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1
Bharadwaj, Arpita S., Meagan Kelly, Dongsoo Kim, and Hengjun Chao. "Induction of immune tolerance to FIX by intramuscular AAV gene transfer is independent of the activation status of dendritic cells." Blood 115, no. 3 (January 21, 2010): 500–509. http://dx.doi.org/10.1182/blood-2009-08-239509.
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Abstract The nature of viral vectors is suggested to be a significant contributor to undesirable immune responses subsequent to gene transfer. Such viral vectors, recognized as danger signals by the host immune system, activate dendritic cells (DCs), causing unwanted antivector and/or transgene product immunity. We recently reported efficient induction of immune tolerance to coagulation factor IX (FIX) by direct intramuscular injection of adeno-associated virus (AAV)–FIX. AAV vectors are nonpathogenic and elicit minimal inflammatory response. We hypothesized that the nonpathogenic nature of AAV plays a critical role in induction of tolerance after AAV gene transfer. We observed inefficient recruitment and activation of DCs subsequent to intramuscular injection of AAV. To further validate our hypothesis, we examined immune responses to FIX after intramuscular injection of AAV with simultaneous activation of DCs. We were able to achieve phenotypic and functional activation of DCs after administration of lipopolysaccharide and anti-CD40 antibody. However, we observed efficient induction of FIX tolerance irrespective of DC activation in mice with different genetic and major histocompatibility complex backgrounds. Furthermore, activation of DCs did not exaggerate the immune response induced after intramuscular injection of AAV serotype 2 vector. Our results demonstrate that induction of FIX tolerance after AAV gene transfer is independent of DC activation status.
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Arruda, Valder R., Hansell H. Stedman, Virginia Haurigot, George Buchlis, Stefano Baila, Patricia Favaro, Yifeng Chen, et al. "Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B." Blood 115, no. 23 (June 10, 2010): 4678–88. http://dx.doi.org/10.1182/blood-2009-12-261156.
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Abstract Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.
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Schuettrumpf, Joerg, Jianxiang Zou, Shin Jen Tai, Alexander Schlachterman, Kian Tian, Shyrie Edmonson, Jianhua Liu, Patricia Andrade-Gordon, Katherine High, and Valder Arruda. "A Novel Role of Coagulation Proteases on Viral-Based Gene Transfer Efficacy." Blood 104, no. 11 (November 16, 2004): 691. http://dx.doi.org/10.1182/blood.v104.11.691.691.
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Abstract Coagulation proteases are crucial for hemostasis and have also been implicated in inflammatory responses, blood vessel formation, and tumor cell metastasis. Cellular responses triggered by proteases are mediated by protease-activated receptors (PAR). Adeno-associated virus (AAV)-2 vectors hold promise for the treatment of several diseases and were already tested in Phase I studies for hemophilia B following intramuscular or hepatic artery deliveries. Previously, we determined an unexpected inhibitory effect (60–70% downregulation) on AAV-2 and adenovirus mediated gene transfer by thrombin- or FXa inhibitors. These results were independent of mouse strain, transgene product, or vector promoter, and gene expression by vectors of alternate serotypes AAV-5 or -8, which do not share cellular receptors with AAV-2, were not affected by any drug. Here we present in vivo evidence of a novel role of coagulation proteases and PARs in modulating gene transfer by viral vectors. We tested AAV-2 gene transfer efficacy in (a) animal models for proteases deficiency [FX and FIX deficient animals], (b) PAR-1 or PAR-2 deficient mice, (c) and following in vivo activation of PARs. FX knockout mice with residual activity of only 1–3% of normal (n=9) were injected with AAV-2-human(h)FIX vector and compared to littermates with FX levels of 50% (n=4). FIX expression levels were 2-fold lower among FX-deficient mice compared to controls (p<0.03). The second model, FIX deficient mice, received AAV expressing α1-antitrypsin (AAT-1). Severe hemophilia B models due to large-gene deletion (n=5) or missense mutation (R180T) in the FIX gene (n=3, <1% FIX) were compared to littermate controls with normal FIX levels (n=6). The results showed that AAT-1 levels among hemophilia B mice were 2-fold lower than in controls (24 vs 48 ng/ml, p<0.05, respectively). Because PAR activation by thrombin enhances αVβ5 (co-receptor for AAV-2 and adenovirus)-dependent cellular function (JBC 276:10952) we hypothesized that PAR modulates AAV-2 gene transfer. Homozygous (−/−) or heterozygous deficient (+/−) PAR-1 (n=24) or PAR-2 (n=25) mice received AAV-2-hF.IX and were compared to littermate controls (+/+). FIX levels among PAR-1 controls (1.9 μg/ml) were comparable to levels obtained among heterozygotes but higher than in homozygotes (1.1 μg/ml, p<0.02). Similarly, PAR-2 deficient mice presented 2-fold lower FIX levels than controls (0.7 vs 1.3 μg/ml, p<0.02) whereas heterozygous mice presented intermediate levels. To further confirm the role of PARs in AAV-2 gene transfer we activated PARs prior to AAV-2 injection. C57BL/6 mice received specific peptide agonists at doses ranging from 10 to 60 μM/kg (n=4 per dose and per peptide) and were compared to controls receiving scramble peptide. FIX levels increased 1.5 to 5-fold in a dose-dependent manner and the activation of PAR-1 and -2 simultaneously was superior to single peptide. Gene copy monitoring revealed low vector uptake by livers of PAR knockout mice while activation of PARs increased uptake. In conclusion, these data demonstrated a novel in vivo role of coagulation proteases and PARs on viral vectors (AAV-2 and adenovirus)-mediated gene expression and provide an alternative target to modulate gene therapy strategies.
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Jiang, Haiyan, Linda B. Couto, Susannah Patarroyo-White, Tongyao Liu, Dea Nagy, Joseph A. Vargas, Shangzhen Zhou, et al. "Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy." Blood 108, no. 10 (November 15, 2006): 3321–28. http://dx.doi.org/10.1182/blood-2006-04-017913.
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Abstract In a clinical study of recombinant adeno-associated virus-2 expressing human factor IX (AAV2-FIX), we detected 2 impediments to long-term gene transfer. First, preexisting anti-AAV neutralizing antibodies (NABs) prevent vector from reaching the target tissue, and second, CD8+ T-cell responses to hepatocyte-cell surface displayed AAV-capsid–terminated FIX expression after several weeks. Because the vector is incapable of synthesizing viral proteins, a short course of immunosuppression, until AAV capsid is cleared from the transduced cells, may mitigate the host T-cell response, allowing long-term expression of FIX. To evaluate coad-ministration of immunosuppression, we studied AAV8 vector infusion in rhesus macaques, natural hosts for AAV8. We administered AAV8-FIX in 16 macaques via the hepatic artery and assessed the effects of (1) preexisting anti-AAV8 NABs, (2) a standard T-cell immunosuppressive regimen, and (3) efficacy and safety of AAV8-FIX. We found that low titers (1:5) of preexisting NABs abrogate transduction, whereas animals with undetectable NABs are safely and effectively transduced by AAV8-FIX. Coadministration of mycophenolate mofetil and tacrolimus with vector does not induce toxicity and does not impair AAV transduction or FIX synthesis. These findings enable a clinical study to assess the effects of immunomodulation on long-term FIX expression in patients with hemophilia B.
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Kao, Chung-Yang, Chia-Ni Lin, I.-Shing Yu, Mi-Hua Tao, Hua-Lin Wu, Guey-Yueh Shi, Yung-Li Yang, Jau-Tsuen Kao, and Shu-Wha Lin. "FIX-Triple, a gain-of-function factor IX variant, improves haemostasis in mouse models without increased risk of thrombosis." Thrombosis and Haemostasis 104, no. 08 (2010): 355–65. http://dx.doi.org/10.1160/th09-11-0792.
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SummaryEngineered recombinant factor IX (FIX) with augmented clotting activity may prove useful for replacement therapy, but it has not been studied for risk of thrombosis. We used three mouse models to evaluate thrombosis risk associated with the FIX variant FIX-Triple, which has a 13-fold higher specific activity than wild-type FIX (FIX-WT). Protein infusion of FIX-Triple into haemophilia B mice was not thrombogenic, even at a dose of 13-fold higher than FIX-WT. Gene knock-in to generate mice that constitutively produce FIX-WT or FIX-Triple protein revealed that all mice expressed equal antigen levels. FIX-Triple knock-in mice that exhibited 10-fold higher FIX clotting activity did not show hypercoagulation. Adeno-associated viral (AAV) delivery of the FIX gene into mice was used to mimic gene therapy. Haemophilia B and inbred C57Bl/6 mice injected with different doses of virus particles carrying FIX-WT or FIX-Triple and expressing up to a nearly 13-fold excess (1289% of normal) of FIX clotting activity did not show increased risk of thrombosis compared with untreated wild-type mice in a normal haemostatic state. When challenged with ferric chloride (FeCl3), the mesenteric venules of AAV-treated C57Bl/6 mice that gave a nearly five-fold excess (474%) of FIX clotting activity were not thrombotic; however, thrombosis became obvious in FeCl3-challenged mice expressing extremely high FIX clotting activities (976–1289%) achieved by AAV delivery of FIX-Triple. These studies suggest that FIX-Triple is not thrombogenic at therapeutic levels and is a potential therapeutic substitute for FIX-WT.
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Kao, Chung-Yang, Shu-Jhu Yang, Mi-Hua Tao, Yung-Ming Jeng, I.-Shing Yu, and Shu-Wha Lin. "Incorporation of the factor IX Padua mutation into FIX-Triple improves clotting activity in vitro and in vivo." Thrombosis and Haemostasis 110, no. 08 (2013): 244–56. http://dx.doi.org/10.1160/th13-02-0154.
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SummaryUsing gain-of-function factor IX (FIX) for replacement therapy for haemophilia B (HB) is an attractive strategy. We previously reported a high-activity FIX, FIX-Triple (FIX-V86A/E277A/R338A) as a good substitute for FIX-WT (wild-type) in protein replacement therapy, gene therapy, and cell therapy. Here we generated a new recombinant FIXTripleL (FIX-V86A/E277A/R338L) by replacing the alanine at residue 338 of FIX-Triple with leucine as in FIX-Padua (FIX-R338L). Purified FIX-TripleL exhibited 22-fold higher specific clotting activity and 15-fold increased binding affinity to activated FVIII compared to FIXWT. FIX-TripleL increased the therapeutic potential of FIX-Triple by nearly 100% as demonstrated with calibrated automated thrombogram and thromboelastography. FIX-TripleL demonstrated a normal clearance rate in HB mice. The clotting activity of FIX-TripleL was consistently 2- to 3-fold higher in these mice than that of FIX-Triple or FIXR338L. Gene delivery of adeno-associated virus (AAV) in HB mice showed that FIX-TripleL had 15-fold higher specific clotting activity than FIX-WT, and this activity was significantly better than FIX-Triple (10-fold) or FIX-R338L (6-fold). At a lower viral dose, FIX-TripleL improved FIX activity from sub-therapeutic to therapeutic levels. Under physiological conditions, no signs of adverse thrombotic events were observed in long-term AAV-FIX-treated C57Bl/6 mice. Hepatocellular adenomas were observed in the high- but not the medium- or the lowdose AAV-treated mice expressing FIX-WT or FIX-Triple, indicating the advantages of using hyperfunctional FIX variants to reduce viral doses while maintaining therapeutic clotting activity. Thus, incorporation of the FIX Padua mutation significantly improves the clotting function of FIX-Triple so as to optimise protein replacement therapy and gene therapy.
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Wang, Lili, Yang Yang, Camilo Ayala Breton, John White, Jia Zhang, Yan Che, Alexei Saveliev, et al. "CRISPR/Cas9-mediated in vivo gene targeting corrects hemostasis in newborn and adult factor IX–knockout mice." Blood 133, no. 26 (June 27, 2019): 2745–52. http://dx.doi.org/10.1182/blood.2019000790.
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Abstract Many genetic diseases, including hemophilia, require long-term therapeutic effects. Despite the initial success of liver-directed adeno-associated virus (AAV) gene therapy for hemophilia in clinical trials, long-term sustained therapeutic effects have yet to be seen. One explanation for the gradual decline of efficacy over time is that the nonintegrating AAV vector genome could be lost during cell division during hepatocyte turnover, albeit at a slow pace in adults. Readministering the same vector is challenging as a result of the AAV-neutralizing antibodies elicited by the initial treatment. Here, we investigated the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated homology-directed gene targeting for sustained treatment of hemophilia B. We developed a donor vector containing a promoterless partial human factor IX (FIX) complementary DNA carrying the hyperactive FIX Padua mutation. A single injection of dual AAV vectors in newborn and adult FIX-knockout (FIX-KO) mice led to stable expression of FIX at or above the normal levels for 8 months. Eight weeks after the vector treatment, we subjected a subgroup of newborn and adult treated FIX-KO mice to a two-thirds partial hepatectomy; all of these animals survived the procedure without any complications or interventions. FIX levels persisted at similar levels for 24 weeks after partial hepatectomy, indicating stable genomic targeting. Our results lend support for the use of a CRISPR/Cas9 approach to achieve lifelong expression of therapeutic proteins.
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Baila, Stefano, Christian Furlan Freguia, Nicholas Iacobelli, Danielle Dunn, Joerg Schuettrumpf, Federico Mingozzi, Patricia Andrade-Gordon, and Valder R. Arruda. "Protease−Activated Receptor 2 (PAR−2) as a Novel Target To Prevent Inhibitor Formation to FIX." Blood 108, no. 11 (November 16, 2006): 763. http://dx.doi.org/10.1182/blood.v108.11.763.763.
