not consistently prevent a presumed capsid immune response in these five participants and four experienced prolonged courses of corticosteroids necessitating steroid-sparing agents (Figure). All five participants who received prophylactic corticosteroids and four participants who received reactive corticosteroids experienced a recurrence of one or more laboratory trigger(s) potentially linked to a presumed capsid immune response during their corticosteroid taper. Subsequent increased corticosteroid use did not consistently resolve these laboratory changes. Conclusions: Overcoming the challenge of capsid immune responses to achieve long-term stable and predictable FVIII expression remains a goal of AAV gene transfer for HA. Reactive oral corticosteroids were well tolerated but did not prevent loss of FVIII expression in all participants. Early prophylactic corticosteroids did not consistently prevent presumed capsid immune responses and resulted in prolonged immunomodulatory courses with associated AEs. Data so far have permitted refinement of laboratory triggers and investigation is ongoing to identify an alternative, safe and optimal immunomodulatory regimen. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

First-in-Human Dose-Finding Study of AAVhu37 Vector-Based Gene Therapy: BAY 2599023 Has Stable and Sustained Expression of FVIII over 2 Years

▪BackgroundGene therapy for hemophilia A has the potential to reduce the treatment burden for patients and their care providers by eliminating the need for regular factor VIII (FVIII) prophylaxis through long-term expression of endogenous FVIII at levels sufficient to provide bleed protection. Ongoing phase 3 gene therapy trials for hemophilia A show promise but can result in unpredictable FVIII expression of uncertain durability. Gene therapy must evolve to meet patient expectations of a durable, efficacious and safe treatment.BAY 2599023 (AAVhu37.hFVIIIco) is the first, clinical stage adeno-associated virus (AAV) gene therapy vector, based on the AAVhu37 serotype. BAY 2599023 is a non-replicating AAV vector and contains a single-stranded DNA genome encoding a B-domain-deleted FVIII, under the control of a liver-specific promoter/enhancer combination, optimized for transgenic expression. The AAVhu37 capsid is a member of the hepatotropic clade E family and was selected based on preclinical studies demonstrating efficient, liver-directed FVIII gene transfer, favorable biodistribution and durable FVIII expression. Here, we report safety and FVIII activity levels achieved to date in this first-in-human, dose-finding study of BAY 2599023.MethodsThe ongoing BAY 2599023 phase 1/2, open-label, dose-finding study (NCT03588299) included male patients aged ≥18 years with severe hemophilia A, no history of FVIII inhibitors, no detectable neutralizing immunity against AAVhu37 (neutralizing antibody titer ≤5), and ≥150 exposure days to FVIII products. Patients received a single intravenous infusion of BAY 2599023 and were enrolled sequentially into three dose cohorts (0.5 × 10 13 GC/kg, 1.0 × 10 13 GC/kg and 2.0 × 10 13 GC/kg), each comprising at least two patients. Patients to be enrolled in a fourth cohort will receive a single infusion of 4 × 10 13 GC/kg (Figure 1). Primary endpoints were adverse events (AEs), serious AEs (SAEs) and AEs/SAEs of special interest (S/AESIs). The secondary endpoint was FVIII activity over time. Informed patient consent and ethics committee approval were obtained.ResultsThree cohorts of ≥2 patients each (N = 9) were enrolled sequentially (Figure 1). At the data cutoff (May 2021), FVIII activity data were available for the first eight patients.BAY 2599023 delivered sustained FVIII expression levels for up to >23 months, with evidence of bleed protection. Patients in Cohorts 2 and 3 have all been off prophylaxis with FVIII products since approximately 6-12 weeks after gene transfer. To date, it has been observed that no spontaneous bleeds requiring treatment have been reported once FVIII levels >11 IU/dL were achieved. Of the 9 patients treated, 5 patients developed an AESI: mild/moderate alanine aminotransferase (ALT) elevations observed in Cohort 2 (n =1) and Cohort 3 (n = 3) were managed with corticosteroid treatment; another ALT elevation was reported as study-drug-related SAE in Cohort 3 (n = 1) but returned to normal a few weeks after interruption of the H2 blocker famotidine. The latest follow-up data for up to 28 months will be presented.ConclusionsBAY 2599023 was designed to enhance efficacy and durability of FVIII expression with a favorable safety profile. Sustained FVIII levels allowed suspension of FVIII prophylaxis in the majority of patients, with asymptomatic ALT elevations that respondedto corticosteroids, making BAY 2599023 a key candidate in the evolution of gene therapy in hemophilia A. [Display omitted] DisclosuresPipe: Genventiv: Consultancy; Regeneron/ Intellia: Consultancy; uniQure: Consultancy, Other; Spark Therapeutics: Consultancy; Takeda: Consultancy; Sanofi: Consultancy, Other; Sangamo Therapeutics: Consultancy; Roche/Genentech: Consultancy, Other; Pfizer: Consultancy; Novo Nordisk: Consultancy; Freeline: Consultancy, Other: Clinical trial investigator; HEMA Biologics: Consultancy; CSL Behring: Consultancy; Catalyst Biosciences: Consultancy; Biomarin: Consultancy, Other: Clinical trial investigator; Bayer: Consultancy; ASC Therapeutics: Consultancy; Apcintex: Consultancy; Grifols: Consultancy; Octapharma: Consultancy; Shire: Consultancy. Sheehan: BioMarin: Consultancy, Research Funding; Roche: Consultancy, Research Funding; Bayer: Consultancy, Research Funding. Coppens: Portola/Alexion: Research Funding; CSL Behring: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Roche: Research Funding; Daiichi Sankyo: Research Funding; Sanquin Blood Supply: Research Funding; uniQure: Research Funding; Medcon International: Consultancy; MEDtalks: Consultancy; Novo Nordisk: Consultancy; Pfizer: Consultancy; Sobi: Consultancy. Eichler: Takeda: Consultancy, Honoraria; BioMarin: Consultancy, Research Funding; Roche: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria; Pfizer: Research Funding; Novo Nordisk: Consultancy, Research Funding; Biotest: Consultancy, Honoraria; Bayer: Consultancy, Research Funding. Linardi: Bayer: Current Employment. Wiegmann: Bayer: Current Employment. Hay: Pfizer: Consultancy, Research Funding; Inspiration: Consultancy, Honoraria; BioMarin: Consultancy, Honoraria; Roche: Consultancy, Research Funding; Bayer: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Spark Therapeutics: Consultancy, Research Funding. Lissitchkov: Bayer: Other: Principal Investigator of Clinical Trials; Catalist: Other: Principal Investigator of Clinical Trials; Grifols: Other: Principal Investigator of Clinical Trials.

