Muscle-directed Gene Transfer Research Articles

Biodistribution of Adeno-Associated Virus Gene Therapy Following Cerebrospinal Fluid-Directed Administration.

Adeno-associated virus (AAV)-based gene therapies, exemplified by the approved therapy for spinal muscular atrophy, have the potential to deliver disease-course-altering treatments for central nervous system (CNS) indications. However, several clinical trials have reported severe adverse events, including patient deaths following high-dose systemic administration for muscle-directed gene transfer, highlighting the need to explore approaches utilizing lower doses when targeting the CNS. Animal models of disease provide insight into the response to new AAV therapies. However, translation from small to larger animals and eventually to humans is hampered by anatomical and biological differences across the species and their impact on AAV delivery. We performed a literature review of preclinical studies of AAV gene therapy biodistribution following cerebrospinal fluid (CSF) delivery (intracerebroventricular, intra-cisterna magna, and intrathecal lumbar). The reviewed literature varies greatly in the reported biodistribution of AAV following administration into the CSF. Differences between studies, including animal model, vector serotype used, method used to assess biodistribution, and route of administration, among other variables, contribute to differing outcomes and difficulties in translating these preclinical results. For example, only half of the published AAV-based gene therapy studies report vector copy number, the most direct readout following administration of a vector; none of these studies reported details such as the empty:full capsid ratio and quality of encapsidated genome. Analysis of the last decade's literature focusing on AAV-based gene therapies targeting the CNS underscores limitations of the body of knowledge and room for continued research. In particular, there is a need to understand the biodistribution achieved by different CSF-directed routes of administration and determining if specific cell types/structures of interest will be transduced. Our findings point to a clear need for a more systematic approach across the field to align the assessments and elements reported in preclinical research to enable more reliable translation across animal models and into human studies.

AAV Vector Dose Determines TLR9 Dependence of CD8+ T Cell Response to Transgene Product

Adeno-associated viral (AAV) vectors are currently evaluated in multiple Phase III clinical trial for the treatment of hemophilia and neuromuscular disorders. A major concern is the potential for immune responses. Viral vectors are initially sensed by the innate immune system, which shapes subsequent adaptive immune responses. Particularly, toll-like receptors (TLRs) have been reported as major sensors of pathogens during innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs). Our previous studies found that cross-priming of AAV capsid-specific CD8+ T cells depended on TLR9-MyD88 pathway. TLR9 is an endosomal DNA receptor that responds most potently to unmethylated CpG motifs as found in bacterial and viral DNA. Similarly, others documented TLR9-dependent CD8+ T cell responses against non-secreted transgene products such as LacZ and hemagglutinin upon muscle-directed AAV gene transfer. Similarly, we published that CD8+ T cell responses to a secreted ovalbumin (ova) transgene product were substantially reduced (although not entirely eliminated) upon muscle gene transfer in TLR9-deficient mice [J Innate Immun. 7:302-14]. For those studies, we had used a self-complementary scAAV genomes, which we found to more strongly activate TLR9 than conventional single-stranded ssAAV vectors. Here, we performed intramuscular injections of 3 doses of ssAAV1-CMV-ova vector (2X1010, 2X1011 and1X1012 vg) in wild-type (WT), TLR9-/-, or MYD88-/- C57BL/6 mice. Using MHC tetramer (H2-Kb -SIINFEKL), ova-specific CD8+ T cell frequencies were monitored in peripheral blood for up to 6 weeks. As expected from prior studies, TLR9-/- mice showed a substantially reduced response (1.2% tetramer+ of CD8) at the low dose when compared to WT (12% tetramer+ of CD8) animals (p<0.0001, n=5/group). To our surprise, CD8+ T cell responses were similar in TLR9-/- and WT mice at the 2 higher doses. TLR9-/- mice displayed 16% and 3.3% tetramer+ of CD8 frequencies at the median and the high doses, respectively; which was comparable to WT mice, where 15% and 4.8% tetramer+ of CD8 frequencies were observed (n=5/group). Therefore, sensing of the AAV genome by TLR9 is more critical for the CD8+ T cell response to the secreted transgene product at lower vector doses (possibly related to the lower levels of transgene expression). Interestingly, transgene product-specific CD8+ T cell responses were much reduced in MyD88-/- mice, in which 0.2% and 1.7% tetramer+ of CD8 frequencies were found for low and median doses. Therefore, an alternative signaling pathway that includes the MyD88 adaptor molecule likely exists that is more critical than TLR9 above a certain level of expression. The reduced strength of the CD8+ T cell response seen at the highest vector dose compared to the medium dose may be explained by a transient increase in FoxP3+ Treg and in PD-1+ T cells that we observed 1 week after gene transfer and that was significantly greater at the highest vector dose. In related experiments, we performed intramuscular gene transfer using a ssAAV1-EF1a-FIX vector in hemophilia B mice (C3H/HeJ F9-/-, 1x1011 vg/mouse). Here, we used either a vector with native sequences or with an expression cassette that was entirely devoid of CpG motifs (and there stimulates TLR9 less effectively). CpG depletion did not have substantial effects on antibody formation against human FIX or the viral capsid. However, CD8+ T cell infiltrates in skeletal muscle were markedly reduced but not entirely eliminated when tissue sections were examined 1 month after gene transfer. In conclusion, TLR9 signaling is one important factor in the activation of transgene product-specific CD8+ T cells in AAV gene transfer, but other pathways exist that may be more critical depending on vector dose or levels of expression. Disclosures Herzog: Takeda Pharmaceuticals: Patents & Royalties.