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Abstract Direct intramuscular injection (IM) of adeno−associated viral (AAV) serotype 2 in humans with hemophilia B (HB) is a promising therapeutic strategy since muscle biopsies obtained >3 years after vector injection demonstrated stable local gene expression. However to achieve therapeutic FIX levels using AAV−2 would required hundreds IM injections. The use of alternate AAV serotypes is an attractive strategy since AAV−1 or AAV−6, resulted in a >10−fold increase in transgene levels compared to AAV−2 in large animals, but the immune response to the transgene product has been consistently demonstrated as a major limitation of this strategy. There is growing evidence that blood proteases play an important role in modulating inflammatory and immune responses through activation of PARs. Mice lacking PAR−1(−/−) or PAR−2(−/−) alleles presented amelioration of immune− or infection−mediated diseases. Here we sought to determine whether inhibition of PARs could be used as a strategy to prevent immune responses to the FIX following AAV−mediated gene transfer to skeletal muscle. We used PAR−1 and PAR−2 knockout mice on C57Bl/6 background and littermate mice received IM injection of AAV1−CMV−hFIX. At dose 5x1011vg/kg, PAR−2 (−/−) mice (n=5) exhibited circulating FIX levels of 500± 99ng/ml (8–10%) which remained stable for the duration of the experiment (10 weeks), and no antibodies for FIX were detected (n=5). In contrast, all PAR−2 (+/+) mice (n=4) developed antibodies to FIX which inhibits FIX clotting activity, as determined by Bethesda assay (2.1± 0.6 BU). However, when similar vector doses were delivered to PAR−1(−/−) or PAR−1(+/+) (n=4/genotype) mice, antibodies to FIX developed in all animals. We next tested a higher vector dose in PAR−2 models. At dose 1x1012vg/kg, PAR−2(−/−) mice(n=7) resulted in FIX levels of 1,500±353ng/ml, and again no antibodies for FIX were detected. At the same dose, 6 out of 10 mice of PAR−2 (+/+)/(+/−) developed inhibitory antibodies (1.8± 0.7 BU). Further increase in the vector dose to 5 x 1012 vg/kg resulted in the development of inhibitor to FIX in both PAR−2 (−/−) (4/11 mice, 36%) and PAR−2(+/+)/(+/−) (10/17 mice, 60%). This suggests a threshold value in the protective effect in the PAR−2 (−/−) model. We sought to assay for FIX−specific T−cell by ELISPOT assay to quantify IFN−γ secretion from splenocytes of PAR−2 (−/−) and PAR−2 (+/+) mice injected at 5x1011 or 1x1012vg/kg. No difference in IFN−γ secretion was observed between PAR−2 (−/−) and their controls. Moreover, upon repeated challenges with FIX protein following vector injection antibody to FIX was detected in only 1/4 PAR−2 (+/+) mouse and none of 5 PAR−2 (−/−). Thus, PAR−2 inhibition does not compromise the tolerance to FIX. In a different model, intravenous injection of FIX protein into normal mice upon simultaneous activation of PAR−2 by using specific agonist peptide the rates of FIX antibody formation were comparable with those of a control peptide group. Thus, PAR−2−mediating antibody formation to FIX may differ among distinct immunologic challenges. Together, these data suggest that PARs play a role in the immune response to FIX and that inhibition of PAR−2 (but not PAR−1) could be a novel target in preventing inhibitor formation in hemophilia gene therapy and potentially for protein−based therapy.
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Kelly, Meagan E., Jiacai Zhuo, Arpita S. Bharadwaj, and Hengjun Chao. "Induction of Immune Tolerance to FIX Following Muscular AAV Gene Transfer Is AAV-dose/FIX-level Dependent." Molecular Therapy 17, no. 5 (May 2009): 857–63. http://dx.doi.org/10.1038/mt.2009.25.
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Markusic, David M., and Brett Palaschak. "Defining the Risk of Suboptimal AAV8-F9 Vector Delivery in Inhibitor Positive Hemophilia B Mice." Blood 128, no. 22 (December 2, 2016): 3508. http://dx.doi.org/10.1182/blood.v128.22.3508.3508.
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Abstract Hemophilia B (HB) is a coagulation disorder where patients lack functional coagulation factor IX (FIX) protein. The recommended treatment is prophylactic infusion of plasma derived or recombinant FIX protein. 3-5% of HB patients develop anti-drug antibodies, termed inhibitors. Patients with high titer inhibitors (> 5 BU) require bypassing agents for management of hemostasis. Immune tolerance induction therapy (ITI), while effective in hemophilia A patients (factor VIII deficiency), is less effective in HB patients due to the development of severe anamnestic responses. Over the last 25 years, gene therapy has emerged as an effective therapy for HB patients. Adeno-associated virus (AAV) vectors have had the best translational success in the clinic. Early clinical studies revealed an unpredicted risk of a vector dose dependent immune response targeting the AAV capsid presented by transduced hepatocytes, resulting in the loss in FIX protein expression. Successive clinical trials have adapted to now include transient immune suppression, alternative capsids, and a naturally occurring hyperactive FIX variant, to reach therapeutic FIX activity with reduced vector doses. Importantly, there has been no report of inhibitors in AAV treated HB patients, with the caveat that patients with a high risk for inhibitors have not been treated. Studies in HB mice and dogs have shown that AAV-F9 gene delivery to and expression in hepatocytes induces immunological tolerance to FIX protein, dependent on a threshold level of FIX protein expression and regulatory T cells (Treg). Therefore, we hypothesized that AAV-F9 liver gene transfer may be an effective ITI therapy. Using an inhibitor and anaphylaxis prone HB mouse strain (C3H/HeJ-F9-/Y), we demonstrated that AAV-F9 gene therapy rapidly eliminated high titer anti-FIX IgE and IgG1 antibodies, provided long-term correction of hemostasis, and protected mice from anamnestic responses following supplemental FIX protein therapy (Markusic et al. EMBO Mol Med 2013). The average reported sustained levels of FIX protein expression in ongoing clinical studies is ~5% normal, at the threshold level for ITI in our model (Markusic et al. EMBO Mol Med 2013), raising concerns regarding the efficacy and safety of gene therapy for inhibitor positive patients. To address this, we set out to identify a suboptimal AAV-F9 vector dose (2x109 - 1x1011 vg) in C3H/HeJ-F9-/Y mice with the following criteria (1) measurable expression of FIX protein, (2) absence of spontaneous inhibitors, and (3) susceptibility to inhibitor formation following FIX protein challenge.Vector treated mice were followed for 4-8 weeks and were then immunized with FIX protein in adjuvant and followed out 20 weeks. As previously shown mice receiving 1x1010 and 1x1011 vg had stable FIX expression that persisted following FIX-adjuvant challenge. We identified a suboptimal vector dose of 6x109 vg with an average FIX level of ~75 ng/mL (1.5% normal) over 8 weeks which became undetectable following adjuvant-FIX challenge. Next we generated three groups of inhibitor positive mice (1) no treatment control, (2) 6x109 vg AAV8-F9, and (3) 1x1011 vg AAV8-F9 treated to determine the impact of suboptimal vector on inhibitor levels and potential risks of anaphylaxis. Mice receiving the suboptimal vector dose were monitored daily for signs of anaphylaxis out to two weeks post vector delivery. No animals in the 6x109 vg group displayed any physical symptoms of anaphylaxis. Positive control mice receiving 1x1011 vg demonstrated a rapid reduction in anti-FIX titers at two weeks post gene delivery and have had sustained FIX levels ~100% of normal. Whereas mice in the 6x109vg treated group have had undetectable FIX levels and sustained and elevated anti-FIX IgG1 levels compared to untreated control inhibitor mice. The study is still ongoing and we have now followed mice out to 12 weeks post vector delivery. This study raises some potential concerns regarding gene therapy in HB patients with inhibitors. The present adoption of hyperactive FIX protein in gene therapy vectors, inefficient human hepatocyte transduction, and immune responses against AAV capsids may compromise tolerance induction by reducing circulating FIX protein levels, as modeled in our present study. These studies were supported by an ASPIRE Hemophilia Research Award from Pfizer. Disclosures Markusic: Pfizer: Research Funding.
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Finn, Jonathan D., Timothy C. Nichols, Nikolaos Svoronos, Elizabeth P. Merricks, Dwight A. Bellenger, Shangshen Zhou, Paolo Simioni, Katherine A. High, and Valder R. Arruda. "The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy." Blood 120, no. 23 (November 29, 2012): 4521–23. http://dx.doi.org/10.1182/blood-2012-06-440123.
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Abstract Studies on gene therapy for hemophilia B (HB) using adeno-associated viral (AAV) vectors showed that the safety of a given strategy is directly related to the vector dose. To overcome this limitation, we sought to test the efficacy and the risk of immunogenicity of a novel factor IX (FIX) R338L associated with ∼ 8-fold increased specific activity. Muscle-directed expression of canine FIX-R338L by AAV vectors was carried out in HB dogs. Therapeutic levels of circulating canine FIX activity (3.5%-8%) showed 8- to 9-fold increased specific activity, similar to humans with FIX-R338L. Phenotypic improvement was documented by the lack of bleeding episodes for a cumulative 5-year observation. No antibody formation and T-cell responses to FIX-R338L were observed, even on challenges with FIX wild-type protein. Moreover, no adverse vascular thrombotic complications were noted. Thus, FIX-R338L provides an attractive strategy to safely enhance the efficacy of gene therapy for HB.
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French, Robert, Nicholas Martin, Timothy C. Nichols, Glenn P. Niemeyer, Clinton D. Lothrop, and Valder R. Arruda. "Complete Correction of Severe Canine Hemophilia B By Skeletal Muscle Directed AAV-Based FIX-Padua Gene Therapy in Inhibitor-Prone Dogs." Blood 126, no. 23 (December 3, 2015): 3487. http://dx.doi.org/10.1182/blood.v126.23.3487.3487.
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Abstract Hemophilia B (HB) is an X-linked bleeding disorder caused by a deficiency in Factor IX (FIX). Ongoing gene therapy clinical trials for HB using adeno-associated viral (AAV) vectors targeting the liver have demonstrated sustained FIX expression at therapeutic levels and improvement of the bleeding phenotype in severe HB patients. However, one of the major safety concerns is the cellular immune response directed against vector capsid antigens that can limit FIX expression if not promptly treated with transient immunosuppression. Emerging evidence suggests that this complication is vector dose dependent, occurring only at doses of 2 x 1012 vg/kg or higher. FIX-Padua, a naturally occurring hyperactive FIX variant we have preciously described (Simioni et al. NEJM 2009), which exhibits ~8-fold increase in specific activity, offers a potential way to decrease the vector dose while maintaining hemostatic efficacy. In preclinical studies, we have recently shown that in severe HB dogs with a high risk for inhibitor formation to canine FIX (cFIX) protein or with preexisting inhibitors to cFIX, liver expression after AAV-cFIX Padua gene therapy resulted in immune tolerance induction and eradication of inhibitory antibodies (Crudele et al., Blood 2015). In addition, the first description of an ongoing clinical trial with AAV-FIX-Padua reported therapeutic FIX levels without the development of FIX inhibitors. However, the success of liver-directed gene therapy approaches is not applicable to those HB patients with underlying liver disease, mostly due to HCV infection that affects >80% of adults with hemophilia treated with plasma-derived products. Thus, we sought to investigate AAV delivery to skeletal muscle as a potential alternative for these patients. Clinical trials and preclinical studies in HB dogs have demonstrated that the therapeutic vector dose for AAV muscle gene therapy was a log higher than liver GT when direct multi-site intramuscular injection was used; however, intravascular delivery to the muscle allows the vector dose to be significantly reduced to 2-3 x 1012 vg/kg. Here, we used a regional peripheral transvenular injection of AAV serotype 6 to express cFIX-Padua in the skeletal muscle of HB dogs. These dogs are at high risk for inhibitor formation due to the null mutation in their canine F9 gene resulting in no mRNA or protein expression; typically, a single injection of cFIX wild-type protein (0.5 mg) results in the formation of a high titer inhibitor. Two adult dogs received gene therapy with 3 x 1012 vg/kg of AAV6-cFIX-Padua under the control of a CMV promoter. One dog (Malani) reached a plateau of 80-100% circulating cFIX activity levels with a follow-up time of 10 months (ongoing). Sustained antigen levels are 7-10%, resulting in ~10-fold higher specific activity than wild type FIX. The second dog (Una) achieved 45-60% sustained activity with corresponding 4-5% antigen levels (~11-fold increase in specific activity) after 14 months (ongoing observation). Neither dog has developed inhibitors to cFIX. Both dogs are also negative for non-neutralizing IgG1 and IgG2 against cFIX. Whole blood clotting time and thromboelastogram parameters have also normalized in both dogs. Notably, immune tolerance has been maintained despite challenge with 0.5 mg of cFIX-wild type protein. Together, these data support the use of skeletal muscle as a target tissue for the expression of FIX Padua and, in contrast to an early direct AAV-FIX intramuscular injection trial, patients with null mutations can now be enrolled. This is the first demonstration of complete correction of HB in large animal models using AAV gene therapy targeting the skeletal muscle and supports the feasibility and safety for potential clinical studies. Disclosures Arruda: Spark Therapeutics: Patents & Royalties; Pfizer: Consultancy, Patents & Royalties, Research Funding.
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High, Katherine, Michael Tigges, Catherine Manno, Denise Sabatino, Valder Arruda, Roland Herzog, Pradip Rustagi, et al. "Human Immune Responses to AAV-2 Capsid May Limit Duration of Expression in Liver-Directed Gene Transfer in Humans with Hemophilia B." Blood 104, no. 11 (November 16, 2004): 413. http://dx.doi.org/10.1182/blood.v104.11.413.413.
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Abstract Based on studies in mice, hemophilic dogs, and non-human primates demonstrating long-term (>5 yrs) expression of Factor IX (FIX) after infusion of an AAV vector expressing FIX into the portal vein or the hepatic artery, we undertook a Phase I dose escalation study of AAV-FIX in humans with severe hemophilia B. The first two doses, 2x1011 vg/kg, and 1x1012 vg/kg, were safe but subtherapeutic. Two subjects treated at a dose of 5x1012 vg/kg showed detectable circulating levels of FIX (up to 11.8% and 3% respectively), but expression was transient and accompanied in one case (subject E) by a transient asymptomatic transaminitis. There was never evidence of a FIX inhibitor. Two differences between the large animal models and humans with the disease were hypothesized to contribute to the difference in duration of expression; long-term in hemophilic dogs, short-term in hemophilic humans. First was pre-existing immunity to wild-type AAV-2, which infects humans, but not dogs; and the other was prior exposure to viral hepatitis , found in humans but not in animals. To further assess the roles of viral hepatitis and of the immune response to AAV-2, we treated an additional subject (subject G) at a dose of 1x1012 vg/kg. This subject was 20 yrs. of age and had never been infected with hepatitis. Nevertheless, his transaminases began to rise 3 weeks after vector injection, peaked 6 weeks after injection, and resolved spontaneously as had been seen in subject E. In subject G, magnitude of the peak ALT response was 5-fold less than that found in subject E (5-fold higher dose). Both subjects had similar and low baseline anti-AAV antibody titers. Immune response to AAV-2 was assessed by ELISpot at serial time points before and after vector injection in subject G. The subject’s PBMCs were incubated with a peptide library arrayed in a matrix of 24 pools, each containing 12 peptides of 15-mers overlapping by 10 and spanning the entire VP-1 protein. There was no detectable IFN- γ secretion in response to AAV-2 peptides at baseline, although there was a strong IFN- γ response to PHA. Two weeks after vector infusion, three pools elicited IFN- γ secretion from the subject’s PBMCs. Response to the same pools of peptides, but not to other pools, was repeatedly detected over the next 6 weeks. By week 12, IFN- γ responses were no longer detectable. The matrix array allowed identification of two specific AAV-2 capsid peptides as the T cell immunoreactive epitopes. These peptides are highly conserved in AAV serotypes 1–8. Similar experiments were conducted with a FIX peptide library and demonstrated no response. These data are consistent with a model in which a T cell response to AAV capsid epitopes results in elimination of the transduced cells. This response is only briefly detectable in PBMCs, and hepatitis is not an important risk factor. These immune responses may limit use of standard serotypes of AAV for gene transfer into human liver. Transient immunomodulation may prevent these responses.