Long-Term AAV-Mediated Factor VIII Expression in Nine Hemophilia A Dogs: A 10 Year Follow-up Analysis on Durability, Safety and Vector Integration

Hemophilia is an X-linked bleeding disorder caused by a deficiency in clotting factor VIII (FVIII)(hemophilia A, HA) or factor IX (FIX)(hemophilia B, HB). While early clinical trials of AAV delivery of FIX for HB have demonstrated stable FIX expression for >8 years, an ongoing clinical trial of AAV-FVIII delivery for HA achieved high levels of transgene expression that unexpectedly declined after 1 year. Here we describe preclinical studies of AAV-canine FVIII (cFVIII) delivery in nine HA dogs with sustained FVIII expression for the duration of the study, as long as 10 years. FVIII was delivered using two delivery approaches: (1) co-administration of two AAV vectors encoding separate cFVIII heavy and light chains driven by the thyroxine binding globulin (TBG) promoter (Two chain approach)(TC) (n=5) at two AAV doses (2.5 x 1013vg/kg; F24, Woodstock, J60) and (1.2 x 1013vg/kg; Linus, H19) or (2) delivery of cFVIII as a single chain driven by the human alpha-1 anti-trypsin (hAAT) promoter (Single chain approach)(SC)(n=4) at two AAV doses (4 x 1013 vg/kg; M50, M06) and (2 x 1013vg/kg; M66, L51) (Sabatino 2011). We demonstrated that both strategies were efficacious; preventing >95% of spontaneous bleeding episodes without toxicity. We now report the long-term follow-up of between 2.2 and 10.1 years for these treated dogs. Dose-dependent cFVIII:C (Coatest SP4 FVIII) was observed. At the final time point, the cFVIII:C was 2.7% (F24), 7.1% (Woodstock), 4.5% (J60), 11.3% (Linus) and 2.5% (H19) for TC dogs. For the SC dogs, the cFVIII:C was 9.4% (M06), 10.3% (M50), 1.9% (L51) and 3.7% (M66). Stable FVIII expression was maintained for seven of the dogs over the course of the study. Two dogs (Linus, M50) had a gradual increase in FVIII:C that began about three years after vector administration and continued for an additional seven years (Linus) and four years (M50), until the termination of the study. Liver function tests, serum alpha-fetoprotein concentrations, fibrinogen levels as well as liver pathology did not suggest altered liver function or tumor development in Linus and M50 compared to the other dogs. Clinically, there was no evidence of malignancy and no tumors were detected at the time of necropsy in any dog. One of the safety concerns for AAV-mediated gene therapy approaches is the potential for AAV integration events to be genotoxic and lead to tumorigenesis. While recombinant AAV primarily remains as an episome, integration events have been observed in mouse models and hepatocellular carcinoma has been observed after neonatal delivery of AAV vectors. In addition, the increase in FVIII expression in Linus and M50 prompted us to investigate integration and clonal expansion as a potential mechanism for these observations. Vector copy number (VCN) analysis was performed on liver samples (5-29 per dog, n=8 dogs) by Q-PCR and detected DNA copy numbers between 0.0 and 7.8 per diploid genome (Fig 1A). We performed integration target site analysis on liver samples (n=3/dog) from six of the AAV-treated HA dogs and naïve HA dogs (n=2) by ligation-mediated PCR, Ilumina paired-end sequencing and analysis using the custom software pipeline, AAVenger. Analysis of the 20 samples identified >2,000 unique AAV integration events (IE). There was a correlation between the DNA copy number and the number of integration events detected. Clonal abundances were estimated by counting the unique genome breaks associated with integration positions, which showed that the maximum clonal abundance ranged from 1 to 138. The integration events were distributed across the canine genome. Clonal expansions were observed with integration near genes previously associated with growth control and transformation in humans (Fig 1B) with the most abundant clones located in DLEU2L (Linus), PEBP4 (J60) and EGR3 (M50). Integration events in EGR3, EGR2, CCND1, LTO1 and ZNF365 were detected in multiple dogs. Validation of integration sites in the most abundant clones was performed using targeted PCR to isolate junction fragments followed by Sanger sequencing. While AAV integration and clonal expansion was observed, the dogs had no evidence for tumorigenesis and it is not clear if the increase in FVIII expression is associated with the clonal expansions detected. Overall, these studies demonstrate long-term sustained FVIII expression for up to 10 years with clonal expansion, but without clinical adverse events after AAV-mediated gene therapy for hemophilia. Disclosures Sabatino: Spark Therapeutics: Patents & Royalties.

