Monogenic diseases, including hemophilia, represent ideal targets for genome-editing approaches aimed at correcting a defective gene. Here we report that systemic adeno-associated virus (AAV) vector delivery of zinc finger nucleases (ZFNs) and corrective donor template to the predominantly quiescent livers of adult mice enables production of high levels of human factor IX in a murine model of hemophilia B. Further, we show that off-target cleavage can be substantially reduced while maintaining robust editing by using obligate heterodimeric ZFNs engineered to minimize unwanted cleavage attributable to homodimerization of the ZFNs. These results broaden the therapeutic potential of AAV/ZFN-mediated genome editing in the liver and could expand this strategy to other nonreplicating cell types.
Self-complementary adeno-associated viral (AAV) vectors expressing human factor IX (hF.IX) have achieved transient or sustained correction of hemophilia B in human volunteers. High doses of AAV2 or AAV8 vectors delivered to the liver caused in several patients an increase in transaminases accompanied by a rise in AAV capsid-specific T cells and a decrease in circulating hF.IX levels suggesting immune-mediated destruction of vector-transduced cells. Kinetics of these adverse events differed in patients receiving AAV2 or AAV8 vectors causing rise in transaminases at 3 versus 8 weeks after vector injection, respectively. To test if CD8(+) T cells to AAV8 vectors, which are similar to AAV2 vectors are fully-gutted vectors and thereby fail to encode structural viral proteins, could cause damage at this late time point, we tested in a series of mouse studies how long major histocompatibility (MHC) class I epitopes within AAV8 capsid can be presented to CD8(+) T cells. Our results clearly show that depending on the vectors genome, CD8(+) T cells can detect such epitopes on AAV8s capsid for up to 6 months indicating that the capsid of AAV8 degrades slowly in mice.
Adeno-associated virus (AAV) vectors delivered through the systemic circulation successfully transduce various target tissues in animal models. However, similar attempts in humans have been hampered by the high prevalence of neutralizing antibodies to AAV, which completely block vector transduction. We show in both mouse and nonhuman primate models that addition of empty capsid to the final vector formulation can, in a dose-dependent manner, adsorb these antibodies, even at high titers, thus overcoming their inhibitory effect. To further enhance the safety of the approach, we mutated the receptor binding site of AAV2 to generate an empty capsid mutant that can adsorb antibodies but cannot enter a target cell. Our work suggests that optimizing the ratio of full/empty capsids in the final formulation of vector, based on a patients anti-AAV titers, will maximize the efficacy of gene transfer after systemic vector delivery.
Immune responses directed against viral capsid proteins constitute a main safety concern in the use of adeno-associated virus (AAV) as gene transfer vectors in humans. Pharmacological immunosuppression has been proposed as a solution to the problem; however, the approach suffers from several potential limitations. Using MHC class II epitopes initially identified within human IgG, named Tregitopes, we showed that it is possible to modulate CD8+ T cell responses to several viral antigens in vitro. We showed that incubation of peripheral blood mononuclear cells with these epitopes triggers proliferation of CD4+CD25+FoxP3+ T cells that suppress killing of target cells loaded with MHC class I antigens in an antigen-specific fashion, through a mechanism that seems to require cell-to-cell contact. Expression of a construct encoding for the AAV capsid structural protein fused to Tregitopes resulted in reduction of CD8+ T cell reactivity against the AAV capsid following immunization with an adenoviral vector expressing capsid. This was accompanied by an increase in frequency of CD4+CD25+FoxP3+ T cells in spleens and lower levels of inflammatory infiltrates in injected tissues. This proof-of-concept study demonstrates modulation of CD8+ T cell reactivity to an antigen using regulatory T cell epitopes is possible.
Severe deficiency of plasma ADAMTS13 activity causes thrombotic thrombocytopenic purpura (TTP), a life-threatening syndrome for which plasma is the only effective therapy currently available. As much as 5% of TTP cases are hereditary, resulting from mutations of the ADAMTS13 gene. Here, we report the efficacy and safety of recombinant adeno-associated virus serotype 8 (AAV8)-mediated expression of a murine ADAMTS13 variant (MDTCS), truncated after the spacer domain, in a murine model of TTP. Administration of AAV8-hAAT-mdtcs at doses greater than 2.6 × 10(11) vg/kg body weight resulted in sustained expression of plasma ADAMTS13 activity at therapeutic levels. Expression of the truncated ADAMTS13 variant eliminated circulating ultralarge von Willebrand factor multimers, prevented severe thrombocytopenia, and reduced mortality in Adamts13(-/-) disease-prone mice triggered by shigatoxin-2. These data support AAV vector-mediated expression of a comparable truncated ADAMTS13 variant as a novel therapeutic approach for hereditary TTP in humans.