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Baila, Stefano, Christian Furlan Freguia, Daniel Orr, Federico Mingozzi, Joerge Schuettrumpf, and Valder R. Arruda. "Protease-Activated Receptor-2 (PAR-2) as a Novel Target for Modulating Immune Responses to Neo Antigens Following In Vivo Gene Transfer." Blood 106, no. 11 (November 16, 2005): 1296. http://dx.doi.org/10.1182/blood.v106.11.1296.1296.
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Abstract The safety of several strategies using novel adeno-associated viral (AAV) vectors (serotypes-1,-5,-7,and -8) to skeletal muscle of large animals have been hampered by the immune response to the transgene (Blood103: 85, 3303, and 3330). Dendritic cells (DC) are potent antigen-presenting cells that govern the immune responses. The involvement of proteases is critical to DC antigen processing and presentation. Proteases’ effects on cells are mediated by protease-activated receptor (PAR) a group of four G-protein-coupled receptors. Bone marrow-derived DC from PAR-2 knockout mice do not spontaneously develop to mature state and only do so upon stimulations of inflammatory signals, therefore, PAR-2 proved critical for DC activation. Work with selective PAR-2 agonists and knockout animals suggests a in vivo contribution of this receptor to several chronic inflammatory diseases and tumor development. In fact, small peptides functioning as PAR-2 antagonists are now being tested as anti-angiogenic agents. We sought to determine whether inhibiting specific targets on the T-cell activation cascade would provide a strategy to prevent the development of antibody following IM injection of AAV. To do this we evaluated the risk of inhibitior formation to clotting factor IX (FIX) in PAR-2 deficient mice compared with heterozygous (+/−) or normal (+/+) genotypes. Mice received IM injection of AAV-1 encoding human FIX (hFIX) at two different doses (low and high cohorts). FIX and antibody levels were monitored weekly. PAR-2(−/ −) mice(n=4) given the low dose exhibited circulating FIX levels of 500± 99ng/ml which remained stable for the duration of the experiment(10 weeks), and no antibody for FIX was detected. In three PAR-2(+/+) mice, FIX expression was transiently detected at week 2 followed by a period of 4 weeks with undetectable levels. This was due to the formation of FIX specific IgG-1 antibody, which results from activation of CD4+ T-cell mainly of Th2 subset, that peaked at week 6. These antibodies inhibit FIX clotting activity as determined by a functional assay at titre of 3–5.5BU (Bethesda Unit) in all 3 mice. High dose AAV-1 treated PAR-2(−/ −) mice(n=6) resulted in continuous expression of FIX(1,500±353ng/ml) in the absence of FIX antibody. In contrast, inhibitors of FIX were detected in all mice(5 per group) PAR-2(+/−) or PAR-2(+/+). All animals develop IgG-1 antibodies to FIX through 10 weeks of observation. At week 6 post-injection, FIX inhibitors were detected in 3/10 mice at levels of ~1 BU. Continuous follow-up showed that a decrease in antibody titers to FIX was associated with slowly increasing FIX antigen. In addition, splenocytes from PAR-2 (+/+) mice exhibit significant increase (p<0.05) in antigen-specific IFN-γ secretion detected by ELISpot whereas in PAR-2 (−/ −) mice no response was detected. Furthermore, in PAR-1(−/ −) and PAR-1(+/+) mice IM injected with AAV-1, FIX inhibitor was detected in all animals (n=4/group). This demonstrates a PAR-2-specific immunomodulatory mechanism. In summary, these data suggest that pharmacological inhibition of PAR-2 may provide a novel strategy to evade the immune response to the transgene in a variety of gene-based strategies.
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Ge, Ying, Sandra Powell, Melinda Van Roey, and James G. McArthur. "Factors influencing the development of an anti–factor IX (FIX) immune response following administration of adeno-associated virus–FIX." Blood 97, no. 12 (June 15, 2001): 3733–37. http://dx.doi.org/10.1182/blood.v97.12.3733.
Full textAbstract:
The present study sought to determine the impact of the route of administration of an adeno-associated virus (AAV) vector encoding human factor IX (hFIX) on the induction of an immune response against the vector and its xenogenic transgene product, hFIX. Increasing doses of AAV-hFIX were administered by different routes to C57Bl/6 mice, which typically demonstrate significant immune tolerance to hFIX. The route of delivery had a profound impact on serum hFIX levels as well as the induction of an anti-hFIX humoral immune response. At all dose levels tested, delivery of AAV-hFIX by an intramuscular (IM) route induced an antibody response against the human FIX protein and no hFIX was detected in the serum of animals even at doses of 2 × 1011 DNA viral particles (vp) of AAV-hFIX. This was in stark contrast to the mice that received AAV-hFIX by intraportal vein (IPV) administration. No anti-hFIX inhibitors were observed in any of these mice and therapeutic levels of hFIX were detected in the serum of all mice that received doses of 2 × 1010 vp AAV-hFIX and higher. When pre-existing neutralizing immunity to AAV was established in mice, AAV-hFIX administration by either the IM or IPV routes did not result in detectable serum hFIX. Although hFIX expression was not observed in mice with pre-existing neutralizing immunity to AAV, an anti-hFIX response was induced in all of the animals that received AAV-hFIX by the IM route. This was not observed in the preimmune mice that received AAV-hFIX by IPV administration. These results suggest that the threshold of inducing an immune response against a secreted transgene product, in this case the xenoprotein hFIX, is lower when the vector is administered by the IM route even in animals with pre-existing immunity to AAV.
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Scallan, Ciaran D., Haiyan Jiang, Tongyao Liu, Susannah Patarroyo-White, Jurg M. Sommer, Shangzhen Zhou, Linda B. Couto, and Glenn F. Pierce. "Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice." Blood 107, no. 5 (March 1, 2006): 1810–17. http://dx.doi.org/10.1182/blood-2005-08-3229.
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Long-term cures of hemophilia B have been achieved using AAV2 delivering the factor IX gene to the liver of adeno-associated virus (AAV)–naive hemophilic animals. However, the clinical success of this approach requires overcoming pre-existing AAV neutralizing antibodies prevalent in humans. To better define the inhibition of neutralizing antibodies on AAV2-mediated liver transduction, we developed an in vivo passive immunity model. SCID mice were first reconstituted to a defined neutralizing titer with pooled plasma-derived human immunoglobulin. AAV2-FIX vectors then were administered to the liver, and the transduction efficiency was measured by plasma FIX levels. Unexpectedly, AAV2 neutralizing titers lower than 1:10 were sufficient to neutralize 4 to 20 × 1012 vg/kg of AAV2 vectors in vivo, a capacity that was underestimated by in vitro neutralizing assays. We also evaluated strategies to evade neutralization, including the use of alternative delivery routes, infusion parameters, empty capsids, and alternative AAV serotypes 6 and 8. The results indicate that low AAV2 neutralizing titers can be inhibitory to the tested human and primate AAV vectors delivered into the circulatory system. Therefore, novel nonprimate AAV vectors or compartmentalized delivery may offer more consistent therapeutic effects in the presence of pre-existing AAV neutralizing antibodies.
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Nathwani, Amit C., Edward GD Tuddenham, Savita Rangarajan, Cecilia Rosales, Jenny H. McIntosh, David C. Linch, Pratima Chowdary, et al. "Adeno-Associated Viral Vector Mediated Gene Transfer for Hemophilia B." Blood 118, no. 21 (November 18, 2011): 5. http://dx.doi.org/10.1182/blood.v118.21.5.5.
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Abstract Abstract 5 Background: Hemophilia B (HB), an X-linked bleeding disorder, is ideally suited for gene therapy. We investigated a novel approach using peripheral vein infusion of a single dose of a serotype-8 pseudotyped self-complementary adeno-associated virus (AAV) vector expressing a codon-optimized coagulation factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco). Methods: Six severe HB subjects (FIX ≤1%) were enrolled sequentially into one of three dose cohorts with two subjects in each group. Vector was administered without immunosuppression. The subjects were followed for 6–16 months post treatment. Results: AAV-mediated expression of FIX at 2–11% of normal was observed in all subjects. Four of the six have discontinued prophylaxis and remain free of spontaneous hemorrhage. The other two have increased the interval between FIX prophylaxes. A high-dose subject developed asymptomatic, transient elevation of serum transaminases associated with detection of AAV8 capsid specific T cells in peripheral blood. The second high-dose subject experienced a slight increase of liver enzymes, of less clear etiology. Treatment of each with a short course of steroids led to rapid normalization of the transaminases and maintenance of FIX levels in the 3–11% range. Conclusion: Peripheral vein administration of scAAV2/8-LP1-hFIXco was well tolerated and resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype. Immune-mediated clearance of AAV-transduced hepatocytes remains a concern but our data suggest that this process may be controlled with a short course of steroids without loss of transgene expression. Hence, our novel approach shows promise for gene therapy of HB and other protein deficiencies. (ClinicalTrials.gov number, NCT00979238) Disclosures: Nathwani: Amsterdam Molecular Therapeutics: Patents & Royalties. Gray:Amsterdam Molecular Therapeutics: Patents & Royalties. Davidoff:Amsterdam Molecular Therapeutics: Patents & Royalties.
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Nathwani, Amit C. "Gene therapy for hemophilia." Hematology 2019, no. 1 (December 6, 2019): 1–8. http://dx.doi.org/10.1182/hematology.2019000007.
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Abstract Gene therapy offers the potential for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX (FIX) following transfer of a functional gene to replace the hemophilic patient’s own defective gene. The hemophilias are ideally suited for gene therapy because a small increment in blood factor levels (≥5% of normal) is associated with significant amelioration of bleeding phenotype in severely affected patients. In 2011, the St. Jude/UCL phase 1/2 trial was the first to provide clear evidence of a stable dose-dependent increase in FIX levels in patients with severe hemophilia B following a single administration of adeno-associated viral (AAV) vectors. Transgenic FIX expression has remained stable at ∼5% of normal in the high-dose cohort over a 7-year follow-up period, resulting in a substantial reduction in spontaneous bleeding and FIX protein usage without toxicity. This study has been followed by unparalleled advances in gene therapy for hemophilia A and B, leading to clotting factor activity approaching normal or near-normal levels associated with a “zero bleed rates” in previously severely affected patients following a single administration of AAV vectors. Thus, AAV gene therapies are likely to alter the treatment paradigm for hemophilia A and B. This review explores recent progress and the remaining limitations that need to be overcome for wider availability of this novel treatment of inherited bleeding disorders.
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Samelson-Jones, Benjamin J., John D. Finn, Rodney M. Camire, and Valder R. Arruda. "The Complete Dependence of Factor IX Padua (R338L) Hyperactivity on Factor VIIIa Cofactor Activity Supports Its Safety As a Transgene for Hemophilia B Gene Therapy." Blood 132, Supplement 1 (November 29, 2018): 3486. http://dx.doi.org/10.1182/blood-2018-99-119270.
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Abstract Factor IX (FIX) Padua (R338L) has been described as a game changer for hemophilia B (HB) gene therapy. The ~8-fold increased specific activity compared to wild-type FIX (FIX WT) in aPTT-based clotting assay has recently allowed for a lowering of adeno-associated virus (AAV) vector dose compared to earlier gene therapy trials using FIX WT, while still achieving sustained near-curative FIX activity levels. The lowered AAV vector dose mitigated the vector dose-dependent hepatotoxicity, which had remained a major safety and efficacy limitation for AAV gene therapy. To date, at least 2 pivotal phase III gene therapy trials for HB using AAV FIX Padua are planned. Despite this enthusiasm, the underlying molecular mechanism of the hyperactivity of FIX Padua is undefined. As such, the safety concerns of unregulated FIX activity and the potential for ensuing thrombotic complications have not been fully addressed. Indeed, recent thrombotic complications in non-gene therapy hemophilia clinical trials evaluating non-factor therapies that promote hemostasis by circumventing regulatory interactions should engender caution. Activated FVIII (FVIIIa) is essential for the biological activity of activated FIX (FIXa), which provides an important regulatory requirement. To determine the role of FVIIIa in the hyperactivity of FIX Padua, we measured the relative specific activity of FIX Padua compared to WT in HB plasma using a unique reagent, ACE910. ACE910 is a bispecific antibody that sufficiently brings together FIXa and its substrate, FX, to efficiently promote hemostasis, even in the absence of FVIIIa molecules. It has been described as a "FVIII-mimetic" and is currently approved as a bypassing agent for hemophilia A patients with FVIII inhibitors. In our assays, plasma FVIII activity is inhibited to <1% normal activity with a cocktail of monoclonal anti-FVIII antibodies. In unadulterated HB plasma, recombinant zymogen FIX Padua has a ~8-fold higher clotting activity compared to FIX WT (Fig). However, when FVIIIa is replaced by ACE910, FIX Padua and WT have comparable clotting activities (Fig). Similarly, we observe that the hyperactivity of activated FIX Padua compared to WT in a clotting assay is completely abrogated by replacing FVIIIa with ACE910. These results suggest that the enhanced clotting activity of FIX(a) Padua compared to FIX(a) WT is wholly dependent on FVIIIa cofactor activity. We also measured the ability of recombinant FIX(a) Padua and WT to restore thrombin production to HB plasma in a thrombin generation assay (TGA). We observe that the EC50 of thrombin generation in HB plasma of zymogen and activated FIX Padua is ~8-fold lower than for FIX(a) WT. However, when FVIIIa is replaced with ACE910, FIX(a) Padua and WT demonstrate comparable EC50s of thrombin generation. Importantly, we observe no difference in the binding of FIXa Padua and FIXa WT with ACE910 in enzyme kinetic studies; this was expected because FIXa Padua and WT have an identical ACE910 binding site in their EGF-domain. Combined, these results demonstrate that the ~8 fold hyperactivity of FIX(a) Padua compared to FIX(a) WT in plasma assays requires FVIIIa cofactor activity. Consistent with this mechanism, we have previously observed in reconstituted systems that FIX(a) Padua and WT have comparable rates of activation by FXIa and inactivation by antithrombin. This result suggests that FIX Padua and WT are regulated similarly, supporting the safety of FIX Padua as a transgene. Enzyme kinetic studies of FIXa Padua and WT without FVIIIa demonstrate similar rates of FX activation, while FIXa Padua demonstrates increased rates of FX activation with FVIIIa compared to FIXa WT. We conclude, therefore, the mechanism of the increased specific activity of FIX(a) Padua is wholly dependent on its enhanced interaction with its cofactor FVIIIa. This enhancement is essential for the increased activity observed in both reconstituted and plasma-based assays. Moreover, the augmented interaction of FIX(a) Padua with FVIIIa is sufficient to account for the ~8-fold pro-hemostatic enhancement compared to FIX(a) WT observed in plasma assays. Combined, these results definitively address previously unanswered safety concerns regarding the potential risk of thrombotic complications with the use of FIX Padua transgene. They strongly support the ongoing use of FIX Padua in HB gene therapy. Disclosures Camire: Pfizer: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.