52. Therapeutic Factor VIII Expression After AAV Delivery in Non-Human Primates

Adeno-associated viral (AAV) vector delivery of factor VIII (FVIII) has been challenging due to its intrinsic properties that result in inefficient expression compared to similarly sized proteins. Early studies of AAV delivery in hemophilia A mice and dogs suggested that the therapeutic vector dose for FVIII will be higher than for factor IX. However, higher vector loads may induce stronger immune responses against capsid antigens, as evidenced in the clinical studies of AAV delivery for hemophilia B. The use of codon-optimization and novel FVIII variants with enhanced biological properties may provide strategies to increase FVIII expression or secretion to support clinical studies for hemophilia A. One published study has reported clinically relevant levels of hFVIII following AAV-hFVIII delivery in non-human primates (NHPs). This study utilized a hFVIII variant that included a 17 amino acid synthetic sequence within the 14 amino acid B-domain region that increased hFVIII expression compared to the parental B-domain deleted FVIII-SQ transgene (McIntosh, 2013). While this and other variants may increase expression after AAV delivery, the use of non-native FVIII sequences may also increase the risk of development of neutralizing antibodies to potential neoantigens. In order to generate an AAV-hFVIII vector capable of expressing therapeutic levels of FVIII at a clinically relevant vector dose without introduction of any neoantigens, 28 hFVIII-SQ sequences were generated and introduced into our optimized expression cassette containing a modified transthyretin (TTRm) promoter. The constructs were initially screened by hydrodynamic delivery of plasmid DNA which identified 11 candidates that expressed FVIII 2-7 fold higher than our first generation codon optimized construct, CO3. AAV vectors (n=9) were generated using a novel AAV capsid, Spark100, with the best performing FVIII constructs. Hem A/CD4 KO mice were administered the vectors alongside CO3 (4×10e12vg/kg). At 8 weeks post vector administration, 2/9 expressed hFVIII similar to CO3, 5/9 were 4-8 fold higher than CO3 while 2/9 (SPK-8003 and SPK-8005) were >10 fold more potent than CO3. SPK-8005 was then evaluated in a dose escalation study in cynomologus macaques (n=3/group) treated with 3 doses: 2×10e12, 5×10e12 and 1×10e13 vg/kg and compared to vehicle controls (n=2). At 2 weeks post AAV administration, average hFVIII levels in the low, mid and high dose cohorts were 12.7 ± 2.1, 22.6 ± 0.8 and 54.1 ± 15.6 percent of normal, respectively. By 3-4 weeks, hFVIII expression started to decline in most of the animals concomitant with generation of antibodies against human FVIII. Of note, this is an expected and well-described observation that occurs in immune competent animal models due to differences between human and endogenous FVIII protein sequences. The 2 macaques that did not develop anti-hFVIII antibodies had sustained FVIII expression through the last time point evaluated. Finally, no vector-related toxicity events were observed. In summary, extensive codon-optimization identified novel AAV-hFVIII constructs capable of achieving therapeutic FVIII levels in macaques at clinically relevant doses. To our knowledge, the hFVIII levels observed in this study are the highest reported in a large animal model after treatment with an AAV vector expressing an unmodified FVIII-SQ protein. These safety and efficacy results in NHPs support the use of SPK-8005 hepatic gene transfer for the potential treatment of hemophilia A.

Novel Gene Therapy Strategy for Hemophilia a By Hydrodynamic Gene Delivery Combined with Non-Viral Piggybac Transposon Vector in Canine Model