Liver-Directed but Not Muscle-Directed AAV-Antibody Gene Transfer Limits Humoral Immune Responses in Rhesus Monkeys

A number of publications have described the use of adeno-associated virus (AAV) for the delivery of anti-HIV and anti-simian immunodeficiency virus (SIV) monoclonal antibodies (mAbs) to rhesus monkeys. Anti-drug antibodies (ADAs) have been frequently observed, and long-term AAV-mediated delivery has been inconsistent. Here, we investigated different AAV vector strategies and delivery schemes to rhesus monkeys using the rhesus monkey mAb 4L6. We compared 4L6 immunoglobulin G1 (IgG1) delivery using the AAV1 versus the AAV8 serotype with a cytomegalovirus (CMV) promoter and the use of a muscle-specific versus a liver-specific promoter. Long-term expression levels of 4L6 IgG1 following AAV8-mediated gene transfer were comparable to those following AAV1-mediated gene transfer. AAV1-mediated gene transfer, using a muscle-specific promoter, showed robust ADAs and transiently low 4L6 IgG1 levels that ultimately declined to below detectable levels. Intravenous AAV8-mediated gene transfer, using a liver-specific promoter, also resulted in low levels of delivered 4L6 IgG1, but those low levels were maintained in the absence of any detectable ADAs. Booster injections using AAV1-CMV allowed for increased 4L6 IgG1 serum levels in animals that were primed with AAV8 but not with AAV1. Our results suggest that liver-directed expression may help to limit ADAs and that re-administration of AAV of a different serotype can result in successful long-term delivery of an immunogenic antibody.

Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle

The pro-tolerogenic environment of the liver makes this tissue an ideal target for gene replacement strategies. In other peripheral tissues such as the skeletal muscle, anti-transgene immune response can result in partial or complete clearance of the transduced fibers. Here, we characterized liver-induced transgene tolerance after simultaneous transduction of liver and muscle. A clinically relevant transgene, α-sarcoglycan, mutated in limb-girdle muscular dystrophy type 2D, was fused with the SIINFEKL epitope (hSGCA-SIIN) and expressed with adeno-associated virus vectors (AAV-hSGCA-SIIN). Intramuscular delivery of AAV-hSGCA-SIIN resulted in a strong inflammatory response, which could be prevented and reversed by concomitant liver expression of the same antigen. Regulatory T cells and upregulation of checkpoint inhibitor receptors were required to establish and maintain liver-mediated peripheral tolerance. This study identifies the fundamental role of the synergy between Tregs and upregulation of checkpoint inhibitor receptors in the liver-mediated control of anti-transgene immunity triggered by muscle-directed gene transfer.

Recombinant AAV Vectors for Enhanced Expression of Authentic IgG.

Adeno-associated virus (AAV) has become a vector of choice for the treatment of a variety of genetic diseases that require safe and long-term delivery of a missing protein. Muscle-directed gene transfer for delivery of protective antibodies against AIDS viruses and other pathogens has been used experimentally in mice and monkeys. Here we examined a number of variations to AAV vector design for the ability to produce authentic immunoglobulin G (IgG) molecules. Expression of rhesus IgG from a single single-stranded AAV (ssAAV) vector (one vector approach) was compared to expression from two self-complementary AAV (scAAV) vectors, one for heavy chain and one for light chain (two vector approach). Both the one vector and the two vector approaches yielded considerable levels of expressed full-length IgG. A number of modifications to the ssAAV expression system were then examined for their ability to increase the efficiency of IgG expression. Inclusion of a furin cleavage sequence with a linker peptide just upstream of the 2A self-cleaving sequence from foot-and-mouth disease virus (F2A) increased IgG expression approximately 2 fold. Inclusion of these sequences also helped to ensure a proper sequence at the C-terminal end of the heavy chain. Inclusion of the post-transcriptional regulatory element from woodchuck hepatitis virus (WPRE) further increased IgG expression 1.5–2.0 fold. IgG1 versions of the two rhesus IgGs that were examined consistently expressed better than the IgG2 forms. In contrast to what has been reported for AAV2-mediated expression of other proteins, introduction of capsid mutations Y445F and Y731F did not increase ssAAV1-mediated expression of IgG as determined by transduction experiments in cell culture. Our findings provide a rational basis for AAV vector design for expression of authentic IgG.

Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

AbstractIn previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

Humoral and Anaphylactic Responses to Factor IX In Murine Hemophilia B Are Genotype Dependent and Can Be Reversed by Hepatic Gene Transfer

AbstractAbstract 2202Hemophilia B is the X-linked bleeding disorder caused by loss of coagulation factor IX (F.IX) function. Current treatment relies on infusion of plasma derived or recombinant F.IX protein. Approximately 2–4% of hemophilia B patients develop inhibitory antibodies to F.IX protein, and those with F.IX gene deletions are at risk for anaphylaxis. We recently described a murine mode for this pathogenic antibody response, namely C3H/HeJ hemophilia B (HB) mice with a F9 gene deletion (PNAS 107:7101). Anaphylactic reactions were the result of a strong Th2-driven antibody response, comprised of IgG1 and IgE. In new experiments, we again found that repeated exposure to F.IX protein from weekly intravenous injections of recombinant human F.IX (1 IU/mouse) resulted in inhibitors, with 53% of the treated mice experiencing fatal anaphylaxis following the third administration. Surviving animals were given three additional intravenous F.IX protein injections (the fifth and sixth injections were given with antihistamine) and resulted in an average inhibitor titer of 6.2 BU/ml corresponding to an IgG1 titer of 5 mg/ml. Previously, we generated C3H/HeJ HB mice expressing non-functional crm- hF.IX variants due a missense mutation analogous to the UNC-Chapel Hill strain of hemophilia B dog (HB-CH) or due to a stop codon at amino acid residue 338 (HB-338). These mice, in contrast to the gene deletion mutation, failed to develop CD8+ T cell responses to hF.IX, but still formed inhibitors (albeit at reduced titers) upon muscle-directed gene transfer with AAV vector (Mol. Ther 17:1733). Furthermore, HB mice transgenic for a crim+ hF.IX missense mutation (HB-180) were entirely unresponsive to functional hF.IX. When challenged by a total of 6 weekly injections of hF.IX protein, all three strains (HB-338, HB-CH, and HB-180) tolerated the hF.IX antigen without any indication of inhibitors/IgG formation or anaphylaxis, which was in sharp contrast to the gene deletion mice. These results indicate that anaphylaxis in the murine model is F.IX genotype dependent, as it is in humans. In other experiments, naïve gene deletion mice were successfully tolerized to hF.IX by hepatic gene transfer. AAV8 or AAV2 (Y444/500/730F) capsid vectors containing a livers-specific expression cassette were injected into the tail vein. AAV8 vector induced tolerance over a wide range of vector doses. For optimized AAV2, hF.IX levels of 8–14% of normal were achieved with a dose of 2×10^11 vg/mouse, which protected from inhibitor formation and anaphylaxis upon subsequent challenge with F.IX protein. Next, we attempted to reverse the inhibitor response that had formed after protein therapy in gene deletion mice. These were repeatedly treated with hF.IX protein resulting in an average inhibitor of 6 BU/ml. Shortly afterwards, AAV8 vector expressing hF.IX (1×10^11 vg/mouse, n=4) was injected via the tail vein for hepatic gene transfer. Strikingly, this resulted in a complete reversal of antibody titers and systemic hF.IX levels of >40% of normal and aPTTs in the normal range. There was no evidence for an amnestic response, and the animals could be challenged with intravenous hF.IX protein without anaphylaxis. These results demonstrate that inhibitor formation resulting from F.IX replacement therapy can be reversed by liver gene transfer. Moreover, gene therapy in pre-immune mice corrected coagulation and protected from subsequent anaphylactic reactions after gene transfer. We are currently testing whether the protocol is safe for even higher pre-existing inhibitor titers. Disclosures:Herzog: Genzyme Corp: Patents & Royalties.