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Mount, Jane D., Roland W. Herzog, D. Michael Tillson, Susan A. Goodman, Nancy Robinson, Mark L. McCleland, Dwight Bellinger, et al. "Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver-directed gene therapy." Blood 99, no. 8 (April 15, 2002): 2670–76. http://dx.doi.org/10.1182/blood.v99.8.2670.
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Abstract Hemophilia B is an X-linked coagulopathy caused by absence of functional coagulation factor IX (FIX). Using adeno-associated virus (AAV)–mediated, liver-directed gene therapy, we achieved long-term (> 17 months) substantial correction of canine hemophilia B in 3 of 4 animals, including 2 dogs with an FIX null mutation. This was accomplished with a comparatively low dose of 1 × 1012 vector genomes/kg. Canine FIX (cFIX) levels rose to 5% to 12% of normal, high enough to result in nearly complete phenotypic correction of the disease. Activated clotting times and whole blood clotting times were normalized, activated partial thromboplastin times were substantially reduced, and anti-cFIX was not detected. The fourth animal, also a null mutation dog, showed transient expression (4 weeks), but subsequently developed neutralizing anti-cFIX (inhibitor). Previous work in the canine null mutation model has invariably resulted in inhibitor formation following treatment by either gene or protein replacement therapies. This study demonstrates that hepatic AAV gene transfer can result in sustained therapeutic expression in a large animal model characterized by increased risk of a neutralizing anti-FIX response.
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Monahan, Paul E., Junjiang Sun, Tong Gui, David G. Wichlan, Scott W. McPhee, and R. Jude Samulski. "Employing Factor IX Variants to Avoid Limitations Imposed by Immune Recognition of AAV Vector in Hemophilia B Gene Therapy." Blood 118, no. 21 (November 18, 2011): 3124. http://dx.doi.org/10.1182/blood.v118.21.3124.3124.
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Abstract Abstract 3124 Persistent factor IX expression and phenotypic improvement have been achieved in a human clinical trial for hemophilia B using liver-directed adeno-associated virus (AAV) gene therapy vectors. An ongoing clinical trial uses a vector incorporating self-complementing AAV (scAAV) genome form, factor IX codon optimization (FIXopt) and AAV serotype 8 capsid. As was seen in a previous single-strand AAV serotype 2 trial, dose escalation has been associated with apparent immune-mediated transient inflammation of vector-transduced liver, although in contrast to the previous trial persistent FIX expression has been maintained for the first time. Taken together, these important trials define a consistent threshold load of AAV capsid that has stimulated capsid-specific cytotoxic lymphocyte recognition and potential transaminitis. To advance the successes achieved in these trials while providing a clear margin of safety so that this immunogenic threshold need not be approached, we have pursued steps to limit further the AAV capsid load. Single amino acid substitutions at arginine 338 in the FIX catalytic domain generate FIX variants with increased specific activity. We separately substituted either R338A, R338Q, or R338L (FIX Padua) into a codon optimized human factor IX cDNA and evaluated F.IX expression in tissue culture following plasmid DNA transfection of HEK 293t cells. Each R338 substitution improved FIX specific activity, up to 10 times increased over wild type using the R338LFIXopt cDNA. We next generated scAAV8 vectors incorporating a liver-specific transthyretin (TTR) promoter to express optimized codon F.IX cDNA with or without the R338L substitution. FIX−/− mice receiving portal vein injection of 1 × 1010 vg/animal (4 ×1011 vg/kg) expressed 86.5% of normal FIX activity at 2 months post-transduction from the WTopt vector and 330% normal from the R338LFIXopt. Incorporation of R338Lopt variant resulted in at least 6 to 10 fold increase in FIX specific activity over a follow-up of > 40 weeks. At ten months following FIX gene delivery, mice underwent a tail transection bleeding challenge. FIX vector mice demonstrated therapeutic protection from this major bleeding challenge and furthermore all survived with no late rebleeding (a hallmark of hemophilic phenotype). Greater than 100% normal human FIX activity was maintained for >40 weeks following treatment with the R338LFIX vector (v. 26.3% at euthanasia in WTopt vector group). The prolonged follow-up permitted extended safety evaluation. Factor IX inhibitor antibodies were not detected in any mice throughout the follow-up; FIX-binding IgG1 and IgG2 were negative also. Thrombin/antithrombin III complexes (TAT) examined at 12 weeks and at >30 weeks of age in R338LFIXopt vector mice did not differ from levels in WTFIXopt vector-treated or age-matched C57Bl/6 hemostatically normal mice. Necropsy at 40–44 weeks after vector (1 year of age) showed only age-related changes with no microvascular or macrovascular thrombosis on H&E staining or specific immunostaining for fibrin/fibrinogen deposition; specific staining for fibrosis within myocardium or other sites was negative. We next synthesized a R338LFIXopt expression cassette containing the LP1 promoter/enhancer/intron sequence being used in the ongoing clinical trial and demonstrated equivalent FIX activity from either promoter construct. We then established that the R338LFIXopt vector gives a predictable dose-response across a range of doses as low as 1x 1010 vg/kg I.V. and as high as 4 × 1012 vg/kg I.V. Hemarthrosis is the most common bleeding complication in hemophilia and leads to chronic joint destruction. Bleeding was induced in the joint of FIX−/− mice that had been transduced 4 weeks earlier with the R338LFIX vector. Joints were collected at 2 weeks after induced bleed and the bleeding-induced joint damage was graded using an established histologic score. I.V. R338LFIXopt vector pretreatment resulted in protection against joint degeneration in a dose-dependent fashion in this most relevant clinical scenario. These preclinical studies demonstrate a safety :efficacy profile to advance hemophilia gene therapy using the scAAV8.R338LFIXopt vector. Disclosures: Monahan: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asklepios BioPharmaceutical: Patents & Royalties, Research Funding; CSL Behring: Honoraria; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaIN: Research Funding; Prolor-Biotech: Research Funding. McPhee:Asklepios Biopharmaceutical: Employment. Samulski:Asklepios Biopharmaceutical: Employment, Patents & Royalties.
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Verhoef, Daniel, Jonathan H. Foley, Andrew Goodale, Emma Macrae, Jenny McIntosh, Romuald Corbau, Keith Gomez, Anne Riddell, and Amit C. Nathwani. "A Novel Lysine to Arginine Substitution at Position 301 Enhances Activity of Factor IX." Blood 132, Supplement 1 (November 29, 2018): 3772. http://dx.doi.org/10.1182/blood-2018-99-112832.
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Abstract Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.
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Chao, Hengjun, Wei Chen, James R. Tunstead, and Christopher E. Walsh. "Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes." Blood 104, no. 11 (November 16, 2004): 3985. http://dx.doi.org/10.1182/blood.v104.11.3985.3985.
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Abstract Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.
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Cao, Ou, Eric Dobrzynski, Lixin Wang, Sushrusha Nayak, Bethany Mingle, Cox Terhorst, and Roland W. Herzog. "Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer." Blood 110, no. 4 (August 15, 2007): 1132–40. http://dx.doi.org/10.1182/blood-2007-02-073304.
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Abstract Gene replacement therapy is complicated by the risk of an immune response against the therapeutic transgene product, which in part is determined by the route of vector administration. Our previous studies demonstrated induction of immune tolerance to coagulation factor IX (FIX) by hepatic adeno-associated viral (AAV) gene transfer. Using a regulatory T-cell (Treg)–deficient model (Rag-2−/− mice transgenic for ovalbumin-specific T-cell receptor DO11.10), we provide first definitive evidence for induction of transgene product-specific CD4+CD25+ Tregs by in vivo gene transfer. Hepatic gene transfer–induced Tregs express FoxP3, GITR, and CTLA4, and suppress CD4+CD25− T cells. Tregs are detected as early as 2 weeks after gene transfer, and increase in frequency in thymus and secondary lymphoid organs during the following 2 months. Similarly, adoptive lymphocyte transfers from mice tolerized to human FIX by hepatic AAV gene transfer indicate induction of CD4+CD25+GITR+ that suppresses antibody formation to FIX. Moreover, in vivo depletion of CD4+CD25+ Tregs leads to antibody formation to the FIX transgene product after hepatic gene transfer, which strongly suggests that these regulatory cells are required for tolerance induction. Our study reveals a crucial role of CD4+CD25+ Tregs in preventing immune responses to the transgene product in gene transfer.
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Niemeyer, Glenn P., Roland W. Herzog, Jane Mount, Valder R. Arruda, D. Michael Tillson, John Hathcock, Frederik W. van Ginkel, Katherine A. High, and Clinton D. Lothrop. "Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy." Blood 113, no. 4 (January 22, 2009): 797–806. http://dx.doi.org/10.1182/blood-2008-10-181479.
Full textAbstract:
AbstractPreclinical studies and initial clinical trials have documented the feasibility of adenoassociated virus (AAV)–mediated gene therapy for hemophilia B. In an 8-year study, inhibitor-prone hemophilia B dogs (n = 2) treated with liver-directed AAV2 factor IX (FIX) gene therapy did not have a single bleed requiring FIX replacement, whereas dogs undergoing muscle-directed gene therapy (n = 3) had a bleed frequency similar to untreated FIX-deficient dogs. Coagulation tests (whole blood clotting time [WBCT], activated clotting time [ACT], and activated partial thromboplastin time [aPTT]) have remained at the upper limits of the normal ranges in the 2 dogs that received liver-directed gene therapy. The FIX activity has remained stable between 4% and 10% in both liver-treated dogs, but is undetectable in the dogs undergoing muscle-directed gene transfer. Integration site analysis by linear amplification–mediated polymerase chain reaction (LAM-PCR) suggested the vector sequences have persisted predominantly in extrachromosomal form. Complete blood count (CBC), serum chemistries, bile acid profile, hepatic magnetic resonance imaging (MRI) and computed tomography (CT) scans, and liver biopsy were normal with no evidence for tumor formation. AAV-mediated liver-directed gene therapy corrected the hemophilia phenotype without toxicity or inhibitor development in the inhibitor-prone null mutation dogs for more than 8 years.
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Niemeyer, Glenn P., Roland W. Herzog, Jane D. Mount, Valder Arruda, Michael Tillson, John Hathcock, Katherine A. High, and Clinton D. Lothrop. "Six-Year Follow-Up of Inhibitor Prone Hemophilia B Dogs Treated with Muscle and Liver-Directed AAV2 Mediated Factor IX Gene Therapy." Blood 108, no. 11 (November 16, 2006): 3282. http://dx.doi.org/10.1182/blood.v108.11.3282.3282.
Full textAbstract:
Abstract Hemophilia B, caused by factor IX (FIX) deficiency, is intensively studied as a model for gene therapy. Preclinical studies in mice, dogs and initial clinical trials have documented the feasibility of adeno-associated virus (AAV) mediated gene therapy for hemophilia B. The purpose of this study is to report 6 year follow-up of inhibitor prone hemophilia B dogs treated with muscle or liver directed AAV2 FIX gene therapy. Short term, one year follow-up of these dogs (n=6) has been previously reported. The dogs treated with liver directed AAV2 FIX have not had a single bleed requiring FIX replacement in the 6 years since vector administration. The dogs undergoing muscle directed gene therapy have continued to have a bleed frequency similar to untreated FIX deficient dogs. The whole blood clotting time (WBCT), activated clotting time (ACT), and activated partial thromboplastin time (APTT) have remained at the upper limits of the normal ranges for the six year follow-up in the two liver treated dogs. The FIX activity has remained stable between 4–10% in both liver treated dogs but undetectable in the dogs undergoing muscle directed therapy. The vector/FIX sequences have persisted in liver biopsies but were undetectable in WBC and sperm DNA. Administration of purified canine FIX protein resulted in an immune response to FIX in the muscle treated dogs but not the dogs treated with liver directed gene therapy, suggesting FIX tolerance had been established in the hemophilia B dogs with liver directed gene therapy. A complete clinical evaluation of the dogs undergoing liver directed gene therapy including CBC, serum chemistries, bile acid profile, hepatic MRI and CT scans and liver biopsy was normal with no evidence for tumor formation. These results demonstrate that AAV mediated liver directed gene therapy completely corrects the hemophilia phenotype without toxicity or inhibitor development in the inhibitor prone null mutation dogs for more than six years.
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Schroeder, Jocelyn A., Juan Chen, Yingyu Chen, Yuanhua Cai, Hongyin Yu, Jeremy G. Mattson, Paul E. Monahan, and Qizhen Shi. "Platelet-targeted hyperfunctional FIX gene therapy for hemophilia B mice even with preexisting anti-FIX immunity." Blood Advances 5, no. 5 (March 1, 2021): 1224–38. http://dx.doi.org/10.1182/bloodadvances.2020004071.
Full textAbstract:
Abstract Gene therapy may lead to a cure for hemophilia B (HB) if it is successful. Data from clinical trials using adeno-associated virus (AAV)–mediated liver-targeted FIX gene therapy are very encouraging. However, this protocol can be applied only to adults who do not have liver disease or anti-AAV antibodies, which occur in 30% to 50% of individuals. Thus, developing a protocol that can be applied to all HB patients is desired. Our previous studies have demonstrated that lentivirus-mediated platelet-specific FIX (2bF9) gene therapy can rescue bleeding diathesis and induce immune tolerance in FIXnull mice, but FIX expression was only ∼2% to 3% in whole blood. To improve the efficacy, we used a codon-optimized hyperfunctional FIX-Padua (2bCoF9R338L) to replace the 2bF9 cassette, resulting in 70% to 122% (35.08-60.77 mU/108 platelets) activity levels in 2bCoF9R338L-transduced FIXnull mice. Importantly, sustained hyperfunctional platelet-FIX expression was achieved in all 2bCoF9R338L-transduced highly immunized recipients with activity levels of 18.00 ± 9.11 and 9.36 ± 12.23 mU/108 platelets in the groups treated with 11 Gy and 6.6 Gy, respectively. The anti-FIX antibody titers declined with time, and immune tolerance was established after 2bCoF9R338L gene therapy. We found that incorporating the proteasome inhibitor bortezomib into preconditioning can help eliminate anti-FIX antibodies. The bleeding phenotype in 2bCoF9R338L-transduced recipients was completely rescued in a tail bleeding test and a needle-induced knee joint injury model once inhibitors dropped to undetectable. The hemostatic efficacy in 2bCoF9R338L-transduced recipients was further confirmed by ROTEM and thrombin generation assay (TGA). Together, our studies suggest that 2bCoF9R338L gene therapy can be a promising protocol for all HB patients, including patients with inhibitors.