Currently, hemophilia A patients are treated with plasma-derived or recombinant factor VIII (FVIII) concentrates. These treatments have substantially improved the management of bleedings in hemophilia and more recently long-acting recombinant FVIII concentrates are available in clinic, but hemophilia A patients still require the repeated intravenous injections frequently 2-3 times per week. Therefore, gene and cell-based therapy is an attractive strategy for the treatment of the disease because continuous expression of FVIII levels as low as 1-5 % of normal have been shown to ameliorate the bleeding phenotype as well as improve the quality of life. Although several attempts have been made for hemophilia A gene therapy, systemic delivery of viral vectors into liver cells in vivo, however, resulted in a transient and low-level expression of FVIII, because the large size of FVIII cDNA prohibited the efficient delivery of conventional viral vectors. Moreover, there remain concerns over the safety of these approaches such as adverse immunological reactions or virus-mediated cytotoxicity. Hydrodynamic gene delivery (HGD) is considered to be an alternative candidate to viral vectors. Previously, we tested the HGD in hemophilia A mouse model with the combination of non-viral piggyBac transposon vectors which can transfer the full-length FVIII transgene. As a result, we confirmed the sustained FVIII expression for over 300 days without any immune responses (Matsui H, et.al. PLos One 9(8) e104597, 2014).While HGD system is effective and safe in small animal such as mouse (20-25g body weight), the major challenge is in applying this functioning procedure in the patients with hemophilia A. For this purpose, the current study focus on an assessment of the safety of liver-target HGD in dogs as the first step toward hydrodynamic gene therapy in clinic. Hemophilia A carrier dog was provided from Dr. Lillicrap group at Queen's University and we have already developed hemophilia A dog colony in our institution. We used both normal and hemophilia A dogs in this study. In brief, under general anesthesia using isoflurane, beagle dog (10-12Kg body weight) was placed on the table of fluoroscopy machine. A 18G peripheral catheter was directly inserted into the saphenous vein followed by insertion of a 0.038 inch guide wire, a long sheath (5Fr) and an injection balloon catheter. The balloon catheter was placed and inflated just below the branch of hepatic vein using the image-guided interventional radiology technique. Liver-targeted HGD was performed into 4 liver lobes, the right lateral, right medial, left medial, and left lateral lobe, separately using piggyBac transposon vector expressing either GFP or FVIII. An injection of saline containing plasmid DNA were performed at the speed of 10mL/second sequentially into 4 liver lobes through the hepatic veins with 20 minutes interval between each injections.Various physiological parameters including heart rate, blood pressure, oxygen saturation were continuously monitored and recorded before, during and after each injection. For the assessment of tissue damage, blood samples were collected at the various time points. For the assessment of GFP expression, the liver samples were collected under echo-guided liver biopsy both 5 and 120 days after HGD. Although, transient 5-10 fold increase in hepatobiliary enzymes, including AST, ALT, were observed, there is no significant impact on physiological parameters, such as systemic circulation, respiration and cardiac function.By histological examination of liver samples, approximately 70-80% of hepatocytes in all 4 lobes were positive with GFP both 5 and 120 days after HGD. These results suggest that HGD system combined with interventional radiology technique is capable for the large animal with safe, efficient and less invasive. Now, we are conducting FVIII gene transfer by HGD in canine hemophilia A model. DisclosuresNo relevant conflicts of interest to declare.

