Therapeutic Levels Of Factor VIII Research Articles

Assessing Patient Experiences with Prophylactic Treatments for Hemophilia a: Concept Elicitation for Gene Therapy

Background: Gene therapy for hemophilia A is designed to be a one-time infusion to deliver functional copies of the defective factor VIII (FVIII) gene, to facilitate the endogenous production of therapeutic FVIII levels. The aim is to achieve long-term protection from bleeds without the burden of regular infusions. Aims: To better understand patients' experiences of living with hemophilia A, the impact of traditional hemophilia A treatments, and patients' perceptions of the potential value of gene therapy versus traditional prophylactic treatments. Methods: Patients were identified from the database of a US rare diseases patient organization, who also recruited and screened patients for the study using materials developed by the outcomes research organization (RTI-HS) and Takeda. Adult males aged ≥18 years with a self-reported diagnosis of moderate or severe hemophilia A, who reported using factor or nonfactor prophylactic treatment and were not currently receiving treatment for inhibitors, were eligible for the study. Eligible patients provided verbal informed consent to participate in a semi-structured, 60-minute telephone interview conducted in English by 2 members of RTI-HS who did not have access to any patient-identifying information at any time during the study. Targeted questions probed perceptions of treatment burden, impact of hemophilia A on daily life, and time spent on treatment. Additionally, questions were posed to assess patients' perceptions of the impact of traditional treatments and the potential benefits they anticipate from gene therapy. Results: Nineteen patients aged 19-55 years with moderate (n = 1) or severe (n = 18) hemophilia A were interviewed. Most (16/19, 89.5%) received prophylactic FVIII therapy, (3/19, 15.8%) were receiving nonfactor prophylactic treatment, of which 1 patient also used FVIII treatment. The aspects of current or past treatments most frequently disliked by patients were lack of efficacy, frequency of infusions, intravenous administration, vein health/scar tissue, and dosing volume. Most patients expressed satisfaction with their current treatment (18/19; 94.7%), though all listed ≥1 negative treatment impact, most frequently related to difficulties with travel (13/19; 68.4%), mood/emotions (12/19; 63.2%), day-to-day activities (10/19; 52.6%), and physical health/activities (7/19; 36.8%), including having to give up or reduce particular activities because of their treatment and needing to be more cautious, especially on nontreatment days. When presented with a hypothetical scenario for gene therapy - a one-time long-acting intravenous infusion to provide a constant level of FVIII that could reduce future bleeds - all patients stated they would choose gene therapy over their current therapy, although several said they would have initial questions regarding safety, efficacy, and duration of protection. Commonly expressed reasons for preferring gene therapy (Figure 1) included fewer infusions and less worry about the need to infuse. All 19 patients said they expected to be highly satisfied with this treatment, largely because of the long-term protection from bleeds, fewer infusions, and less concern about inhibitors. The most commonly anticipated improvements were in mood/emotions (15/19; 78.9%), specifically related to reduced concern about infusions and bleed protection. Other commonly anticipated improvements included gain in time usually spent infusing (13/19; 68.4%), easier travel (12/19; 63.2%), and improved physical health and ability to perform activities (10/19; 52.6%). Conclusions: This study identified specific patient priorities, including treatment convenience, long-lasting bleed protection, frequency of intravenous infusions, and infusion volumes. The results suggest that gene therapy clinical trials should consider evaluating patient concerns in relation to the level of patient confidence in bleed protection. A study limitation is that, at the time of the survey, data on the efficacy and safety of gene therapy were limited. In the future, the study will be expanded to include a larger population of patients with hemophilia. Disclosures Sun: Takeda Pharmaceutical Company Ltd.: Current Employment. Olayinka-Amao:Takeda Pharmaceutical Company Ltd: Other: RTI-HS was contracted by Takeda Pharmaceutical Company Ltd to conduct this work; RTI Health Solutions: Current Employment. DiBenedetti:RTI Health Solutions: Current Employment; Takeda Pharmaceutical Company Ltd: Other: RTI-HS was contracted by Takeda Pharmaceutical Company Ltd to conduct this work.