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Schlachterman, Alexander, Jianhua Liu, Majed Aljamali, Valder R. Arruda, and Katherine A. High. "Assessing Hemostatic Efficacy of Continuous Expression of Factor VIIa Following Administration of AAV Vectors into Hemophilia B Mice." Blood 104, no. 11 (November 16, 2004): 3184. http://dx.doi.org/10.1182/blood.v104.11.3184.3184.
Full textAbstract:
Abstract In previous work, we have shown that portal vein infusion of an AAV vector expressing an engineered secreted activated murine factor VIIa (mFVIIa) results in long-term phenotypic correction of mFVIIa and phenotypic correction in hemophilia B mice, as judged by shortening of the prothrombin time (PT) and of the activated partial thromboplastin time (aPTT), and correction of the tail-clip bleeding time (J Clin Invest2004 Apr; 113(7): 1025–31). We sought to compare endpoints for hemostasis in hemophilic mice infused with AAV-mFVIIa, analyzing the tail-clip assay, the FeCl3-carotid artery model, and real-time imaging of clot formation utilizing the cremasteric muscle arteriole. Thirty-five (n=35) hemophilia B mice were injected with 1.2 x 1012 vg/mouse AAV-mFVIIa via the portal vein. Eight weeks post AAV injection the mFVIIa antigen levels were measured by ELISA at levels ranging from 2–2.5 ug/ml (~2-fold the baseline values). Shortening of the PT mean values from 23 to 17 seconds post-AAV injection was documented in all animals as well as the aPTT mean value from 86 to 43 seconds. A tail-clip challenge was performed by transecting the tail at a diameter of 3 mm, submerging the tail in warm saline then measuring blood loss by determining the hemoglobin content via optical density at 575nm. This documented a statistically significant reduction in blood loss in AAV-mFVIIa-treated mice (N=5) relative to the untreated hemophilia B mice (N=10) * [P-value < 0.0001]. To further investigate the in vivo effects of mFVIIa, we utilized real-time imaging of clot formation in the cremaster muscle arterioles. We first documented that untreated HB mice showed no clot formation upon extensive laser-induced endothelial damage (N=3). Infusion of plasma-derived human FIX (>25 ug/kg) or recombinant human FVIIa (90 ug/kg) proteins resulted in clot formation following laser-induced endothelial damage in these HB mice. Mice treated with AAV-mFVIIa showed similar kinetics and composition of clot formation using this method. We also performed FeCl3-carotid artery experiments and tested for a complete and/or stable occlusion of the vessel post-FIX or FVIIa purified protein infusion and compared this to AAV-treated mice. The results demonstrated that post-FIX infusion (>25 ug/kg) animals developed complete, albeit transient, occlusion but neither FVIIa protein (>90 ug/kg) nor AAV-mFVIIa mice reached levels that lead to occlusion formation. Together these data suggest that 1) AAV-mFVIIa is functional in supporting hemostasis as judged by some but not all in vivo hemostasis challenges; and 2) results in these hemostasis assays may vary as a function of the nature of the vessel injury, and/or the diameter of the vessel that is injured. Which of these endpoints most faithfully reflects hemostasis in humans is not yet clear. We are currently investigating performance in these assays as a function of dose of vector delivered. These studies provide clear evidence of improvement of in vivo hemostasis and support continuous expression of FVIIa as an alterative strategy for the treatment of hemophilia.
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High, Katherine A. "The gene therapy journey for hemophilia: are we there yet?" Blood 120, no. 23 (November 29, 2012): 4482–87. http://dx.doi.org/10.1182/blood-2012-05-423210.
Full textAbstract:
AbstractSince the isolation and characterization of the genes for FVIII and FIX some 30 years ago, a longstanding goal of the field has been development of successful gene therapy for the hemophilias. In a landmark study published in 2011, Nathwani et al demonstrated successful conversion of severe hemophilia B to mild or moderate disease in 6 adult males who underwent intravenous infusion of an adeno-associated viral (AAV) vector expressing factor IX. These 6 subjects have now exhibited expression of FIX at levels ranging from 1% to 6% of normal for periods of > 2 years. This review discusses obstacles that were overcome to reach this goal and the next steps in clinical investigation. Safety issues that will need to be addressed before more widespread use of this approach are discussed. Efforts to extend AAV-mediated gene therapy to hemophilia A, and alternate approaches that may be useful for persons with severe liver disease, who may not be candidates for gene transfer to liver, are also discussed.
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Cohn, Ellen F., Meagan E. Kelly, Jiacai Zhuo, and Hengjun Chao. "Efficient Induction of Tolerance to FIX by Direct Intramuscular Delivery of AAV1." Blood 108, no. 11 (November 16, 2006): 3287. http://dx.doi.org/10.1182/blood.v108.11.3287.3287.
Full textAbstract:
Abstract Hemophilia B is an X-linked recessive genetic disease resulting from deficiency in coagulation factor IX (FIX). The current therapy for hemophilia B is life-long replacement of FIX through recombinant FIX or purified blood products in response to bleeding events. However, this replacement therapy is non-prophylactic, costly, and can be complicated by formation of inhibitory anti-FIX antibodies in up to 5% of patients. While somatic gene therapy is expected to provide a final cure for hemophilia B, it may also cause high incidence of FIX antibodies formation and other adverse immune responses following gene delivery. Direct intramuscular injection of adeno-associated virus (AAV) is a safe and promising procedure for hemophilia B gene therapy. This treatment, however, elicits anti-FIX antibodies in immune competent animal models. We have previously reported that intramuscular injection of AAV1 expressed high levels of canine FIX and induced FIX tolerance in a mouse model of hemophilia B, but AAV2 elicited anti-FIX antibodies. Here, we report efficient induction of human FIX (hFIX) tolerance in naive as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1×1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected close to 1000ng/ml of hFIX antigen by ELISA 8 weeks post AAV injection (n=5). No significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. In addition, subsequent challenge with recombinant hFIX in complete Freund’s adjuvant did not cause anti-FIX antibodies to be produced and the level of hFIX in the blood remained constant. However, anti-FIX antibodies, but not hFIX antigen, were measured in the mice injected with the same dose of AAV2 (n=7). Subsequent injection of AAV1 vector into the skeletal muscle of these AAV2-injected mice resulted in the disappearance of anti-FIX antibodies and emergence of FIX antigen at similar levels to AAV1-injected naive mice in the circulation of these mice. In addition, direct intramuscular injection of AAV1 also induced FIX tolerance in mice that developed anti-FIX antibodies after exposure to recombinant FIX proteins (n=6). Similar experiments in mice with different genetic and MHC backgrounds have also demonstrated efficient induction of tolerance to FIX, implying that AAV1-hFIX can induce tolerance regardless of MHC haplotype. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. To elucidate the mechanism of different immune responses to FIX following intramuscular injection of AAV1 and AAV2, we are examining variations in antigen presentation, interaction between antigen presenting cells and antigen-specific T cells, and fate of antigen-specific T cells following intramuscular injection of AAV1 and AAV2 vectors. In summary, our results demonstrate efficient induction of FIX following direct intramuscular injection of AAV1 vectors. Investigations to elucidate the underlying mechanism are ongoing in our lab.
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Crudele, Julie M., Jonathan D. Finn, Nicholas B. Martin, Joshua I. Siner, Yifeng Chen, Shangzhen Zhou, Glenn Niemeyer, et al. "Tolerance Induction To FIX Padua With AAV Liver Gene Transfer In Inhibitor-Prone Hemophilia B Dogs." Blood 122, no. 21 (November 15, 2013): 4203. http://dx.doi.org/10.1182/blood.v122.21.4203.4203.
Full textAbstract:
Abstract Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX [FIX] deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB. We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies. Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months. An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections). The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases. Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity. Disclosures: High: Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.
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High, Katherine A. "The gene therapy journey for hemophilia: are we there yet?" Hematology 2012, no. 1 (December 8, 2012): 375–81. http://dx.doi.org/10.1182/asheducation.v2012.1.375.3797885.
Full textAbstract:
Abstract Since the isolation and characterization of the genes for FVIII and FIX some 30 years ago, a longstanding goal of the field has been development of successful gene therapy for the hemophilias. In a landmark study published in 2011, Nathwani et al demonstrated successful conversion of severe hemophilia B to mild or moderate disease in 6 adult males who underwent intravenous infusion of an adeno-associated viral (AAV) vector expressing factor IX. These 6 subjects have now exhibited expression of FIX at levels ranging from 1% to 6% of normal for periods of > 2 years. This review discusses obstacles that were overcome to reach this goal and the next steps in clinical investigation. Safety issues that will need to be addressed before more widespread use of this approach are discussed. Efforts to extend AAV-mediated gene therapy to hemophilia A, and alternate approaches that may be useful for persons with severe liver disease, who may not be candidates for gene transfer to liver, are also discussed.
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Davidoff, Andrew, Edward GD Tuddenham, Savita Rangarajan, Cecilia Rosales, Jenny McIntosh, Pratima Chowdary, Anne Riddell, et al. "Stable Factor IX Activity Following AAV-Mediated Gene Transfer in Patients with Severe Hemophilia B." Blood 120, no. 21 (November 16, 2012): 752. http://dx.doi.org/10.1182/blood.v120.21.752.752.
Full textAbstract:
Abstract Abstract 752 Introduction: We are conducting a phase I/II clinical trial of factor IX gene transfer for severe hemophilia B. In the trial we are using a serotype-8 pseudotyped self-complementary adeno-associated virus (scAAV) vector expressing a codon-optimized coagulation factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco). We have previously reported the early safety and efficacy of our novel gene transfer approach in six patients with severe hemophilia B following a single peripheral vein infusion of one of three vector doses (low [2×1011 vector particles (vp)/kilogram weight (kg)], intermediate [6×1011 vp/kg], or high dose [2×1012 vp/kg]) (Nathwani et al, NEJM 365:2357–65, 2011). AAV-mediated expression of FIX at 1–6% of normal was established in all six participants with an initial follow-up of between 6–14 months following gene transfer. We now report longer follow-up of these participants, as well as data from two additional participants recently enrolled at the high dose level. Methods: We have now infused scAAV2/8-LP1-hFIXco in eight subjects with severe hemophilia B (FIX activity, <1% of normal values). Vector was administered without immunosuppressive therapy, and participants have now been followed for 3 months to 2½ years. FIX activity, serum transaminases, vector genomes in secretions/excretions, antibodies to FIX and AAV8, and AAV8 capsid-specific T-cells were monitored during the follow-up. Results: Each of the participants currently has AAV-mediated activity of FIX at 1 to 6% of normal levels. These levels have been stable in each during the follow-up period which is now greater than 1½ years for the first six participants. Five of the eight participants have discontinued FIX prophylaxis and remain free of spontaneous hemorrhage; in the other three, the interval between prophylactic injections has increased. None of the participants in the low or intermediate dose cohorts had evidence of transaminitis; each currently has FIX activity of 1–3% for over 1½ years. Of the four participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; two others had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these three participants received a short course of glucocorticoid therapy, which rapidly normalized their aminotransferase levels and maintained FIX levels in the range of 4 to 6% of normal values. The fourth participant has not had transaminitis three months after vector administration. Conclusions: This represents the first successful, long-term, gene therapy-mediated expression of a therapeutic protein from an AAV vector delivered to human liver. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. Larger numbers of patients followed for longer periods of time are necessary to fully define the benefits and risks and to optimize dosing. However, this gene therapy approach, even with its risk of mild, transient transaminitis, has the potential to convert the bleeding phenotype of patients with severe hemophilia B into a mild form of the disease or to reverse it entirely for a prolonged period of time following vector administration. (ClinicalTrials.gov number, NCT00979238). Disclosures: Chowdary: Novo Nordisk: Consultancy. High:Amsterdam Molecular Therapeutics: ; Baxter Healthcare: Consultancy; Biogen Idec: Consultancy; bluebird bio, Inc.: Membership on an entity's Board of Directors or advisory committees; Genzyme, Inc.: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: ; Sangamo Biosciences: ; Shire Pharmaceuticals: Consultancy.
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Hui, Daniel J., Etiena Basner-Tschakarjan, Gary C. Pien, William D. Martin, Annie S. DeGroot, Katherine A. High, and Federico Mingozzi. "Peptide-Induced Antigen-Specific CD4+CD25+FoxP3+ T Cells Suppress Cytotoxicity T Cell Responses Directed Against the AAV Capsid." Blood 116, no. 21 (November 19, 2010): 3769. http://dx.doi.org/10.1182/blood.v116.21.3769.3769.
Full textAbstract:
Abstract Abstract 3769 Recent advances in adeno-associated viral (AAV) vector-mediated gene transfer continue to offer hope for the correction of monogenic disorders such as hemophilia B. However, unanticipated T cell responses directed against viral capsid epitopes may limit the efficacy of AAV gene transfer. A phase I clinical study in which an AAV2 vector expressing human factor IX (FIX) was delivered systemically provided the first evidence that AAV vector administration at high doses may trigger the expansion of memory CD8+ T cells directed against AAV capsid epitopes. This response was associated with the loss of FIX transgene expression and a transient increase in liver enzymes. Additional studies in human subjects undergoing AAV gene transfer suggest that the capsid antigen load is an important determinant of capsid-specific T cell activation. Thus, strategies for the modulation of capsid T cell responses could contribute to achieving sustained transgene expression following high dose delivery of AAV. MHC class II peptide ligands identified within the human IgG Fc fragment (Tregitopes, Blood 2008;112:3303) have been shown to expand regulatory T cells (Tregs). Restimulation of human peripheral blood mononuclear cells (PBMC) in vitro with AAV capsid antigen in the presence of Tregitopes resulted in the suppression of capsid-specific CD8+ T cells and in the expansion of CD4+CD25+FoxP3+ Tregs. To better define the nature of Tregitope-induced Tregs, we depleted CD25+ cells from PBMC prior to in vitro restimulation. This completely prevented capsid-specific CTL suppression and the expansion of Tregs, suggesting that Tregitopes act by expanding natural Tregs. Cytokine ELISA on conditioned media from PBMC co-cultured with AAV antigen and Tregitopes showed a 50% decrease in IL-2 levels and a >500-fold increase in IL-10 levels. These results suggest that the effect of Tregitopes may be cytokine mediated. To test this hypothesis, we used a transwell system in which the CD4+ T cell fraction of Tregitope-restimulated PBMC was co-cultured with the capsid-specific CD8+ T cells. Without cell contact, a nearly 50% suppression of anti-capsid CD8+ T cell responses was observed. Further evidence supporting the role of cytokine-mediated suppression came from the observation that Tregitope-treated capsid-specific CD8+ T cells appeared to be anergic, and depletion of CD4+ T cells (Tregs) followed by a 24-hour incubation of CD8+CD4− T cells with IL-2 restored >80% of CTL activity. Finally, antigen specificity of Tregitope-induced Tregs was tested by expanding PBMC in vitro with HLA-B*0702-restricted epitopes from either the AAV capsid or the Epstein-Barr Virus (EBV). After in vitro restimulation, negatively-isolated CD4+ T cells expanded in the presence of EBV antigen and Tregitopes were co-incubated with either CD8+ T cells expanded against the AAV capsid or against EBV. Suppression of CTL activity was observed only when EBV Tregs were co-incubated with EBV CD8+ T cells. Similarly, Tregs isolated from AAV and Tregitope cultures suppressed AAV-specific CD8+ T cells but not EBV-specific CD8+T cells. These results suggest that inhibition of CD8+ T cell responses is antigen-specific. We conclude that Tregitopes induce the expansion of Tregs, which can mediate a potent antigen-specific inhibition of CD8+ T cell responses directed to the AAV capsid. Disclosures: Hui: Children's Hospital of Philadelphia: Patents & Royalties. Martin:EpiVax: Employment, Equity Ownership, Patents & Royalties. DeGroot:EpiVax: Employment, Equity Ownership. High:Children's Hospital of Philadelphia: Patents & Royalties. Mingozzi:Children's Hospital of Philadelphia: Patents & Royalties.