Diabetes is associated with severe secondary complications, largely caused by poor glycemic control. Treatment with exogenous insulin fails to prevent these complications completely, leading to significant morbidity and mortality. We previously demonstrated that it is possible to generate a "glucose sensor" in skeletal muscle through coexpression of glucokinase and insulin, increasing glucose uptake and correcting hyperglycemia in diabetic mice. Here, we demonstrate long-term efficacy of this approach in a large animal model of diabetes. A one-time intramuscular administration of adeno-associated viral vectors of serotype 1 encoding for glucokinase and insulin in diabetic dogs resulted in normalization of fasting glycemia, accelerated disposal of glucose after oral challenge, and no episodes of hypoglycemia during exercise for >4 years after gene transfer. This was associated with recovery of body weight, reduced glycosylated plasma proteins levels, and long-term survival without secondary complications. Conversely, exogenous insulin or gene transfer for insulin or glucokinase alone failed to achieve complete correction of diabetes, indicating that the synergistic action of insulin and glucokinase is needed for full therapeutic effect. This study provides the first proof-of-concept in a large animal model for a gene transfer approach to treat diabetes.
Recombinant canine B-domain deleted (BDD) factor VIII (FVIII) is predominantly expressed as a single-chain protein and exhibits greater stability after activation compared with human FVIII-BDD. We generated a novel BDD-FVIII variant (FVIII-RH) with an amino acid change at the furin cleavage site within the B domain (position R1645H) that mimics the canine sequence (HHQR vs human RHQR). Compared with human FVIII-BDD, expression of FVIII-RH protein revealed a 2.5-fold increase in the single-chain form. Notably, FVIII-RH exhibited a twofold increase in biological activity compared with FVIII-BDD, likely due to its slower dissociation of the A2-domain upon thrombin activation. Injection of FVIII-RH protein in hemophilia A (HA) mice resulted in more efficacious hemostasis following vascular injury in both the macro- and microcirculation. These findings were successfully translated to adeno-associated viral (AAV)-based liver gene transfer in HA mice. Expression of circulating FVIII-RH was approximately twofold higher compared with AAV-FVIII-BDD-injected mice. Moreover, FVIII-RH exhibits superior procoagulant effects compared with FVIII-BDD following a series of hemostatic challenges. Notably, the immunogenicity of FVIII-RH did not differ from FVIII-BDD. Thus, FVIII-RH is an attractive bioengineered molecule for improving efficacy without increased immunogenicity and may be suitable for both protein- and gene-based strategies for HA.
Recent clinical trials have shown that evasion of CD8(+) T-cell responses against viral capsid is critical for successful liver-directed gene therapy with adeno-associated viral (AAV) vectors for hemophilia. Preclinical models to test whether use of alternate serotypes or capsid variants could avoid this deleterious response have been lacking. Here, the ability of CD8(+) T cells ("cap-CD8," specific for a capsid epitope presented by human B*0702 or murine H2-L(d) molecules) to target AAV-infected hepatocytes was investigated. In a murine model based on adoptive transfer of ex vivo expanded cap-CD8, AAV2-transduced livers showed CD8(+) T-cell infiltrates, transaminitis, significant reduction in factor IX transgene expression, and loss of transduced hepatocytes. AAV8 gene transfer resulted in prolonged susceptibility to cap-CD8, consistent with recent clinical findings. In contrast, using an AAV2(Y-F) mutant capsid, which is known to be less degraded by proteasomes, preserved transgene expression and largely avoided hepatotoxicity. In vitro assays confirmed reduced major histocompatibility complex class I presentation of this capsid and killing of human or murine hepatocytes compared with AAV2. In conclusion, AAV capsids can be engineered to substantially reduce the risk of destruction by cytotoxic T lymphocytes, whereas use of alternative serotypes per se does not circumvent this obstacle.