Patient Preferences for Hemophilia a Treatments: A Discrete Choice Experiment

Background: Gene therapy is being investigated as a potential new treatment for patients with hemophilia A. This is designed to provide functional copies of the factor VIII (FVIII) gene via a 1-time infusion to facilitate sustained endogenous production of therapeutic FVIII levels. As part of the patient-focused development of new hemophilia treatments, gene therapy is expected to have a number of different characteristics from current hemophilia A treatments, which are likely to influence patient treatment preferences. Aims: To assess patients' preferences for different hemophilia A treatments, including emerging treatments such as gene therapy. Methods: Male patients aged ≥18 years with moderate or severe hemophilia A and no history of FVIII inhibitors, who had been receiving prophylaxis in the previous month, were recruited in the US from December 2019 to January 2020. Patients who provided informed consent to participate in the online survey were screened and those eligible took part in a discrete choice experiment to quantify the relative importance of different treatment attributes on patient treatment preferences. Patients were presented with 14 choice cards and were asked to select the preferred treatment profile from 2 treatments with varying attribute levels on each card. Attributes and their levels to describe hemophilia A treatments were selected on the basis of a targeted literature review and feedback from 15 patients interviewed by telephone. One attribute level for out-of-pocket cost (a cost not covered by medical insurance) was hypothetical (1-time cost of $3000 for 5 years). Attributes focused on efficacy, safety, and administration-related measures. A conditional logit regression model was used to analyze patient preferences. Odds ratios quantified the impact of specific attribute levels on patients' treatment choices. Estimates of relative attribute importance (RAI) quantified the influence of each attribute on patients' decision making. Results: The survey was completed by 95 patients, 20 (21.1%) with moderate and 75 (78.9%) with severe hemophilia A, who had received prophylactic treatment for a mean (standard deviation [SD]) 14.0 (10.2) years. Most patients (75/95 [78.9%]) were receiving recombinant or plasma-derived FVIII prophylaxis administered once per month, once every 2 weeks, once per week, twice per week, or every other day (2, 3, 4, 33, and 33 patients, respectively). Twenty patients (21.1%) received nonfactor prophylaxis once per month, once every 2 weeks, once per week, twice per week, and with regular use of concomitant FVIII (2, 8, 7, 2, and 1 patient, respectively). Mean (SD) patient age at the survey date was 36.0 (11.3) years. Administration-related measures were the most important class of attributes for patients when selecting treatments (71.5% RAI): route and frequency of administration (30.3% RAI), out-of-pocket cost (24.1% RAI), and place of administration, eg, home versus clinic (17.1% RAI; Figure 1A). The risk of serious side effects was the least important attribute (2.2% RAI), lacking a significant impact on patient treatment choice. The directionality of odds ratios (ORs) for all attribute levels aligned with expectations. Patients exhibited a clear preference for infusion treatments administered with low versus high frequency (OR 3.3; P<0.001) and for treatments administered at home versus the clinic (OR 2.0; P<0.001; Figure 1B). Conclusions: Frequency, route, and place of administration and out-of-pocket costs were the treatment attributes found to have the greatest impact on patient preferences for hemophilia A treatment. Study limitations include potential for selection bias associated with the use of an online panel-based survey. Exclusion of patients with FVIII inhibitors also reduced generalizability, possibly leading to the reduced influence of safety-related treatment attributes. In future studies, design of efficacy attribute levels may better reflect the observed variability in FVIII expression between patients and over time with gene therapy. Insights from this study improve our understanding of preferred treatment regimens for patients with hemophilia A, and could help physicians, stakeholders, and decision makers to evaluate patient acceptance of emerging treatments for hemophilia A, such as gene therapies. Disclosures Sun: Takeda Pharmaceutical Company Ltd.: Current Employment. Zhao:Analysis Group Inc.: Current Employment; Takeda Pharmaceutical Company Ltd: Research Funding. Yang:Analysis Group Inc.: Current Employment; Takeda Pharmaceutical Company Ltd: Research Funding. Wu:Analysis Group Inc.: Current Employment; Takeda Pharmaceutical Company Ltd: Research Funding.

Defining the Optimal FVIII Transgene for Placental Cell-Based Gene Therapy to Treat Hemophilia A

The delivery of factor VIII (FVIII) through gene and/or cellular platforms has emerged as a promising hemophilia A treatment. Herein, we investigated the suitability of human placental cells (PLCs) as delivery vehicles for FVIII and determined an optimal FVIII transgene to produce/secrete therapeutic FVIII levels from these cells. Using three PLC cell banks we demonstrated that PLCs constitutively secreted low levels of FVIII, suggesting their suitability as a transgenic FVIII production platform. Furthermore, PLCs significantly increased FVIII secretion after transduction with a lentiviral vector (LV) encoding a myeloid codon-optimized bioengineered FVIII containing high-expression elements from porcine FVIII. Importantly, transduced PLCs did not upregulate cellular stress or innate immunity molecules, demonstrating that after transduction and FVIII production/secretion, PLCs retained low immunogenicity and cell stress. When LV encoding five different bioengineered FVIII transgenes were compared for transduction efficiency, FVIII production, and secretion, data showed that PLCs transduced with LV encoding hybrid human/porcine FVIII transgenes secreted substantially higher levels of FVIII than did LV encoding B domain-deleted human FVIII. In addition, data showed that in PLCs, myeloid codon optimization is needed to increase FVIII secretion to therapeutic levels. These studies have identified an optimal combination of FVIII transgene and cell source to achieve clinically meaningful levels of secreted FVIII.

Gene Therapy with BMN 270 Results in Therapeutic Levels of FVIII in Mice and Primates and Normalization of Bleeding in Hemophilic Mice.