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Li, Hojun, Nirav Malani, Shari R. Hamilton, Alexander Schlachterman, Jim Z. Zhang, Giulio Bussadori, Shyrie A. Edmonson, et al. "Assessment of Insertional Mutagenesis Risk Following AAV Vector-Mediated Factor IX Gene Transfer in Mice." Blood 114, no. 22 (November 20, 2009): 2465. http://dx.doi.org/10.1182/blood.v114.22.2465.2465.
Full textAbstract:
Abstract Abstract 2465 Poster Board II-442 Gene therapy for Hemophilia B is a promising alternative to recombinant protein therapy. Long-term expression of coagulation factor IX via stable gene transfer could reduce costs and risks associated with intravenous recombinant factor IX infusions. Liver-directed, adeno-associated virus (AAV) vector-mediated gene transfer of factor IX (FIX) is a gene therapy strategy currently used in two human clinical trials. Our group is performing one of these trials using an AAV vector (AAV-hFIX16) expressing human FIX from a liver-specific promoter. Hepatic artery delivery of AAV-hFIX16 has shown short-term efficacy and disease correction. A fundamental issue facing clinical gene transfer has to do with risk related to vector integration. Though AAV vector DNA is predominantly episomal in transduced cells, chromosomal integration can occur, and consequences of AAV genotoxicity at the molecular level in in vivo systems require further analysis. Here we present the results of a large-scale longitudinal study in which we followed 120 male, wild-type C57BL/6 mice for 18 months after portal vein injection of either 5e12 vg/kg of AAV-hFIX16 (60 mice), 1e14 vg/kg of AAV-hFIX16 (20 mice), 1e14 vc/kg of empty AAV capsid (20 mice), or saline (20 mice). At 18 months we found circulating hFIX levels of 12 ug/mL in mice receiving the lower AAV dose, and 25 ug/mL in mice receiving the higher AAV dose, with no detectable hFIX in mice receiving empty capsid or saline. To assess the risk of insertional mutagenesis in cells transduced by AAV vector, we analyzed the incidence of hepatocellular carcinoma (HCC). We found an HCC incidence rate of 9.1% in untreated mice, 0% in mice receiving saline, 0% in mice receiving empty capsid, 3.8% in mice receiving the lower AAV dose, and 12.5% in mice receiving the higher AAV dose, resulting in a p-value of 0.64 when comparing both AAV groups to controls. We quantified vector genome copy number by qPCR in the HCC's occurring in mice treated with AAV and compared them to vector genome copy number in normal tissue adjacent to the tumors and found no statistically significant difference (p=0.3). We then used LM-PCR to recover vector-chromosome integration junctions from tumor tissue and normal adjacent tissue. We were able to clone 249 unique integrants from tumor tissue and 862 unique integrants from normal adjacent tissue. This yields an average of 0.00018 unique integrants per diploid genome in tumor tissue and 0.00064 unique integrants per diploid genome in normal adjacent tissue. When determining integration profiles within the mouse genome, we found that integrants in both tumor tissue and normal adjacent tissue had a preference for integrating within genes, particularly within exons, as well as within CpG-rich regions. In addition, we found that integrants in both tumor tissue and normal adjacent tissue were more likely than not to be located within 50kb of the 5′ end of an oncogene. While this preference was stronger for integrants in normal adjacent tissue (p=6.3e-5) than integrants in tumor tissue (p=7.7e-2), it was even more likely for the HIV virus to integrate within 50kb of the 5′ end of an oncogene (p=4.1e-11). Functional consequences of these sites of AAV vector integration are not yet known, but are currently being investigated further. In conclusion, our large-scale prospective study of mice receiving AAV-hFIX16 did not demonstrate a statistically significant difference in HCC incidence between AAV-treated groups and control groups, although there was a trend of increasing HCC incidence with very high AAV doses. When analyzing tumors from mice treated with AAV, we found no relationship between vector genome copy number and tumor formation. When analyzing integrated vector in tumor tissue and normal adjacent tissue, we found that integrants in both normal adjacent tissue and tumor tissue had less likelihood for localizing within 50kb of the 5' end of oncogenes than HIV. Disclosures: Wright: Genzyme: Consultancy; Tacere: Consultancy.
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Crudele, Julie M., Jonathan D. Finn, Joshua I. Siner, Nicholas B. Martin, Glenn P. Niemeyer, Shangzhen Zhou, Federico Mingozzi, Clinton D. Lothrop, and Valder R. Arruda. "AAV liver expression of FIX-Padua prevents and eradicates FIX inhibitor without increasing thrombogenicity in hemophilia B dogs and mice." Blood 125, no. 10 (March 5, 2015): 1553–61. http://dx.doi.org/10.1182/blood-2014-07-588194.
Full textAbstract:
Key Points Liver-restricted expression of FIX-Padua induces immune tolerance to the transgene in hemophilia B inhibitor dog models. Long-term toxicity studies show no increased risk of thrombogenicity of FIX-Padua in mice and dogs.
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Nguyen, Giang N., John K. Everett, Hayley Raymond, Samita Kafle, Elizabeth P. Merricks, Haig H. Kazazian, Timothy C. Nichols, Frederic D. Bushman, and Denise E. Sabatino. "Long-Term AAV-Mediated Factor VIII Expression in Nine Hemophilia A Dogs: A 10 Year Follow-up Analysis on Durability, Safety and Vector Integration." Blood 134, Supplement_1 (November 13, 2019): 611. http://dx.doi.org/10.1182/blood-2019-126007.
Full textAbstract:
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.
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Dobrzynski, E., F. Mingozzi, L. Wang, B. Mingle, O. Cao, and R. W. Herzog. "Hepatic Tolerance Induction Prevents Inflammatory Responses To Factor IX Expressing Cells at Supplementary Sites of Gene Transfer." Blood 104, no. 11 (November 16, 2004): 3185. http://dx.doi.org/10.1182/blood.v104.11.3185.3185.
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Abstract The use of gene replacement therapy is an attractive approach for the treatment of the genetic bleeding disorder hemophilia B (caused by mutations in the coagulation factor IX, FIX, gene). A major concern with this type of procedure is the potential for a host immune response to the therapeutic gene product, which would render treatment ineffective. Previously, we observed inflammatory, cytotoxic T lymphocyte, and antibody responses to a human FIX (hFIX) transgene product after intramuscular (IM) delivery via an E1/E3-deleted adenoviral vector (Ad-hFIX) in C57BL/6 mice. Different from this Th1-biased immune response, IM injection of adeno-associated viral (AAV) vector, a Th2-biased, non-inflammatory response led to antibody-mediated neutralization of hFIX expression, without CTL activation. In contrast to these observations on muscle-directed vector administration, hepatic AAV-hFIX gene transfer induced immune tolerance to the transgene product (JCI 111:1347). Lack of anti-hFIX formation was demonstrated even after challenge with hFIX in adjuvant. In order to examine the effect of tolerance induction on CD8+ T cell-mediated cellular immune responses, we performed the following experiments. C57BL/6 mice (n=4 per experimental group) received IM injections of AAV-hFIX vector (serotype 1) in one hind limb and/or Ad-hFIX vector in the contra-lateral leg. In the latter case, inflammation (as determined by H&E histological evaluation), CD8+ T cell infiltrate and destruction of hFIX expressing muscle fibers were obvious in both legs because of the Ad-hFIX mediated activation of CTL to hFIX. CD8+ T cell responses were strongest in Ad-hFIX transduced muscle at day 14 and in the AAV-hFIX leg at day 30. Expression of hFIX as determined by immunohistochemistry became undetectable in Ad-hFIX injected muscle by day 30, but was not completely eliminated in AAV-hFIX transduced muscle. Injection of AAV-hFIX only, did not cause inflammation of muscle tissue or CD8+ cell infiltrate. When the identical experiment was carried out in C57BL/6 mice that were expressing hFIX from hepatic gene transfer via the AAV serotype 2 vector (performed 6 weeks earlier), a substantial increase in systemic hFIX expression was observed after IM administration of the Ad and AAV-1 vectors (again injected into contra-lateral legs). However, a portion of the increased expression was subsequently lost, which correlated with inflammation and CD8+ T cell infiltrate of the Ad-hFIX transduced muscle. Interestingly, no (3/4 mice) or only minor (1/4 mice) infiltrate was observed in AAV-hFIX injected muscles. Consequently, hFIX expression persisted in the AAV, but not the Ad transduced legs. Presumably, CTL responses to adenoviral antigens were sufficient to target Ad-hFIX transduced muscle despite tolerance to the transgene product. In contrast to control mice, hepatic tolerized animals failed to form anti-hFIX after challenge by IM injection of these viral vectors. Moreover, inflammatory and destructive cellular immune responses to the transgene product were successfully prevented by hepatic tolerance induction, indicating that tolerance induced by gene transfer to the liver affects cellular as well as antibody-mediated responses and extents to tissues other than liver.
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Finn, Jonathan D., Daniel J. Hui, Downey Harre, Danielle Dunn, Federico Mingozzi, Shangzhen Zhou, and Katherine A. High. "Proteasome Inhibitors Decrease AAV2 Capsid-Derived Peptide Epitope Presentation On MHC Class I Following Transduction." Blood 114, no. 22 (November 20, 2009): 695. http://dx.doi.org/10.1182/blood.v114.22.695.695.
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Abstract Abstract 695 Adeno-associated viral (AAV) vectors are one of the most extensively studied and highly used vector platforms for gene therapy applications. We have recently provided evidence for AAV capsid-derived antigen presentation through MHC class I on the surface of AAV-transduced cells, supporting the hypothesis that in the first clinical trial using AAV to treat Hemophilia B, AAV capsid proteins were presented on the surface of transduced hepatocytes, resulting in clearance by antigen-specific CD8+ T cells and consequent loss of therapeutic transgene expression. Proteasome inhibitors are small molecule compounds that are able to specifically inhibit the activity of the proteasome, resulting in a buildup of ubiquitinated proteins, increased intracellular reactive oxygen species, and a general decrease in presentation of MHCI-peptide complexes. It has previously been shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here we describe using the FDA approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, while at the same time, enhancing gene expression in vivo. Using an AAV capsid specific T cell reporter line to analyze effects of proteasome inhibitor on antigen presentation, we demonstrated capsid antigen presentation at low MOI's, as well as inhibition of antigen presentation at clinically relevant levels of bortezomib. We also demonstrate that bortezomib can enhance FIX expression from an AAV2 vector in C57Bl/6 mice, however does not appear to enhance expression of AAV8. Based on the data presented here, it appears as if future studies using proteasome inhibitors in large animal models may be warranted. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease antigen presentation would be a promising solution to many of the obstacles to successful translation of AAV-mediated, liver-directed gene transfer to the clinic. Disclosures: No relevant conflicts of interest to declare.
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Sun, Junjiang, Narine Hakobyan, Leonard A. Valentino, Brian L. Feldman, R. Jude Samulski, and Paul E. Monahan. "Intraarticular factor IX protein or gene replacement protects against development of hemophilic synovitis in the absence of circulating factor IX." Blood 112, no. 12 (December 1, 2008): 4532–41. http://dx.doi.org/10.1182/blood-2008-01-131417.
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AbstractHemophilic bleeding into joints causes synovial and microvascular proliferation and inflammation (hemophilic synovitis) that contribute to end-stage joint degeneration (hemophilic arthropathy), the major morbidity of hemophilia. New therapies are needed for joint deterioration that progresses despite standard intravenous (IV) clotting factor replacement. To test whether factor IX within the joint space can protect joints from hemophilic synovitis, we established a hemophilia B mouse model of synovitis. Factor IX knockout (FIX−/−) mice received a puncture of the knee joint capsule with a needle to induce hemarthrosis; human factor IX (hFIX) was either injected through the needle into the joint space (intraarticularly) or immediately delivered IV. FIX−/− mice receiving intraarticular FIX protein were protected from synovitis compared with mice receiving same or greater doses of hFIX IV. Next, adeno-associated virus (AAV) gene transfer vectors expressing hFIX were injected into knee joints of FIX−/− mice. Joints treated with 1010 vector genomes (vg)/joint AAV2-, AAV5-, or AAV8-hFIX or 2.5 × 109 vg/joint AAV5-hFIX developed significantly fewer pathologic changes 2 weeks after injury compared with the pathology of control injured contralateral hind limbs. Extravascular factor activity and joint-directed gene transfer may ameliorate hemophilic joint destruction, even in the absence of circulating FIX.
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Weiller, Markus, Helen Wang, Sogué Coulibaly, Maria Schuster, Hanspeter Rottensteiner, Kefeng Sun, Marinee K. Chuah, Thierry Vandendriessche, Friedrich Scheiflinger, and Werner Höllriegl. "Evaluation of the Human Factor IX Gene Therapy Vector TAK-748 in Hemophilia: Results from Non-Clinical Studies in Factor IX Knockout Mice and Rhesus Monkeys." Blood 134, Supplement_1 (November 13, 2019): 4633. http://dx.doi.org/10.1182/blood-2019-124734.