PiggyBac-mediated phenotypic correction of factor VIII deficiency

Hemophilia A, caused by a deficiency in factor VIII (FVIII), is the most severe inherited bleeding disorder. Hemophilia A is an attractive gene therapy candidate because even small increases in FVIII levels will positively alter the phenotype. While several vectors are under investigation, gene addition from an integrated transgene offers the possibility of long term expression. We engineered the DNA transposon-based vector, piggyBac (PB), to carry a codon-optimized B-domain deleted human FVIII cDNA. Evaluation of gene transfer efficiency in FVIII null mice demonstrated that PB containing the FVIII cDNA, delivered via hydrodynamic injection to immunocompetent hemophilia mice, conferred persistent gene expression, attaining mean FVIII activity of approximately 60% with 3/19 developing inhibitors. In addition to efficacious expression, a goal of gene transfer-based therapies is to develop vectors with low toxicity. To assess endoplasmic reticulum stress in hepatocytes stably expressing the transgene, we evaluated levels of ER stress markers via qPCR and found no evidence of cell stress. To evaluate phenotypic correction, a tail clip assay performed at the end of the study revealed reduced blood loss. These data demonstrate that PB can be used to achieve sustained FVIII expression and long-term therapeutic benefit in a mouse model.

Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.

Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.

Sustained FVIII Expression and Phenotypic Correction of Hemophilia A in Neonatal Mice Using an Endothelial-Targeted Sleeping Beauty Transposon

Hemophilia A, deficiency of coagulation factor VIII (FVIII), is an attractive candidate for gene therapy as expression of modest amounts of FVIII can provide therapeutic benefit. Most gene transfer approaches for hemophilia have focused on the liver, as this is the major source of endogenous FVIII; however, increasing evidence suggests that endothelial cells are capable of synthesis and release of FVIII. Here the Sleeping Beauty (SB) transposon is employed to target long-term expression of the human B-domain-depleted FVIII gene (approved gene symbol F8) within lung endothelia of hemophilic mice. As the formation of inhibitory antibodies to FVIII has been a significant impediment toward achieving therapeutic plasma levels after gene or protein therapy, we chose to perform gene transfer in neonatal mice, which are more likely to be immune tolerant. Using this approach, low therapeutic levels of FVIII (∼10%), as well as phenotypic correction of the bleeding disorder, were achieved in all animals that received the FVIII transposon and functional transposase throughout the duration of the study (24 weeks). Rechallenge of these animals with additional gene transfer did not result in significant increases in FVIII levels, due mainly to increases in inhibitory antibodies. These studies demonstrate the feasibility of using endothelial-targeted SB transposons for the treatment of hemophilia A.