Hemophilia A is an X-linked bleeding disorder caused by mutations in the gene encoding the factor VIII (FVIII) coagulation protein. Bleeding episodes in patients are reduced by prophylactic therapy or treated acutely using recombinant or plasma-derived FVIII. We have made an adeno-associated virus 5 vector containing a B domain-deleted (BDD) FVIII gene (BMN 270) with a liver-specific promoter. BMN 270 injected into hemophilic mice resulted in a dose-dependent expression of BDD FVIII protein and a corresponding correction of bleeding time and blood loss. At the highest dose tested, complete correction was achieved. Similar corrections in bleeding were observed at approximately the same plasma levels of FVIII protein produced either endogenously by BMN 270 or following exogenous administration of recombinant BDD FVIII. No evidence of liver dysfunction or hepatocyte endoplasmic reticulum stress was observed. Comparable doses in primates produced similar levels of circulating FVIII. These preclinical data support evaluation of BMN 270 in hemophilia A patients.

Circumventing furin enhances factor VIII biological activity and ameliorates bleeding phenotypes in hemophilia models.

Processing by the proprotein convertase furin is believed to be critical for the biological activity of multiple proteins involved in hemostasis, including coagulation factor VIII (FVIII). This belief prompted the retention of the furin recognition motif (amino acids 1645-1648) in the design of B-domain-deleted FVIII (FVIII-BDD) products in current clinical use and in the drug development pipeline, as well as in experimental FVIII gene therapy strategies. Here, we report that processing by furin is in fact deleterious to FVIII-BDD secretion and procoagulant activity. Inhibition of furin increases the secretion and decreases the intracellular retention of FVIII-BDD protein in mammalian cells. Our new variant (FVIII-ΔF), in which this recognition motif is removed, efficiently circumvents furin. FVIII-ΔF demonstrates increased recombinant protein yields, enhanced clotting activity, and higher circulating FVIII levels after adeno-associated viral vector-based liver gene therapy in a murine model of severe hemophilia A (HA) compared with FVIII-BDD. Moreover, we observed an amelioration of the bleeding phenotype in severe HA dogs with sustained therapeutic FVIII levels after FVIII-ΔF gene therapy at a lower vector dose than previously employed in this model. The immunogenicity of FVIII-ΔF did not differ from that of FVIII-BDD as a protein or a gene therapeutic. Thus, contrary to previous suppositions, FVIII variants that can avoid furin processing are likely to have enhanced translational potential for HA therapy.

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.

Potential for cellular stress response to hepatic factor VIII expression from AAV vector

Hemophilia A and B are coagulation disorders resulting from the loss of functional coagulation factor VIII (FVIII) or factor IX proteins, respectively. Gene therapy for hemophilia with adeno-associated virus vectors has shown efficacy in hemophilia B patients. Although hemophilia A patients are more prevalent, the development of therapeutic adeno-associated virus vectors has been impeded by the size of the F8 cDNA and impaired secretion of FVIII protein. Further, it has been reported that over-expression of the FVIII protein induces endoplasmic reticulum stress and activates the unfolded protein response pathway both in vitro and in hepatocytes in vivo, presumably due to retention of misfolded FVIII protein within the endoplasmic reticulum. Engineering of the F8 transgene, including removal of the B domain (BDD-FVIII) and codon optimization, now allows for the generation of adeno-associated virus vectors capable of expressing therapeutic levels of FVIII. Here we sought to determine if the risks of inducing the unfolded protein response in murine hepatocytes extend to adeno-associated virus gene transfer. Although our data show a mild activation of unfolded protein response markers following F8 gene delivery at a certain vector dose in C57BL/6 mice, it was not augmented upon further elevated dosing, did not induce liver pathology or apoptosis, and did not impact FVIII immunogenicity.

Recombinant Porcine Factor VIII, Obi-1, Successfully Controlled Gastrointestinal Bleeding in a Patient with Acquired Hemophilia A

