Academic literature on the topic 'AAV-FIX'

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.

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.

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.

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.

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.

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.

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.

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 &gt;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 &gt;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.

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.

Hemophilia B is an X-linked bleeding disorder that prevents the blood from clotting normally. It is caused by mutations in the factor IX (FIX) gene leading to low levels or complete loss of FIX expression/activity. It is a rare disease that affects about 1 in 20,000 male births in the USA and approximately 400,000 individuals across the world. The severity of this disease depends on the level of FIX protein expression in the patients and has been classified as mild (5-40% FIX activity), moderate (1-5% FIX activity) or severe (< 1% FIX activity). Patients with mild disease usually bleed only after serious injury, trauma or surgery; and in such patients, the first bleeding episode usually doesn’t occur until adulthood. Individuals with moderate disease, who account for about 15% of the hemophilia population, have bleeding episodes after minor injuries and often have spontaneous bleeding episodes without an obvious cause. People with severe disease also bleed following an injury and have frequent spontaneous bleeding episodes in joints and muscle. Such patients account for about 60% of the hemophilia B population (National Hemophilia Foundation, USA and Centre for Disease Control and Prevention, USA). Replacement therapy for the missing FIX is the only drug modality currently available for the treatment of haemophilia B. It requires frequent infusion of plasma derived or recombinant FIX protein to the patients. Importantly, infusion of plasma derived FIX might entail the risk of patient infection. This study was carried out to develop adeno associated viral vector based gene therapy product for the treatment of Hemophilia B (AAV-FIX). AAV-FIX vectors were expressed in HEK293 cells by triple plasmid co-transfection. Transfected cells were lysed to release the vector in cell lysate which was clarified and subjected to affinity based purification. The purified vectors were analysed by SDS-PAGE and ELISA for the purity and quantity.