Choroideremia (CHM) is an X- linked retinal degeneration that is symptomatic in the 1(st) or 2(nd) decade of life causing nyctalopia and loss of peripheral vision. The disease progresses through mid-life, when most patients become blind. CHM is a favorable target for gene augmentation therapy, as the disease is due to loss of function of a protein necessary for retinal cell health, Rab Escort Protein 1 (REP1).The CHM cDNA can be packaged in recombinant adeno-associated virus (rAAV), which has an established track record in human gene therapy studies, and, in addition, there are sensitive and quantitative assays to document REP1 activity. An animal model that accurately reflects the human condition is not available. In this study, we tested the ability to restore REP1 function in personalized in vitro models of CHM: lymphoblasts and induced pluripotent stems cells (iPSCs) from human patients. The initial step of evaluating safety of the treatment was carried out by evaluating for acute retinal histopathologic effects in normal-sighted mice and no obvious toxicity was identified. Delivery of the CHM cDNA to affected cells restores REP1 enzymatic activity and also restores proper protein trafficking. The gene transfer is efficient and the preliminary safety data are encouraging. These studies pave the way for a human clinical trial of gene therapy for CHM.
Intramuscular (IM) administration of an adeno-associated viral (AAV) vector represents a simple and safe method of gene transfer for treatment of the X-linked bleeding disorder hemophilia B (factor IX, F.IX, deficiency). However, the approach is hampered by an increased risk of immune responses against F.IX. Previously, we demonstrated that the drug cocktail of immune suppressants rapamycin, IL-10, and a specific peptide (encoding a dominant CD4(+) T cell epitope) caused an induction of regulatory T cells (Treg) with a concomitant apoptosis of antigen-specific effector T cells (Nayak et al., 2009). This protocol was effective in preventing inhibitory antibody formation against human F.IX (hF.IX) in muscle gene transfer to C3H/HeJ hemophilia B mice (with targeted F9 gene deletion). Here, we show that this protocol can also be used to reverse inhibitor formation. IM injection of AAV1-hF.IX vector resulted in inhibitors of on average 8-10?BU within 1?month. Subsequent treatment with the tolerogenic cocktail accomplished a rapid reduction of hF.IX-specific antibodies to <2?BU, which lasted for >4.5?months. Systemic hF.IX expression increased from undetectable to >200?ng/ml, and coagulation times improved. In addition, we developed an alternative prophylactic protocol against inhibitor formation that did not require knowledge of T cell epitopes, consisting of daily oral administration of rapamycin for 1-month combined with frequent, low-dose intravenous injection of hF.IX protein. Experiments in T cell receptor transgenic mice showed that the route and dosing schedule of drug administration substantially affected Treg induction. When combined with intravenous antigen administration, oral delivery of rapamycin had to be performed daily in order to induce Treg, which were suppressive and phenotypically comparable to natural Treg.
Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.
Catalytic domain variants of activated factor VII (FVIIa) with enhanced hemostatic properties are highly attractive for the treatment of bleeding disorders via gene-based therapy. To explore this in a hemophilic mouse model, we characterized 2 variants of murine activated FVII (mFVIIa-VEAY and mFVIIa-DVQ) with modified catalytic domains, based on recombinant human FVIIa (rhFVIIa) variants. Using purified recombinant proteins, we showed that murine FVIIa (mFVIIa) and variants had comparable binding to human and murine tissue factor (TF) and exhibited similar extrinsic coagulant activity. In vitro in the absence of TF, the variants showed a 6- to 17-fold enhanced proteolytic and coagulant activity relative to mFVIIa, but increased inactivation by antithrombin. Gene delivery of mFVIIa-VEAY resulted in long-term, effective hemostasis at 5-fold lower expression levels relative to mFVIIa in hemophilia A mice or in hemophilia B mice with inhibitors to factor IX. However, expression of mFVIIa-VEAY at 14-fold higher than therapeutic levels resulted in a progressive mortality to 70% within 6 weeks after gene delivery. These results are the first demonstration of the hemostatic efficacy of continuous expression, in the presence or absence of inhibitors, of a high-activity gene-based FVIIa variant in an animal model of hemophilia.