▪Introduction: Acquired hemophilia A (AHA) is a rare, life-threatening bleeding diathesis categorized by low levels of Factor VIII (FVIII) due to an acquired auto-antibody against FVIII. Treatment for AHA requires both control of the bleeding and eradication of the inhibitor. Recombinant activated factor VII (rFVIIa) maybe used to control the bleeding, but with only mixed results in controlling gastrointestinal bleeding (GIB). rFVIIa is associated with thrombosis risk and lack of ability to monitor. The recombinant porcine FVIII (rpFVIII), OBI-1, recently obtained regulatory approval based on the results of Phase 2/3 trial, but the patients in this cohort did not have GIBs. Here we present a patient with AHA admitted with a GIB and successfully treated with OBI-1.Case: A 69 year old African American female with T1N0M0 infiltrative ductal carcinoma of the left breast and Stage IVA Squamous cell carcinoma of the cervix, treated in 1997 and 2007 respectively, presented to the emergency department with two days of painless bright red blood per rectum. She was orthostatic (supine: HR 79, BP 112/77 and standing: HR 111, BP 100/72) with hemoglobin (Hb) of 5.7gms/dl. An EGD demonstrated a bleeding vessel and multiple arteriovenous malformations (AVM) in the gastric body that were secured with clipping. Over the next 5 days EGD was repeated twice and multiple endoclips were placed in an attempt to achieve hemostasis. She subsequently underwent angiography to identify a target vessel for embolization, but one could not be found. On hospital day 11, hematology was consulted for a prolonged aPTT (113.8 seconds) and inadequate correction with mixing studies (63.7 seconds). FVIII level was <1% with factor VIII inhibitor titer of 245 Bethesda Units/ml (BU). Prior to starting OBI-1, she received 9 units of packed red blood cells and 2 units of fresh frozen plasma. OBI-1 was started at 200u/kg q4h for first 2 days and then decreased to q8h, with cessation of bleeding within 24 hours. Upon completion of the bolus of OBI-1, her FVIII level was 14%, then 38% at 8 hours, 48% at 16 hours and 62% at 24 hours. Rituximab (375mg/m2 weekly) and corticosteroids (prednisone 1mg/kg daily x7 days) were given in an attempt to eliminate the inhibitor. She remained stable for 6 days but subsequently developed abdominal pain. A CT of the abdomen revealed free air prompting emergent surgical intervention. She had internal hernia in the pelvis, ischemic small bowel and perforated jejunum requiring extensive resection. During this time she remained on rpFVIII (200u/kg q8h) with FVIII pre-infusion levels ranging from 2% - 14% and post-infusion levels from 80% to 217% and had only 25ml estimated blood loss during surgery. Unfortunately, 1 day after surgery she developed multi-organ failure and expired on hospital day 19.Discussion: OBI-1 is a glycosylated, B-domain deleted recombinant porcine factor VIII that retains the ability to interact with Factor IXa and activate Factor X, but it is less inhibited by auto-antibodies to human FVIII. For FVIII inhibitor titers >5 BU, an inhibitor bypassing agent is usually necessary because human FVIII products are not able to overcome the inhibitor. Given the high FVIII inhibitor titers in this patient, administration of rFVIIa or OBI-1 was necessary. Our own institutional experience of treating GIBs in patients with AHA had poor outcomes. The efficacy of rFVIIa in treating GIB is inconclusive, with response rate ranging from 53% to 71%. However, rFVIIa is associated with a risk of thrombosis approaching 9.4%, especially in older patients and those with vasculopathy, a common demographic in AHA. Unlike rFVIIa, OBI-1 allows close monitoring of FVIII and in the aforementioned Phase 2/3 trial, there were no thrombotic complications or serious adverse events. We were successful in controlling the bleeding within 24 hours with OBI-1 and were able to achieve therapeutic levels of FVIII despite high Bethesda titers. Unfortunately, our patient died from an ischemic bowel and perforated jejunum that was likely related to hernia or repeated instrumentation in an attempt to stop the bleeding prior to the diagnosis of AHA.Conclusion: OBI-1 appears to effectively and rapidly overcome a high titer FVIII inhibitor and control bleeding in a patient with AHA and GIB. While this is a promising therapy, additional study is necessary to determine safety, efficacy, and cost-effectiveness. DisclosuresNo relevant conflicts of interest to declare.