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Introduction: Adeno-associated virus (AAV)-based factor IX (FIX) gene therapy has the potential to provide clinical benefit in patients with hemophilia B. TAK-748 is a novel next-generation AAV vector for FIX gene therapy. The vector design includes the insertion of 3 hepatocyte-specific cis-regulatory elements to increase the strength of the liver-specific transthyretin promoter driving expression of a human FIX transgene. Aims: The objectives of this study were to investigate the TAK-748 dose-response relationship for FIX activity, and evaluate its efficacy, in FIX knockout (KO) mice and rhesus monkeys. Methods: Male FIX KO mice (N=12/group) received single intravenous doses of TAK-748 (7.4×1010, 1.5×1011, 7.4×1011, or 1.5×1012 vector genomes [vg]/kg) or buffer. Blood samples were taken on days 7, 14, 28, 42, and 56 for the analysis of plasma FIX activity. At the end of the observation period, the bleeding phenotype was assessed by a tail-tip bleeding assay. The viral transduction efficiency of TAK-748 in liver tissue was analyzed by quantitative real-time polymerase chain reaction. Safety assessments included monitoring for clinical signs, and histopathological analysis of selected organs (liver, spleen, kidney, and heart). Male rhesus monkeys (N=3/group) were administered single TAK-748 intravenous bolus injections (3.8×1011, 9.5×1011, or 1.9×1012 vg/kg). Blood samples were collected before dosing and weekly after dosing up to week 18. Plasma FIX activity, human (hu)FIX antigen, and anti-hu-Padua FIX neutralizing antibodies were analyzed. Results: No clinical signs or deaths were recorded in animals treated with TAK-748, and there were no TAK-748-related histopathological findings in the tissues collected from the mice. FIX activity levels in plasma from FIX KO mice treated with buffer were below the lower limit of quantification. Administration of TAK-748 resulted in a dose-dependent increase in mean plasma FIX activity, and supraphysiologic mean FIX levels up to 41.0 IU/mL (1.5×1012 vg/kg). In the tail-tip bleeding assay, blood loss was significantly reduced in the TAK-748 groups at dose levels above 7.4×1010 vg/kg (P<0.05 vs. buffer control). Administration of TAK-748 to rhesus monkeys resulted in a dose-dependent increase in mean plasma FIX activity and antigen, and peak levels of huFIX expression were detected 2-4 weeks after treatment. Mean huFIX activity was 0.3, 0.6, and 1.9 IU/mL after treatment with 3.8×1011 vg/kg, 9.5×1011 vg/kg, and 1.9×1012 vg/kg TAK-748, respectively. A significant reduction in FIX activity and huFIX protein was observed in most of the animals starting about 4 weeks after dosing with TAK-748. In most animals, anti-huFIX Padua neutralizing antibody titers were detected at about week 6 of the study and correlated with the preceding reductions in huFIX expression. Conclusions: Treatment with TAK-748 resulted in dose-dependent increases in plasma FIX activity and was efficacious in FIX KO mice and rhesus monkeys. There were no treatment-related safety findings. Disclosures Weiller: Baxalta Innovations GmbH, a Takeda company: Employment. Wang:Shire US Inc., a Takeda company: Employment, Equity Ownership. Coulibaly:Baxalta Innovations GmbH, a Takeda company: Employment. Schuster:Baxalta Innovations GmbH, a Takeda company: Employment. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Sun:Shire US Inc., a Takeda company: Employment. Chuah:Shire, a Takeda company: Consultancy, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding. Vandendriessche:Shire, a Takeda company: Consultancy, Honoraria, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding; Biotest: Honoraria. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Höllriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.
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Giangrande, Paul. "The Future of Hemophilia Treatment: Longer-Acting Factor Concentrates versus Gene Therapy." Seminars in Thrombosis and Hemostasis 42, no. 05 (May 5, 2016): 513–17. http://dx.doi.org/10.1055/s-0036-1579637.
Full textAbstract:
Gene therapy is the only novel technology that currently offers the prospect of a lasting cure for hemophilia and freedom from the burden of repeated injections. Recent data from a handful of patients who have undergone gene therapy for hemophilia B are very encouraging with a sustained factor IX (FIX) level of 0.05 IU/mL maintained for over 4 years. While this level is above the current usual target trough levels, it falls well short of the level that patients on prophylaxis with longer-acting products can expect. Prophylaxis is also associated with high peak levels, which permits patients to maintain an active lifestyle. A major barrier to widespread adoption of gene therapy is a high seroprevalence of antibodies to adeno-associated virus (AAV) vectors in the general population. Young children would be the best candidates for gene therapy in view of much lower seroprevalence to AAV in infants. A stable level of FIX early in life would prevent the onset of joint bleeds and the development of arthropathy. The recent experience with apolipoprotein tiparvovec (Glybera; uniQure, Amsterdam, the Netherlands) indicates that gene therapy is unlikely to prove to be a cheap therapeutic option. It is also quite possible that other new technologies that do not require viral vectors (such as stem cell therapy) may overtake gene therapy during development and make it redundant.
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Nathwani, Amit C., John T. Gray, Catherine Y. C. Ng, Junfang Zhou, Yunyu Spence, Simon N. Waddington, Edward G. D. Tuddenham, et al. "Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver." Blood 107, no. 7 (April 1, 2006): 2653–61. http://dx.doi.org/10.1182/blood-2005-10-4035.
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AbstractTransduction with recombinant adeno-associated virus (AAV) vectors is limited by the need to convert its single-stranded (ss) genome to transcriptionally active double-stranded (ds) forms. For AAV-mediated hemophilia B (HB) gene therapy, we have overcome this obstacle by constructing a liver-restricted mini–human factor IX (hFIX) expression cassette that can be packaged as complementary dimers within individual AAV particles. Molecular analysis of murine liver transduced with these self-complementary (sc) vectors demonstrated rapid formation of active ds-linear genomes that persisted stably as concatamers or monomeric circles. This unique property resulted in a 20-fold improvement in hFIX expression in mice over comparable ssAAV vectors. Administration of only 1 × 1010 scAAV particles led to expression of hFIX at supraphysiologic levels (8I U/mL) and correction of the bleeding diathesis in FIX knock-out mice. Of importance, therapeutic levels of hFIX (3%-30% of normal) were achieved in nonhuman primates using a significantly lower dose of scAAV than required with ssAAV. Furthermore, AAV5-pseudotyped scAAV vectors mediated successful transduction in macaques with pre-existing immunity to AAV8. Hence, this novel vector represents an important advance for hemophilia B gene therapy.
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Finn, Jonathan D., Paolo Simioni, Nicholas Iacobelli, Shangzhen Zhou, Timothy C. Nichols, Katherine A. High, and Valder R. Arruda. "FIX-R338L (FIX Padua) as a Successful Alternative for the Treatment of Canine Severe Hemophilia B." Blood 114, no. 22 (November 20, 2009): 694. http://dx.doi.org/10.1182/blood.v114.22.694.694.
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Abstract Abstract 694 Hemophilia B (HB) is a severe bleeding disorder affecting 1 in 30,0000 men and results from the deficiency of FIX clotting activity. The current treatment involves infusion of either plasma derived or recombinant FIX. We have recently described a novel naturally occurring gain-of-function mutation in the FIX gene found associated with hyperfunctional activity of the protein (N Engl J Med, 2009). The proband presented with 776% FIX clotting activity (FIX:C), but normal FIX antigen levels (FIX:Ag of 92% of normal), and after detailed analysis the causative mutation was found to be a transversion of R338L in FIX (FIX-Padua) that segregates in a classical X-linked fashion. Furthermore, in vitro expression of FIX-Padua confirmed that the protein had a specific activity that was 8 fold higher than FIX-wild type. Arginine at 338 is highly conserved in FIX from mammals but is unique among other human vitamin K-dependent proteins. We sought to determine whether FIX-Padua would be an attractive strategy for novel therapy of hemophilia B by introducing this mutation in the canine FIX gene (cFIX) and testing in HB dog models. The HB colony at Chapel Hill is an outbred, immunocompetent large animal model that have a missense mutation in the cFIX gene and have no detectable circulating levels of cFIX antigen or activity, and its disease phenotype closely correlates with that seen in humans. Adeno-associated viral (AAV) vector encoding the cFIX-Padua and cFIX-wt under control of the CMV promoter was generated and administered to HB dogs. AAV6 was chosen as the serotype to overcome the high prevalence of neutralizing antibodies to AAV2 in the general population. Delivery of AAV6-cFIX wild type by intravascular delivery to the skeletal muscle via anterograde limb perfusion (ALP) coupled with transient immunosuppression (duration of 5 weeks) resulted in FIX:Ag and FIX:C of 2-5% of normal with partial shortening the whole blood clotting time. Post AAV6-cFIX Padua injection we observed a remarkable correction of the WBCT to the normal range (8-10 min) within the first 7 days that has continued for the duration of the study. One dog's (M55) FIX:Ag levels were ∼250 ng/mL (5% of normal) at the peak and 180 ng/mL (3.6%) at the plateau. Notably, his FIX:C levels were 49% at peak and 35% at plateau, indicating a specific activity ∼10 fold higher than wild type cFIX. Treatment of a second dog (M59) resulted in FIX:Ag levels of 175 ng/mL (3.5%) at the peak and 60 ng/mL (1.2%) at plateau. This is in contrast with his FIX:C levels that were 35% at peak and 12% at plateau levels, respectively. Like M55, the specific activity was 10 fold higher than wild type cFIX. An important consideration is the potential for an immune response against the cFIX-Padua neo-antigen. Notably, there is no evidence of formation of inhibitor to cFIX in these dogs for a cumulative observation period of 6 months post-immunosuppression. We have demonstrated that the cFIX-Padua mutant protein has 10-fold higher specific activity in a canine model of HB. This is significant as it allows for lower doses of AAV vectors to be used for the treatment of HB without compromising efficacy, thereby increasing the safety profile of clinical gene therapy for HB. In addition, FIX Padua provides an excellent alternative to overcome the limited ability of skeletal muscle to synthesize fully posttranslational modified functional FIX (Blood, 2001). Thus, FIX-Padua improves both the efficacy and safety of skeletal muscle-mediated FIX resulting in FIX:C levels sufficient to convert the severe HB phenotype to mild hemophilia (10-35% of normal) at vector doses already tested clinical trials. This strategy has the potential of improving other gene and/or cell therapy for HB targeting ectopic targets as well as the liver. Disclosures: High: Genzyme: Patent licensed to Genzyme, but Dr. High has waived all financial interest, Patents & Royalties.
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Wang, Xiaomei, Alexandra Sherman, Jin Su, Henry Daniell, and Roland W. Herzog. "Mechanism Of Oral Tolerance Induced By Bioencapsulated Coagulation Factor IX In Hemophilia B Mice." Blood 122, no. 21 (November 15, 2013): 30. http://dx.doi.org/10.1182/blood.v122.21.30.30.
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Abstract Currently, the most serious complication in hemophilia therapy is the development of neutralizing antibodies (inhibitors) against the therapeutic coagulation factor. Inhibitor formation in treatment of hemophilia B (deficiency in factor IX, FIX) is less frequent than in hemophilia A. However, FIX inhibitors tend to be high-titer, and 25-50% of such patients develop anaphylactic reactions against FIX. Clinical immune tolerance induction (ITI) protocols (to eliminate the inhibitors) are lengthy, expensive, and are often terminated in hemophilia B due to anaphylaxis or nephrotic syndrome. In order to establish a protocol for prevention of FIX inhibitors in high-risk patients (i.e. those with gene deletions or other severe mutations) while avoiding immune suppressive drugs or genetic manipulation, we have developed a source of bioencapsulated human FIX. Oral delivery of plant leaf material, expressing CTB-FIX in chloroplasts, effectively suppresses inhibitor formation and prevents anaphylaxis in FIX replacement therapy in hemophilia B mice of the C3H/HeJ F9-/- strain (PNAS 107:7101, 2010). This is accomplished by delivery of the fusion molecule to the epithelium of Peyer’s Patches (PP), which expresses high levels of GM1, the receptor for the CTB transmucosal carrier. Subsequently, FIX antigen is transferred to dendritic cells (DCs) in the PP and also into the circulation. Mice fed twice per week for 2 months fail to form inhibitors during a 2-month treatment phase (1 IU rFIX, IV, once/week), lack IgE formation and fatal anaphylactic reactions (which occur in control mice), but still form non-inhibitory IgG1 at reduced titers. An oral dose of FIX antigen equivalent to ∼1.5 IU rFIX/kg is sufficient to achieve tolerance. To define the underlying mechanism, we analyzed DC and lymphocyte populations in different compartments (n=4-6 for all experimental groups and data sets). Compared to control mice, which received FIX only or were fed with WT plant material and treated with FIX, tolerized mice had a marked increase in the frequencies of CD103+ DC and pDC in mesenteric lymph nodes (MLN) and PP. Both DC subsets are known to play crucial roles in oral tolerance and Treg induction. A minor, non-significant increase pDCs was also seen in the spleen. Interestingly, tolerized mice also showed a significant increase in the frequencies of CD4+ T cells expressing gut homing receptors (CCR9+ and α4β7), including in the spleen and thus indicating interaction between the gut and systemic immune systems. The spleen is a major site for systemic immune surveillance and critical for responses to intravenous FIX. Splenocytes re-stimulated in vitro with hF.IX were anayzed by RT-PCR array. We found that Th1 and Th2 cytokines (IL2, IL-4, IL-13, IFN-γ) were downregulated by oral FIX delivery, whereas immune suppressive cytokines (IL-10, TGF-β), and the Treg marker Foxp3 were upregulated, suggesting a switch from an effector to a suppressive response. Moreover, the frequency of LAP+CD4+CD25-FoxP3- T cells (which suppress responses via TGF-β) was dramatically increased in FIX-fed mice in PP (from <5% to 10-20%), and to lesser extent in spleen and MLN. Although the overall frequency of CD4+CD25+FoxP3+ Treg was not altered, tolerized mice had a significant increase in LAP+ cells in that population, indicating Treg activation. Interestingly, we failed to prevent anaphylaxis or inhibitor formation in C3H/HeJ [F9-/- x IL-10-/-] mice (n=7 per group, 42% mortality after 4th FIX injection, >5 BU in surviving animals). Therefore, both IL-10 and TGF-βare integral components of the tolerance mechanism. In summary, oral delivery of CTB-FIX antigen bioencapsulated in plant cells prevents anaphylaxis and inhibitor formation against FIX via induction of a complex immune regulatory network, involving increases in Treg-promoting subsets of DC, induction/activation of LAP+ Treg, and active cytokine-mediated suppression. Preliminary data suggest that the regimen can also be used to desensitize mice from FIX. While animal experiments performed thus far have utilized transgenic tobacco cells, we have now generated and characterized FIX-transplastomic lettuce expressing CTB-FIX, which will be tested towards translation of this approach. Disclosures: Daniell: Bayer: Research Funding. Herzog:Genzyme: AAV technology, AAV technology Patents & Royalties.
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Lillicrap, David. "Genetic Characterization of Hemophilia and Implications for Novel Therapies." Blood 116, no. 21 (November 19, 2010): SCI—9—SCI—9. http://dx.doi.org/10.1182/blood.v116.21.sci-9.sci-9.