Developing adeno-associated viral (AAV)-mediated gene therapy for hemophilia A (HA) has been challenging due to the large size of the factor VIII (FVIII) complementary DNA and the concern for the development of inhibitory antibodies to FVIII in HA patients. Here, we perform a systematic study in HA dogs by delivering a canine FVIII (cFVIII) transgene either as a single chain or two chains in an AAV vector. An optimized cFVIII single chain delivered using AAV serotype 8 (AAV8) by peripheral vein injection resulted in a dose-response with sustained expression of FVIII up to 7% (n = 4). Five HA dogs administered two-chain delivery using either AAV8 or AAV9 via the portal vein expressed long-term, vector dose-dependent levels of FVIII activity (up to 10%). In the two-chain approach, circulating cFVIII antigen levels were more than fivefold higher than activity. Notably, no long-term immune response to FVIII was observed in any of the dogs (1/9 dogs had a transient inhibitor). Long-term follow-up of the dogs showed a remarkable reduction (>90%) of bleeding episodes in a combined total of 24 years of observation. These data demonstrate that both approaches are safe and achieve dose-dependent therapeutic levels of FVIII expression, which supports translational studies of AAV-mediated delivery for HA.
Inhibitory antibodies to factor VIII (FVIII) are a major complication in the treatment of hemophilia A, affecting approximately 20% to 30% of patients. Current treatment for inhibitors is based on long-term, daily injections of large amounts of FVIII protein. Liver-directed gene therapy has been used to induce antigen-specific tolerance, but there are no data in hemophilic animals with pre-existing inhibitors. To determine whether sustained endogenous expression of FVIII could eradicate inhibitors, we injected adeno-associated viral vectors encoding canine FVIII (cFVIII) in 2 strains of inhibitor hemophilia A dogs. In 3 dogs, a transient increase in inhibitor titers (up to 7 Bethesda Units [BU]) at 2 weeks was followed by continuous decline to complete disappearance within 4-5 weeks. Subsequently, an increase in cFVIII levels (1.5%-8%), a shortening of clotting times, and a reduction (> 90%) of bleeding episodes were observed. Immune tolerance was confirmed by lack of antibody formation after repeated challenges with cFVIII protein and normal protein half-life. A fourth dog exhibited a strong early anamnestic response (216 BU), with slow decline to 0.8 BU and cFVIII antigen detection by 18 months after vector delivery. These data suggest that liver gene therapy has the potential to eradicate inhibitors and could improve the outcomes of hemophilia A patients.
RNA interference (RNAi) is being evaluated as an alternative therapeutic strategy for hepatitis C virus (HCV) infection. The use of viral vectors encoding short hairpin RNAs (shRNAs) has been the most common strategy employed to provide sustained expression of RNAi effectors. However, overexpression and incomplete processing of shRNAs has led to saturation of the endogenous miRNA pathway, resulting in toxicity. The use of endogenous microRNAs (miRNAs) as scaffolds for short interfering (siRNAs) may avoid these problems, and miRNA clusters can be engineered to express multiple RNAi effectors, a feature that may prevent RNAi-resistant HCV mutant generation. We exploited the endogenous miRNA-17-92 cluster to generate a polycistronic primary miRNA that is processed into five mature miRNAs that target different regions of the HCV genome. All five anti-HCV miRNAs were active, achieving up to 97% inhibition of Renilla luciferase (RLuc) HCV reporter plasmids. Self-complementary recombinant adeno-associated virus (scAAV) vectors were chosen for therapeutic delivery of the miRNA cluster. Expression of the miRNAs from scAAV inhibited the replication of cell culture-propagated HCV (HCVcc) by 98%, and resulted in up to 93% gene silencing of RLuc-HCV reporter plasmids in mouse liver. No hepatocellular toxicity was observed at scAAV doses as high as 5 × 10(11) vector genomes per mouse, a dose that is approximately five-fold higher than doses of scAAV-shRNA vectors that others have shown previously to be toxic in mouse liver. Conclusion: We have demonstrated that exogenous anti-HCV miRNAs induce gene silencing, and when expressed from scAAV vectors inhibit the replication of HCVcc without inducing toxicity. The combination of an AAV vector delivery system and exploitation of the endogenous RNAi pathway is a potentially viable alternative to current HCV treatment regimens.
Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector-mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-gamma enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4(+)FoxP3(+)IL-10(+) T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle.
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.
Adeno-associated viral (AAV) vectors are an extensively studied and highly used vector platform for gene therapy applications. We hypothesize 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. It has been previously shown that proteasome inhibitors can have a dramatic effect on AAV transduction in vitro and in vivo. Here, we describe using the US Food and Drug Administration-approved proteasome inhibitor, bortezomib, to decrease capsid antigen presentation on hepatocytes in vitro, whereas at the same time, enhancing gene expression in vivo. Using an AAV capsid-specific T-cell reporter (TCR) line to analyze the effect of proteasome inhibitors on antigen presentation, we demonstrate capsid antigen presentation at low multiplicities of infection (MOIs), and inhibition of antigen presentation at pharmacologic levels of bortezomib. We also demonstrate that bortezomib can enhance Factor IX (FIX) expression from an AAV2 vector in mice, although the same effect was not observed for AAV8 vectors. A pharmacological agent that can enhance AAV transduction, decrease T-cell activation/proliferation, and decrease capsid antigen presentation would be a promising solution to obstacles to successful AAV-mediated, liver-directed gene transfer in humans.
Immune responses to factor IX (F.IX), a major concern in gene therapy for hemophilia, were analyzed for adeno-associated viral (AAV-2) gene transfer to skeletal muscle and liver as a function of the F9 underlying mutation. Vectors identical to those recently used in clinical trials were administered to four lines of hemophilia B mice on a defined genetic background [C3H/HeJ with deletion of endogenous F9 and transgenic for a range of nonfunctional human F.IX (hF.IX) variants]. The strength of the immune response to AAV-encoded F.IX inversely correlated with the degree of conservation of endogenous coding information and levels of endogenous antigen. Null mutation animals developed T- and B-cell responses in both protocols. However, inhibitor titers were considerably higher upon muscle gene transfer (or protein therapy). Transduced muscles of Null mice had strong infiltrates with CD8+ cells, which were much more limited in the liver and not seen for the other mutations. Sustained expression was achieved with liver transduction in mice with crm(-) nonsense and missense mutations, although they still formed antibodies upon muscle gene transfer. Therefore, endogenous expression prevented T-cell responses more effectively than antibody formation, and immune responses varied substantially depending on the protocol and the underlying mutation.
Continuous expression of activated factor VII (FVIIa) via gene transfer is a potential therapeutic approach for hemophilia patients with or without inhibitory antibodies to human factor VIII (FVIII) or IX (FIX). Here, we investigate whether gene transfer of an engineered canine FVIIa (cFVIIa) transgene can affect hemostasis in a canine model of hemophilia, a good predictor of efficacy of hemophilia treatments. Purified recombinant cFVIIa exhibited 12-fold higher tissue factor-dependent activity than purified recombinant zymogen cFVII. Subsequently, we generated a serotype 8 recombinant adeno-associated viral vector expressing cFVIIa from a liver-specific promoter. Vector delivery via the portal vein in hemophilia A and B dogs was well tolerated, and long-term expression of cFVIIa resulted in a shortening of the prothrombin time, partial correction of the whole blood clotting time and thromboelastography parameters, and a complete absence of spontaneous bleeding episodes. No evidence of hepatotoxicity, thrombotic complications, or inhibitory immune response was found. These data provide the first evidence for in vivo efficacy and safety of continuously expressed FVIIa as a FVIII/FIX-bypassing agent in a large animal model of hemophilia, avoiding the risk of inhibitor formation associated with bolus FVIII or FIX infusion.
Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid-derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.
The assessment of the risk of germline transmission of vector-coded sequences is critical for clinical translation of gene transfer strategies. We used rabbit models to analyze the risk of germline transmission of adeno-associated viral (AAV) vectors. Intravenous injection of AAV-2 or AAV-8 resulted in liver-mediated, long-term expression of therapeutic levels of human factor IX (hFIX) in a dose-dependent manner. In high-dose cohorts, AAV-8 resulted in twofold higher levels of circulating hFIX and of vector DNA in liver compared to AAV-2. Vector sequences were found in the semen of all rabbits. The kinetics of vector clearance from semen was dose- and time-dependent but serotype-independent. No late recurrence of AAV-8 sequences was found in the semen over several consecutive cycles of spermatogenesis. In a novel rabbit model, AAV-2 or AAV-8 sequences were detected in the semen of vasectomized animals that lack germ cells. Therefore, structures of the genitourinary (GU) tract, as well as the testis, contribute significantly to vector shedding in the semen. Collectively, data from these two models suggest that the risk of inadvertent germline transmission in males by AAV-8 vectors is low, similar to that of AAV-2, and that AAV dissemination to the semen is in part modulated by host-dependent factors.