244. Factor VIII Immunogenicity Is a Barrier to AAV Gene Therapy for Hemophilia A

Liver-directed adeno-associated virus (AAV) gene therapy utilizing a bioengineered, high-expression coagulation factor VIII (FVIII) transgene (ET3) has been demonstrated by our laboratory to produce therapeutic FVIII levels in a naive, immune-competent murine model of hemophilia A (HA). Recently, other groups have shown that similar liver directed gene therapy approaches also have the potential to reverse pre-existing immunity, including inhibitors, to coagulation factor IX (FIX) in murine models of hemophilia B (HB). In the current study, we sought to determine if, similarly, ET3-AAV8 could reverse pre-existing FVIII immunity in HA mice. Cohorts of mice were immunized with either recombinant ET3 or human FVIII (hFVIII). Robust anti-FVIII immune responses were elicited as expected and characterized by ELISA (total IgG) and Bethesda (inhibitor) titers ranging from 6000-15,400 arbitrary units (AU) and 22-286 BU, respectively, for ET3 immunized mice, while hFVIII immunized mice showed respective ELISA and Bethesda titers ranging from 460-1480 AU and 23-63 BU. Subsequently, the mice were administered ET3-AAV8 at a dose (4e12 vp/kg) previously shown to produce complete correction of the FVIII deficiency (>50% normal human FVIII activity) and monitored for 16 weeks. No significant change was observed in either ELISA or Bethesda titers for either cohort indicating that the pre-existing immunity was not modulated substantively by FVIII-AAV8 (p>0.5). As Arruda and colleagues previously showed that liver-directed FVIII-AAV8 can eradicate pre-existing anti-FVIII antibodies in a canine model of HA harboring low Bethesda titers (£12 BU), we hypothesized that the capacity for ET3-AAV8 to eliminate pre-existing antibodies may exist under a certain immune threshold quantitatively linked to the inhibitor titer. To test this hypothesis, cohorts of HA mice were established with pre-existing Bethesda titers ranging from 0-25 BU (n=10). Following administration of ET3-AAV8, no significant changes in titers have been observed to date. A corollary question then arose regarding the immune modulation potential of FVIII-AAV8 in naive HA mice, which has been studied extensively for FIX-AAV in HB mice. Specifically, we tested if a mid (4e12vp/kg) or high (2e13vp/kg) dose of ET3-AAV8 could tolerize naive HA mice to recombinant FVIII infusions. FVIII production was dose dependent with the mid cohort averaging 70% normal human levels (0.7 IU/ml) and the high dose cohort averaging 200% normal levels (2 IU/ml) over 5 months. Mice were then challenged with weekly intravenous injections of ET3. All mice treated with the mid AAV dose developed anti-FVIII ELISA and Bethesda titers similar to challenged controls with a concomitant loss of detectable plasma FVIII activity. However, mice in the high dose cohort maintained FVIII activity indicating a possible antigen production requirement for immune modulation consistent with current models of active tolerance. Collectively, these preclinical data indicate that current clinical FIX-AAV gene therapy data may not predict clinical outcomes for similar FVIII-AAV products, especially as trials progress into previously untreated and inhibitor populations.

Omental implantation of BOECs in hemophilia dogs results in circulating FVIII antigen and a complex immune response

AbstractEx vivo gene therapy strategies avoid systemic delivery of viruses thereby mitigating the risk of vector-associated immunogenicity. Previously, we delivered autologous factor VIII (FVIII)-expressing blood outgrowth endothelial cells (BOECs) to hemophilia A mice and showed that these cells remained sequestered within the implanted matrix and provided therapeutic levels of FVIII. Prior to translating this strategy into the canine (c) model of hemophilia A, we increased cFVIII transgene expression by at least 100-fold with the use of the elongation factor 1 alpha (EF1α) promoter and a strong endothelial enhancer element. BOECs isolated from hemophilia A dogs transduced with this lentiviral vector express levels of cFVIII ranging between 1.0 and 1.5 U/mL per 106 cells over 24 hours. Autologous BOECs have been implanted into the omentum of 2 normal and 3 hemophilia A dogs. These implanted cells formed new vessels in the omentum. All 3 hemophilia A dogs treated with FVIII-expressing autologous BOECs developed anti-FVIII immunoglobulin G2 antibodies, but in only 2 of the dogs were these antibodies inhibitory. FVIII antigen levels >40% in the absence of FVIII coagulant function were detected in the circulation for up to a year after a single gene therapy treatment, indicating prolonged cellular viability and synthesis of FVIII.

Overexpression of Factor VIII Is Associated with Immune Responses to Factor VIII and Cellular Stress in Hemophilia A Mice