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Abstract Abstract SCI-9 The genes encoding factor VIII (FVIII) and factor IX (FIX) were cloned 25 years ago. Since then, substantial translational application of this knowledge has been witnessed. First, genetic analysis of hemophilia has enabled a detailed understanding of the types and patterns of mutation responsible for these conditions. This knowledge has provided important biological insights into the mechanisms underlying mutation generation and has also formed the basis for precise molecular diagnosis for hemophilia carriers and potentially affected fetuses. In addition, the hemophilic genotype is the best-characterized and strongest predictor for the development of inhibitor generation in previously untreated patients. The second benefit deriving from the genetic characterization of the hemophilias has been the production of recombinant coagulation factor concentrates. Over the past 20 years, the use of recombinant factor concentrates has increased dramatically and we are currently witnessing a flurry of activity to develop second-generation, enhanced concentrates. The major objective of these projects is to facilitate clotting factor prophylaxis through the production of concentrates whose half-lives are extended, thus reducing the frequency of factor administration. These endeavors are utilizing a variety of approaches, but most are focused on either chemical modification of the factors through polymer conjugates or the generation of fusion proteins with immunoglobulin or albumin to take advantage of the recycling function of the neonatal Fc receptor. The first wave of these new products is now undergoing early phase clinical studies, and while substantial benefits for FIX half-life extension appear achievable, FVIII half-life modification may be more challenging. Finally, molecular genetic knowledge of the hemophilias has resulted in the pursuit of strategies to utilize genetic approaches to effect long-term “cures” of the disease. These initiatives have resulted in several small phase I/II trials of viral vector-based gene transfer but have also formed the basis of mutation-specific therapies such as a translational read-through approach being undertaken in patients with nonsense mutations. Currently, two phase I/II trials are in progress with liver-directed AAV FIX gene transfer in which two different AAV serotypes are being evaluated. These studies follow on from two previous AAV clinical trials in which the major obstacle to clinical benefit appears to have been the host immune response to the vector. There is considerable interest in the outcomes of these new studies. Disclosures: No relevant conflicts of interest to declare.
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Dane, Allison, Jenny McIntosh, Doyoung Lee, Rose Sheridan, Mark Maginn, Romuald Corbau, Andrew M. Davidoff, and Amit C. Nathwani. "Preclinical Evaluation of an Engineered AAV Capsid in Non-Human Primates for the Treatment of Haemophilia B." Blood 132, Supplement 1 (November 29, 2018): 2197. http://dx.doi.org/10.1182/blood-2018-99-117969.
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Abstract Introduction Over recent years, proof-of-concept academic and early phase commercial trials have demonstrated long-term therapeutic benefit of liver directed AAV-mediated gene transfer of human FIX (hFIX). Extending this success to a greater number of haemophilia B patients is hampered by the need for large vector doses required for efficient gene transfer and associated liver toxicity. To improve potency, we focused on developing a novel synthetic capsid, AAVS3, to increase human hepatocyte transduction. Here we present the preclinical in vitro and in vivo evaluation of AAVS3 pseudotyped vectors. In addition to showing strong liver tropism we demonstrate clinically relevant expression levels of hFIX expression in non human primates. Methods Primary human hepatocyte cultures were used to compare transduction efficiency of our novel capsid with AAV5 and AAV8, variants currently in the clinic. To extend this data in vivo, a humanised mouse liver model was utilised. Subsequently, fourteen Rhesus macaques were dosed with AAVS3 pseudotyped FIX vectors, via saphenous vein injection. Levels of hFIX were determined using an ELISA assay that is able to distinguish hFIX protein from the native Rhesus macaque FIX protein. At harvest biodistribution of vector was detected by quantitative PCR. Results We demonstrate that gene transfer efficiency with AAVS3 was up to 10-fold higher in primary human hepatocytes, when compared to vectors pseudotyped with AAV serotype 5 or 8 capsids. In addition, studies in a chimeric human-murine liver model revealed AAVS3 was 6-fold more efficient at transducing human hepatocytes compared with an AAV8 vector. Using two distinct optimised FIX expression cassettes, we demonstrate highly efficient gene transfer in Rhesus macaques. Indeed, AAVS3 FIX vectors injected at clinically relevant doses of 0.93 x1012 to 2.56 x1013 vg/kg achieved mean peak levels of 0.546 and 21.7µg/ml of hFIX respectively (~11% to ~430% of normal levels). Biodistribution studies showed strong liver tropism following peripheral vein administration with minimal distribution to other organs at or below the level of detection. No vector associated lesions or adverse events were observed in the liver. Conclusion Collectively, these data support further evaluation of an AAVS3 pseudotyped vector in humans with the aim of achieving transduction of a larger proportion of hepatocytes using a lower, potentially safer dose of vector than achieved with AAV8. Disclosures Dane: Freeline: Employment. Sheridan:Freeline: Employment, Equity Ownership. Maginn:Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; BioMarin: Consultancy, Patents & Royalties; UniQure: Patents & Royalties.
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Chen, Yingyu, Jocelyn A. Schroeder, Erin L. Kuether, Guowei Zhang, Robert R. Montgomery, and Qizhen Shi. "Lentivirus-Mediated Platelet Gene Therapy Corrects Bleeding Diathesis and Induces Immune Tolerance in Murine Hemophilia B Mice." Blood 120, no. 21 (November 16, 2012): 1101. http://dx.doi.org/10.1182/blood.v120.21.1101.1101.
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Abstract Abstract 1101 While data from the clinical trials using AAV vector expression FIX in hemophilia B gene therapy in humans are very encouraging, for individuals with severe liver disease or neutralizing antibodies to AAV, an alternative gene therapy approach might be desired. Our previous studies have demonstrated that lentivirus-mediated platelet gene therapy can correct murine hemophilia A phenotype, but this approach has not been explored for hemophilia B. In the current study, we developed a clinical translatable approach for platelet gene therapy of hemophilia B. Platelet-FIX (2bF9) expression in hemophilia B (FIXnull) mice was introduced by transplantation of hematopoietic stem cells (HSCs) transduced with 2bF9 lentivirus (LV). The recipients were analyzed beginning at 3 weeks after bone marrow (BM) transplantation. Expression of the 2bF9 product was detected by PCR in all recipients that received 2bF9 LV-transduced BM cells, indicating viable engraftment of BM genetically modified with the 2bF9 LV transfer vector. The expression of the hFIX transgene protein in the transduced platelets was confirmed by immunofluorescent confocal microscopy. Flow cytometry showed that there were 20.8 ± 12.1% (n = 7) and 14.8 ± 10.7% (n = 6) 2bF9 LV-transduced platelets respectively in the recipients preconditioned with 1100 cGy or 660 cGy. The antigen levels of FIX (FIX:Ag) were 2.89 ± 1.75 mU/108 platelets (n = 9) in the recipients preconditioned with 1100 cGy and 1.87 ± 1.30 mU/108 platelets (n = 7) in the 660 cGy group, while the activity (FIX:C) levels were 1.67 ± 1.15 and 1.13 ± 0.85 mU/108 platelets respectively. There was a small amount of FIX detected in the 2bF9 LV-transduced recipient plasma with the average levels of 2.22 mU/ml in 1100 cGy group and 1.44 mU/ml in 660 cGy group. To analyze the distribution of the FIX between platelets and plasma, we normalized FIX levels to total whole blood FIX content. The results demonstrated that 90% to 95% of whole blood FIX was stored in platelets. The tail clip survival test demonstrated that 15 out of 16 mice that received 2bF9 LV-transduced HSCs survived the tail clip challenge, while 8 out of 10 FIXnull control mice died after tail clipping. Nine months after transplantation, sequential transplantation was performed on some of the primary recipients. Platelet-hFIX expression in the secondary recipients was sustained, leading to phenotypic correction and confirming that long-term engrafting HSCs were successfully transduced with 2bF9 LV. Notably, none of the transduced recipients developed anti-FIX antibodies after platelet gene therapy. To investigate whether immune tolerance was induced in 2bF9 LV-transduced recipients, we challenged the recipients with recombinant human FIX (rhFIX) in the presence of adjuvant. Only 1 out of 9 2bF9 LV-transduced recipients developed a low titer of inhibitory antibodies (1.6 BU/ml) as measured by a modified Bethesda assay. In contrast, all of the FIXnull controls developed inhibitory antibodies ranging from 17 – 37 BU/ml after the same challenge (n = 5). To ensure that the immune system was not defective in the 2bF9 LV-transduced recipients and that the tolerance induction is FIX antigen-specific, we further challenged the animals with ovalbumin (OVA) absorbed on Alum. Both the 2bF9 LV-transduced and FIXnull control mice developed high-titer of anti-OVA antibodies. The levels of anti-OVA IgG in the 2bF9 transduced recipients were not significantly different from FIXnull mice after the OVA immunization, confirming that tolerance induction in 2bF9 LV-transduced mice is FIX-specific. Taken together, our data suggest that lentivirus-mediated bone marrow transduction and transplantation can not only provide sustained phenotypic correction, but also induce immune tolerance in hemophilia B mice, indicating that this approach may be a promising strategy for gene therapy of hemophilia B in humans. Disclosures: No relevant conflicts of interest to declare.
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Arruda, Valder R. "Hemophilia Gene Therapy." Blood 118, no. 21 (November 18, 2011): SCI—48—SCI—48. http://dx.doi.org/10.1182/blood.v118.21.sci-48.sci-48.
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Abstract SCI-48 Over the last decade, five clinical studies on gene therapy for hemophilia A and B using viral and nonviral vectors for liver-restricted or ectopic expression of clotting factor demonstrated safety with no inhibitor formation. Although efficacy in these early phase trials was not achieved because of sustained levels of clotting factor below 1%, these studies form the basis for the second generation of clinical trials. To date, two studies on adeno-associated viral (AAV) vector encoding a FIX transgene for liver-restricted expression are ongoing. In one study using a modified AAV genome pseudotyped with serotype 8, emerging data are encouraging and sustained therapeutic levels of FIX have been obtained in a dose-dependent manner. Novel approaches for hemophilia have been explored targeting hematopoietic stem cells (HSC) using lentiviral vectors for expressing FVIII or FIX genes. Transgene expression under the control of either a non-lineage specific promoter or a platelet-specific promoter showed biological activity and improvement of the disease phenotype. The platelet-restricted approaches did not increase circulating plasma clotting factor levels, but they resulted in enrichment of the factor at the injury site upon platelet activation. An advantage of targeting FVIII expression and storage in platelets is the protection of FVIII from neutralizing antibodies (inhibitors). In this model, platelet-FVIII provided superior hemostasis than elevated plasma FVIII levels upon hemostatic challenges in the microcirculation and macrocirculation. Recently, platelet-restricted expression of human FVIII gene using a lentiviral vector for ex vivo transduction of canine HSC resulted in improvement of the disease phenotype in severe hemophilia A dogs without unwanted immune responses to the transgene. Additional strategies to optimize gene- and/or cell-based approaches have also focused on use of clotting factors with enhanced biological activities will be discussed. Together with the generation of novel vectors, further enhancement of both the efficacy the safety of these approaches is envisioned. Disclosures: Arruda: Pfizer: Research Funding.
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Lin, Chia-Ni, Chung-Yang Kao, Chia-Lun Hong, Peiqing Ye, Carol H. Miao, Nobuko Hamaguchi, Hua-Lin Wu, et al. "Engineered Factor IX with Augmented Clotting Activities in a Hemophilia B Mouse Model." Blood 112, no. 11 (November 16, 2008): 2025. http://dx.doi.org/10.1182/blood.v112.11.2025.2025.
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Abstract Hemophila B is an X-linked inherited disorder caused by the lack of a functional Factor IX (FIX) and characterized by a bleeding diathesis of variable severity. Currently, treatment of hemophilia B is performed by intravenous infusion of plasma-derived or recombinant FIX. However, the high cost associated with the protein concentrates poses a financial burden to protein replacement therapy. Other treatments, such as gene therapy and tissue implant techniques, are also under study but not yet developed for clinical use. An alternative approach is to develop a FIX molecule with higher specific activity that will reduce the amount of the clotting factor consumed in treatment. For this purpose, we used alanine-scanning mutagenesis and generated 7 alanine-replaced FIX variants that were expressed in HEK-293 cells and purified. These variants have either single (n=3), double (n=3) or triple (n=1) amino acid changes and each was characterized by ELISA and aPTT assay. We observed that the FIX triple mutation variant (R86A/E277A/R338A, FIX-Triple) exhibited 10-12 times higher clotting activity than wild-type FIX (FIX-WT). The affinity of IX-Triple to human Factor VIIIa increased 10-fold (Kd = 0.19 nM vs. 2.4 nM, FIX-Triple vs. FIX-WT) in a Factor Xa-generation assay, consistent with the fact that the amino acid substitutions were in two domains important for factor VIII interaction. Protein infusion into hemophilia B (HB) mice showed that recombinant FIX-Triple was also more effective than FIX-WT in shortening the clotting time in these HB mice. To further evaluate the medium and long-term efficacy of FIX-Triple expression, we used two methods of gene delivery in (HB) mice. For medium-term expression, we performed tail vein hydrodynamic plasmid injections of either FIX-WT or FIX-Triple, expressed from a liver-specific promoter. We assayed specific activity (clotting activity/antigen, U/mg) 24h post-delivery and found that FIX-Triple had a 3.5-fold higher specific activity than FIX-WT. For long-term expression, we performed tail vein administration of a serotype 8 recombinant Adeno-associated vector (AAV8) expressing either FIX-WT or FIX-Triple from a liver-specific promoter at vector doses of 4x1012 (high dose) and 8x1010 (low dose) vector genomes (v.g.)/kg. These mice were sacrificed 8 weeks post vector administration and blood samples were analyzed for FIX antigen (ELISA) and clotting activity (aPTT). Corroborating our previous observations, we found that the FIX-Triple variant had 7 times higher specific activity vs. FIX-WT, for either vector dose used. More importantly, mice that received AAV8-FIX-Triple exhibited activity of 23% of normal pooled human plasma (12-38%, n=6), in contrast to mice that received FIX-WT that only reached 4% (1-8%, n = 4), following 8x1010 v.g./kg vector administration. Activity and antigen level for both transgenes was dose-dependent. Lastly, we generated knock-in mice for FIX-WT and FIX-Triple (FIX-KI-WT and FIX-KI-Triple) by exchanging the mouse sequence for the human, keeping the endogenous promoter intact. Confirming our observations with hydrodynamic injection and AAV administration, mice with FIX-KI-Triple exhibited 7 fold higher specific activity than those with FIX-KI-WT (2040 ± 253 vs 279 ± 33 U/mg). Collectively, our results indicate that FIX-Triple variant exhibits significantly enhanced clotting activity relative to FIX-WT due to tighter binding to Factor VIIIa, as demonstrated both in vitro and in vivo. Therefore, Factor IX-Triple is a good candidate for further evaluation in protein replacement therapy as well as other, gene-based therapeutic strategies.