There is considerable interest in the use of adeno-associated virus serotype 9 (AAV9) for neurological gene therapy partly because of its ability to cross the blood-brain barrier to transduce astrocytes and neurons. This raises the possibility that AAV9 might also transduce antigen-presenting cells (APC) in the brain and provoke an adaptive immune response. We tested this hypothesis by infusing AAV9 vectors encoding foreign antigens, namely human aromatic L-amino acid decarboxylase (hAADC) and green fluorescent protein (GFP), into rat brain parenchyma. Over ensuing weeks, both vectors elicited a prominent inflammation in transduced brain regions associated with upregulation of MHC II in glia and associated lymphocytic infiltration. Transduction of either thalamus or striatum with AAV9-hAADC evinced a significant loss of neurons and induction of anti-hAADC antibodies. We conclude that AAV9 transduces APC in the brain and, depending on the immunogenicity of the transgene, can provoke a full immune response that mediates significant brain pathology. We emphasize, however, that these observations do not preclude the use of AAV serotypes that can transduce APC. However, it does potentially complicate preclinical toxicology studies in which non-self proteins are expressed at a level sufficient to trigger cell-mediated and humoral immune responses.
The hepatitis C virus (HCV) chronically infects 2% of the world population and effective treatment is limited by long duration and significant side-effects. Here, we describe a novel drug, intended as a "single-shot " therapy, which expresses three short hairpin RNAs (shRNAs) that simultaneously target multiple conserved regions of the HCV genome as confirmed in vitro by knockdown of an HCV replicon system. Using a recombinant adeno-associated virus (AAV) serotype 8 vector for delivery, comprehensive transduction of hepatocytes was achieved in vivo in a nonhuman primate (NHP) model following a single intravenous injection. However, dose ranging studies performed in 13 NHP resulted in high-expression levels of shRNA from wild-type (wt) Pol III promoters and dose-dependent hepatocellular toxicity, the first demonstration of shRNA-related toxicity in primates, establishing that the hepatotoxicity arises from highly conserved features of the RNA interference (RNAi) pathway. In the second generation drug, each promoter was re-engineered to reduce shRNA transcription to levels that circumvent toxicity but still inhibit replicon activity. In vivo testing of this modified construct in 18 NHPs showed conservation of hepatocyte transduction but complete elimination of hepatotoxicity, even with sustained shRNA expression for 50 days. These data support progression to a clinical study for treatment of HCV infection.
Liver gene transfer for hemophilia B has shown very promising results in recent clinical studies. A potential complication of gene-based treatments for hemophilia and other inherited disorders, however, is the development of neutralizing antibodies (NAb) against the therapeutic transgene. The risk of developing NAb to the coagulation factor IX (F.IX) transgene product following adeno-associated virus (AAV)-mediated hepatic gene transfer for hemophilia is small but not absent, as formation of inhibitory antibodies to F.IX is observed in experimental animals following liver gene transfer. Thus, strategies to modulate antitransgene NAb responses are needed. Here, we used the anti-B cell monoclonal antibody rituximab (rtx) in combination with cyclosporine A (CsA) to eradicate anti-human F.IX NAb in rhesus macaques previously injected intravenously with AAV8 vectors expressing human F.IX. A short course of immunosuppression (IS) resulted in eradication of anti-F.IX NAb with restoration of plasma F.IX transgene product detection. In one animal, following IS anti-AAV6 antibodies also dropped below detection, allowing for successful AAV vector readministration and resulting in high levels (60% or normal) of F.IX transgene product in plasma. Though the number of animals is small, this study supports for the safety and efficacy of B cell-targeting therapies to eradicate NAb developed following AAV-mediated gene transfer.
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