Abstract 22For many years it has been evident that it is challenging to express recombinant factor VIII (FVIII) in vitro and in vivo. In vitro studies suggest that high levels of FVIII expression can lead to cellular stress. We have demonstrated that adeno-associated viral (AAV) vector delivery results in long-term expression of therapeutic levels of FVIII in hemophilia A (HA) dogs up 10% of normal. In this model transgene expression is restricted to a portion of liver cells resulting in sustained FVIII levels. Notably, in dogs FVIII expression is sustained for periods longer than 7 years. Conceivably, the increased load per cell for cellular processing of FVIII may impact the cell’s ability to synthesize and secrete functional FVIII, activation of cellular stress and the potential for immune responses. We sought to determine whether cellular stress and immune responses to the transgene are increased by overexpression of FVIII in mice by AAV vectors. Using AAV to deliver FVIII as a B-domain deleted single chain (single chain approach) or as two separate chains (light and heavy chains) (two chain approach), we analyzed dose-dependent FVIII synthesis and its cellular impact. We hypothesized that high levels of FVIII expression after AAV-mediated delivery of FVIII may induce cellular stress. HA mice were administered AAV serotype 8 (AAV8) vector in 3 groups: (1) two-chain approach (2) single chain approach or (3) AAV empty capsid (control). Three total AAV doses were administered: 1×1010 vector genomes/mouse (low dose), 5×1010 vg/mouse (mid-dose) and 2.5×1011 vg/mouse (high dose). FVIII antigen levels (heavy and light chain) and anti-cFVIII IgG antibodies were detected by ELISA at a series of time points post vector administration until the terminal time points (1, 2, 4, 8, 12 and 24 wks). As we previously observed, after single chain delivery we detect equivalent amounts of heavy chain and light chain in the circulation by ELISA that correlates with activity. However, in the two-chain approach we observe that the light chain is >2-fold higher than the heavy chain in the circulation and that the activity correlates with the amount of heavy chain. We observe a dose-dependent increase in FVIII expression and dose-dependent anti-cFVIII IgG antibody titers. The kinetics of FVIII expression and antibody formation are different for the two-chain delivery compared to the single chain delivery. For the two-chain delivery, we observe AAV dose-dependent peak FVIII expression within 1 week (30, 100, or 400 ng/ml, respective of dose) followed by the onset of anti-FVIII IgG2 antibodies by 4 weeks (16, 27, 52 μg/ml, respective of dose). For single chain delivery, we observe FVIII expression within 1 week and low level antibody titers by 4 weeks that increase dramatically by 12 weeks (14, 21, 38 μg/ml, respective of dose). Thus, we observe a more gradual increase in antibody titers in the single chain compared to the two-chain delivery but at late time points the immunogenicity of both approaches is indistinguishable. RNA was isolated from the livers of the mice at the terminal time points for quantitative PCR analysis of two key components of the unfolded protein response signaling network, BiP and CHOP. We compared the two-chain and single chain AAV delivery of FVIII alongside animals injected with AAV empty capsid that did not express FVIII. A tunicamycin treated positive control had an increase in BiP (54-fold) and CHOP (74-fold) expression compared to untreated HA mice. There were mild elevations (2–3-fold) of BiP and/or CHOP in some animals observed at week 1 in all three treatment groups. Only at 12 weeks post vector administration were significant increases in BiP (>70 fold) and CHOP (>4 fold) observed in the single chain treated group at the high dose expressing >300% but not the two-chain approach or empty capsid. These studies suggest that supraphysiological expression (>100%) of BDD-cFVIII may increase the likelihood of an immune response to FVIII and cellular stress. Further studies may determine if there is a relationship between high FVIII expression, cellular stress and immune responses to FVIII. Thus, there is a threshold of FVIII expression levels that may prove unsafe. Moreover, these data are in agreement with the lack of evidence of cellular toxicity and immunogenicity in HA dogs expressing therapeutic levels of FVIII by AAV vectors. Disclosures:Lange:Bayer Healthcare: Research Funding. Altynova:Bayer Healthcare: Research Funding. Sabatino:Bayer Healthcare: Research Funding.

A MicroRNA-regulated and GP64-pseudotyped Lentiviral Vector Mediates Stable Expression of FVIII in a Murine Model of Hemophilia A

The objective to use gene therapy to provide sustained, therapeutic levels of factor VIII (FVIII) for hemophilia A is compromised by the emergence of inhibitory antibodies that prevent FVIII from performing its essential function as a cofactor for factor IX (FIX). FVIII appears to be more immunogenic than FIX and an immune response is associated more frequently with FVIII than FIX gene therapy strategies. We have evaluated a modified lentiviral delivery strategy that facilitates liver-restricted transgene expression and prevents off-target expression in hematopoietic cells by incorporating microRNA (miRNA) target sequences. In contrast to outcomes using this strategy to deliver FIX, this modified delivery strategy was in and of itself insufficient to prevent an anti-FVIII immune response in treated hemophilia A mice. However, pseudotyping the lentivirus with the GP64 envelope glycoprotein, in conjunction with a liver-restricted promoter and a miRNA-regulated FVIII transgene resulted in sustained, therapeutic levels of FVIII. These modifications to the lentiviral delivery system effectively restricted FVIII transgene expression to the liver. Plasma levels of FVIII could be increased to around 9% that of normal levels when macrophages were depleted prior to treating the hemophilia A mice with the modified lentiviral FVIII delivery system.

Factor VIII delivered by haematopoietic stem cell-derived B cells corrects the phenotype of haemophilia A mice.

The main impediments to clinical application of haematopoietic stem cell (HSC) gene therapy for treatment of haemophilia A are the bone marrow transplant-related risks and the potential for insertional mutagenesis caused by retroviral vectors. To circumvent these limitations, we have adapted a non-myeloablative conditioning regimen and directed factor VIII (FVIII) protein synthesis to B lineage cells using an insulated lentiviral vector containing an immunoglobulin heavy chain enhancer-promoter. Transplantation of lentiviral vector-modified HSCs resulted in therapeutic levels of FVIII in the circulation of all transplanted mice for the duration of the study (six months). Immunostaining of spleen cells showed that the majority of FVIII was synthesised by B220+ B cells and CD138+ plasma cells. Subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII antibody response, demonstrating induction of immune hyporesponsiveness. All transplant recipients exhibited clot formation and survived tail clipping, indicating correction of their haemophilic phenotype. Therapeutic levels of FVIII could be transferred to secondary recipients by bone marrow transplantation, confirming gene transfer into long-term repopulating HSCs. Moreover, short-term therapeutic FVIII levels could also be achieved in secondary recipients by adoptive transfer of HSC-derived splenic B cells. Our findings support pursuit of B cell-directed protein delivery as a potential clinical approach to treat haemophilia A and other disorders correctable by systemically distributed proteins.