Persistent expression of factor VIII in vivo following nonprimate lentiviral gene transfer

AbstractHemophilia A is a clinically important coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)–based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudo-typed FIV efficiently and showed excellent hepatocyte tropism. The GP64-pseudotyped vector was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human α1-antitrypsin promoter) further enhanced transgene expression in hepatocytes. We generated a GP64-pseudotyped FIV vector encoding the B domain–deleted human FVIII coding region driven by the liver-specific promoter, with 2 beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti–human FVIII antibodies and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64-pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

601. Persistent Expression of Factor VIII In Vivo Following FIV Lentiviral Gene Transfer

Top of pageAbstract Hemophilia A is a common X-linked coagulation disorder caused by the lack or abnormality of plasma coagulation factor VIII (FVIII). Gene transfer of the FVIII cDNA to hepatocytes using lentiviral vectors is a potential therapeutic approach. We investigated the efficacy of feline immunodeficiency virus (FIV)-based vectors in targeting hepatocytes and correcting FVIII deficiency in a hemophilia A mouse model. Several viral envelope glycoproteins were screened for efficient FIV vector pseudotyping and hepatocyte transduction. The GP64 glycoprotein from baculovirus Autographa californica multinuclear polyhedrosis virus pseudotyped FIV efficiently and showed excellent hepatocyte tropism. The GP64 pseudotyped FIV was stable in the presence of human or mouse complement. Inclusion of a hybrid liver-specific promoter (murine albumin enhancer/human 1-antitrypsin promoter), further enhanced transgene expression in hepatoctyes. We generated a GP64-pseudotyped FIV vector encoding the B domain-deleted human FVIII coding region driven by the liver-specific promoter, with two beneficial point mutations in the A1 domain. Intravenous vector administration conferred sustained FVIII expression in hemophilia A mice for several months without the generation of anti-human FVIII antibodies, and resulted in partial phenotypic correction. These findings demonstrate the utility of GP64 pseudotyped FIV lentiviral vectors for targeting hepatocytes to correct disorders associated with deficiencies of secreted proteins.

Efficient adenoviral vector transduction and expression of functional human factor VIII in cultured primary human hepatocytes.

Hemophilia A is a severe bleeding disorder caused by a deficiency in blood coagulation factor VIII (FVIII). Adenoviral vectors containing a potent human FVIII expression cassette encoding a truncated FVIII cDNA were developed that mediated sustained FVIII expression in normal and haemophiliac mice and complete phenotypic correction of the bleeding disorder in haemophiliac mice and dogs (Connelly and Kaleko, Haemophilia, 1998; 4: 380-8). Here, we evaluated two E1/E2a/E3-deleted adenoviral vectors encoding human FVIII, one containing the full-length cDNA and the second containing a truncated cDNA lacking the B-domain. Viral vectors encoding the human full-length FVIII cDNA have not been described previously. Hepatocyte transduction was efficient and dose dependent, ranging from 50% to 100%. High levels of functional FVIII were secreted from transduced cells at amounts up to 6000 mU-1 10(6)cells-1 60 h. B-domain deleted FVIII was expressed at levels at least 8-fold higher than the full-length FVIII protein, whereas FVIII RNA levels were similar with both vectors. These data provide the first demonstration of FVIII adenoviral vector function in primary human cells and verify the potential clinical utility of adenoviral vectors for the treatment of haemophilia A.

Evaluation of an Adenoviral Vector Encoding Full-Length Human Factor VIII in Hemophiliac Mice

Adenoviral vectors provide a promising gene therapy system for the treatment of hemophilia A. Potent vectors encoding a human factor VIII (FVIII) cDNA were developed that mediated sustained FVIII expression in normal and hemophiliac mice and complete phenotypic correction of the bleeding disorder in hemophiliac mice and dogs (Connelly and Kaleko, Haemophilia 1998; 4: 380-8). However, these studies utilized vectors encoding a truncated version of the human FVIII cDNA lacking the B-domain (BDD FVIII). In this work, an adenoviral vector encoding the human full-length (FL) FVIII cDNA was generated and characterized. While functional FL FVIII was secreted in vitro, expression of the FL protein was not detected in the plasma of vector-treated hemophiliac mice. Unexpectedly, the FL FVIII vector-treated animals demonstrated phenotypic correction of the bleeding defect as measured by a tail-clip survival study. FL FVIII protein was visualized in the mouse livers using human FVIII-specific immunohistochemical analyses. These data demonstrate that adenoviral vector-mediated in vivo expression of BDD FVIII is more efficient than that of the FL protein and that phenotypic correction can occur in the absence of detectable levels of FVIII.