Ex Vivo Gene Therapy for Hemophilia A That Enhances Safe Delivery and Sustained In Vivo Factor VIII Expression from Lentivirally Engineered Endothelial Progenitors

Novel therapeutic strategies for hemophilia must be at least as effective as current treatments and demonstrate long-term safety. To date, several small clinical trials of hemophilia gene transfer have failed to show the promise of preclinical evaluations. Therefore, we wanted to develop and evaluate the feasibility of a novel ex vivo gene transfer strategy whereby cells derived from progenitor cells are engineered to express factor VIII (FVIII) and then implanted subcutaneously to act as a depot for FVIII expression. Circulating blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine FVIII transgene. To enhance safety, these cells were implanted subcutaneously in a Matrigel scaffold, and the efficacy of this strategy was compared with i.v. delivery of engineered BOECs in nonhemophilic nonobese diabetic/severe combined immunodeficiency mice. Therapeutic levels of FVIII persisted for 15 weeks, and these levels of stable expression were extended to 20 weeks when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Subsequent studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels. This loss of transgene expression appears to be due to the disappearance of the implanted cells. The animals treated with either of the two tolerizing regimens did not develop anti-FVIII antibodies. Biodistribution analysis demonstrated that BOECs were retained inside the subcutaneous implants. These results indicate, for the first time, that genetically modified endothelial progenitor cells implanted in a subcutaneous scaffold can provide sustained therapeutic levels of FVIII and are a promising and safe treatment modality for hemophilia A. Disclosure of potential conflicts of interest is found at the end of this article.

Multiyear therapeutic benefit of AAV serotypes 2, 6, and 8 delivering factor VIII to hemophilia A mice and dogs

Hemophilia A, a deficiency of functional coagulation factor VIII (FVIII), is treated via protein replacement therapy. Restoring 1% to 5% of normal blood FVIII activity prevents spontaneous bleeding, making the disease an attractive gene therapy target. Previously, we have demonstrated short-term activity of a liver-specific AAV2 vector expressing canine B-domain-deleted FVIII (cFVIII) in a hemophilia canine model. Here, we report the long-term efficacy and safety of AAV-cFVIII vectors of serotypes 2, 5, 6, and 8 in both hemophilia A mice and dogs. AAV6-cFVIII and AAV8-cFVIII restored physiologic levels of plasma FVIII activity in hemophilia A mice. The improved efficacy is attributed to more efficient gene transfer in liver compared with AAV2 and AAV5. However, supraphysiologic cFVIII levels correlated with the formation of cFVIII-neutralizing antibodies in these mice. Of importance, hemophilia A dogs that received AAV2-cFVIII, AAV6-cFVIII, and AAV8-cFVIII have persistently expressed therapeutic levels of FVIII, without antibody formation or other toxicities, for more than 3 years. However, liver transduction efficiencies are similar between AAV2, AAV6, and AAV8 serotypes in hemophilia A dogs, in contrast to mice. In summary, this is the first report demonstrating multiyear therapeutic efficacy and safety of multiple AAV-cFVIII vectors in hemophilia A dogs and provides the basis for human clinical studies.

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.

Correction of murine hemophilia a by hematopoietic stem cell gene therapy

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally delivered FVIII transgene offers the possibility of permanently correcting the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based FVIII oncoretroviral vector into immunocompetent hemophilia A mice that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy), or a nonmyeloablative preparative regimen involving busulfan. Therapeutic levels of FVIII (42, 18, and 11% of normal, respectively) were detected in the plasma of the transplant recipients for the duration of the study (over 6 months). Moreover, subsequent challenge with recombinant FVIII elicited at most a minor anti-FVIII inhibitor antibody response in any of the experimental animals, in contrast to the vigorous neutralizing humoral reaction to FVIII that was stimulated in naive hemophilia A mice. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

Correction of Murine Hemophilia A by Hematopoietic Stem Cell Gene Therapy.

A serious complication of current protein replacement therapy for hemophilia A patients with coagulation factor VIII (FVIII) deficiency is the frequent development of anti-FVIII inhibitor antibodies that preclude therapeutic benefit from further treatment. Induction of tolerance by persistent high-level FVIII synthesis following transplantation with hematopoietic stem cells expressing a retrovirally-delivered FVIII transgene offers the possibility to permanently correct the disease. Here, we transplanted bone marrow cells transduced with an optimized MSCV-based oncoretroviral vector encoding a secretion-enhanced B domain-deleted human FVIII transgene linked to a downstream EGFP reporter gene into immunocompetent hemophilia A mice (FVIII exon 16 knockout mice on a C57BL/6 background) that had been conditioned with a potentially lethal dose of irradiation (800 cGy), a sublethal dose of irradiation (550 cGy) or a nonmyelablative preparative regimen involving busulfan (two intraperitoneal doses of 10 mg/kg). Both groups of irradiated mice were transplanted with 2 × 106 sorted EGFP+ bone marrow cells. At 26 weeks, 48 ± 24% (n = 10) and 18 ± 11% (n = 12) EGFP+ nucleated peripheral blood cells were detected in mice conditioned with 800 and 550 cGy irradiation, respectively. The busulfan-conditioned mice (n = 4) were transplanted with 15–20 × 106 transduced unsorted bone marrow cells. One mouse died at 4 weeks posttransplant due to an unknown cause. The reconstitution kinetics of the remaining three mice was very similar to the group of mice conditioned with 550 cGy irradiation (18 ± 7% of their nucleated peripheral blood cells were EGFP+ at 26 weeks posttransplant). Broad transcriptional activity of the vector was observed in cells belonging to both the myeloid and lymphoid lineages in peripheral blood, and in donor-derived cells residing within the bone marrow, spleen and thymus. Importantly, therapeutic levels of FVIII (42%, 18% and 11% of normal, respectively, by COATEST assay) were detected in the plasma of all recipients 22–26 weeks posttransplant. When the mice were subsequently challenged with high doses of recombinant human FVIII (up to eight intravenous injections of 5–10 IU of recombinant full-length human FVIII at weekly intervals) to investigate the durability of tolerance induction, only minimal levels of inhibitor antibodies were detected in a subset of the corrected animals (0.8 ± 0.6, 0.7 ± 0.5 and 3 ± 4 Bethesda units per ml) in contrast to the robust anti-FVIII inhibitor response seen following immunization of naive hemophilia A mice (98 ± 48 Bethesda units per ml; n = 11). Suppression of the immune response to human FVIII was specific, as gene-treated mice mounted a normal humoral immune reaction to an unrelated antigen, tetanus toxoid. While we make no claims as to the nature of the hyporesponsive states elicited in each case, the results obtained in the small cohort of busulfan-conditioned animals are particularly exciting since the experimental protocol more closely approximates a clinically-acceptable situation, both in terms of a mild conditioning regimen as well as the lack of a preselection step for transduced bone marrow cells. These findings represent an encouraging advance toward potential clinical application and long-term amelioration or cure of this progressively debilitating, life-threatening bleeding disorder.

Preclinical gene therapy studies for hemophilia using adenoviral vectors.

Hemophilia A and B are hereditary coagulation defects resulting from a deficiency of factor VIII (FVIII) and factor IX (FIX), respectively. Introducing a functional FVIII or FIX gene could potentially provide a cure for these bleeding disorders. Adenoviral vectors have been used as tools to introduce potentially therapeutic genes into mammalian cells and are by far the most efficient vectors for hepatic gene delivery. Long-term expression of both FVIII and FIX has been achieved in preclinical (hemophilic) mouse models using adenoviral vectors. Therapeutic levels of FVIII and FIX also have been achieved in hemophilic dogs using adenoviral vectors and in some cases expression was long-term. The performance of earlier generation adenoviral vectors, which retained residual viral genes, was compromised by potent acute and chronic inflammatory responses that contributed to significant toxicity and morbidity and short-term expression of FVIII and FIX. The development of improved adenoviral vectors devoid of viral genes (gutless or high-capacity adenoviral vectors) was therefore warranted, which led to a significant reduction in acute and chronic toxicity and more prolonged expression of FVIII and FIX. Strategies aimed at making these vectors safer and less immunogenic and their implications for hemophilia gene therapy are discussed in this review.

Nonviral Gene Therapy Approaches to Hemophilia

The goal of hemophilia gene therapy is to obtain long-term therapeutic levels of factor VIII (FVIII) or factor IX (FIX) without stimulating an immune response against the transgene product or the vector. The success of gene therapy is largely dependent on the development of appropriate gene delivery vectors. Both viral vectors and nonviral vectors have been considered for the development of hemophilia gene therapy. In general, viral vectors are far more efficient than nonviral gene delivery approaches and resulted in long-term therapeutic levels of FVIII or FIX in preclinical animal models. However, there are several reasons why a nonviral treatment would still be desirable, particularly because some viral vectors are associated with inflammatory reactions, that render transgene expression transient, or with an increased risk of insertional oncogenesis when random integrating vectors are used. Nonviral vectors may obviate some of these concerns. Since nonviral vectors are typically assembled in cell-free systems from well-defined components, they have significant manufacturing advantages over viral vectors. The continued development of improved nonviral gene delivery approaches offers new perspectives for gene therapy of chronic diseases including hemophilia.