AAV-directed muscular dystrophy gene therapy

Importance of the field: Muscle-directed gene therapy for genetic muscle diseases can be performed by the recombinant adeno-associated viral (rAAV) vector delivery system to achieve long-term therapeutic gene transfer in all affected muscles.Areas covered in this review: Recent progress in rAAV-vector-mediated muscle-directed gene transfer and associated techniques for the treatment of muscular dystrophies (MD). The review covers literature from the past 2 – 3 years.What the reader will gain: rAAV-directed muscular dystrophy gene therapy can be achieved by mini-dystrophin replacement and exon-skipping strategies. The additional strategies of enhancing muscle regeneration and reducing inflammation in the muscle micro-environment should be useful to optimize therapeutic efficacy. This review compares the merits and shortcomings of different administration methods, promoters and experimental animals that will guide the choice of the appropriate strategy for clinical trials.Take home message: Restoration of muscle histopathology and function has been performed using rAAV systemic gene delivery. In addition, the combination of gene replacement and adjuvant therapies in the future may be beneficial with regard to improving muscle regeneration and decreasing myofiber necrosis. The challenges faced by large animal model studies and in human trials arise from gene transfer efficiency and immune response, which may be overcome by optimizing the rAAV vectors utilized and the administration methods.

Impact of the Underlying Mutation and the Route of Vector Administration on Immune Responses to Factor IX in Gene Therapy for Hemophilia B

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.

Prophylactic immune tolerance induced by changing the ratio of antigen-specific effector to regulatory T cells.

Gene and protein replacement therapies for inherited protein deficiencies such as hemophilia or lysosomal storage disorders are limited by deleterious immune responses directed against their respective therapeutic proteins. Therefore, the development of protocols preventing such responses is key to providing successful long-term therapy. We sought to develop a protocol, utilizing a drug/peptide cocktail, that would effectively shift the antigen-specific CD4+ T-cell population, tipping the balance from effector T cells (Teffs) towards regulatory T cells (Tregs). Treg-deficient (DO11.10-tg Rag2(-/-)) BALB/c mice were used to screen for an optimal protocol addressing the aforementioned goal and to study the mechanisms underlying in vivo changes in T-cell populations. Muscle-directed gene transfer to hemophilia B mice was also performed in order to test the optimal protocol in a therapeutically relevant setting. Specific antigen administration (4-week repeated dosing) combined with rapamycin and interleukin-10 led to substantial reductions in Teffs, via activation-induced cell death, and induced CD4+CD25+FoxP3+ Tregs to a large extent in multiple organs. The proportion of apoptotic T cells also increased over time, whereas Teffs and Tregs were differentially affected. When applied to a model of protein deficiency (gene therapy for hemophilia B), the protocol successfully prevented inhibitor formation, whereas non-specific immunosuppression was only marginally effective. It is feasible to provide a short-term, prophylactic protocol allowing for the induction of immune tolerance. This protocol may provide a marked advance in efforts seeking to improve clinical outcomes in disorders involving therapeutic protein replacement.

Long-term correction of inhibitor-prone hemophilia B dogs treated with liver-directed AAV2-mediated factor IX gene therapy

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.

A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs

The success of many gene therapy applications hinges on efficient whole body transduction. In the case of muscular dystrophies, a therapeutic vector has to reach every muscle in the body. Recent studies suggest that vectors based on adeno-associated virus (AAV) are capable of body-wide transduction in rodents. However, translating this finding to large animals remains a challenge. Here we explored systemic gene delivery with AAV serotype-9 (AAV-9) in neonatal dogs. Previous attempts to directly deliver AAV to adult canine muscle have yielded minimal transduction due to a strong cellular immune response. However, in neonatal dogs we observed robust skeletal muscle transduction throughout the body after a single intravenous injection. Importantly, systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and it lasted for at least 6 months (the duration of study). We also observed several unique features not predicted by murine studies. In particular, cardiac muscle was barely transduced in dogs. Many muscular dystrophy patients can be identified by neonatal screening. The technology described here may lead to an effective early intervention in these patients.

255. Cross Presentation of AAV2 Capsids Activates Cytotoxic T Cells and Hinders Muscle-Directed Gene Transfer but Does Not Render Hepatocytes Effective Cytolytic Targets

255. Cross Presentation of AAV2 Capsids Activates Cytotoxic T Cells and Hinders Muscle-Directed Gene Transfer but Does Not Render Hepatocytes Effective Cytolytic Targets

977. Differential Host T Cell Response to the Capsid of AAV2 and 8 in Muscle-Directed Gene Transfer in Murine and Non-Human Primate Models

Top of pageAbstract Host T cell response to the capsid proteins, if exists, may interfere with the much hoped long-term transgene expression as seen in the recent phase I clinical trial on AAV-mediated and liver-directed gene therapy for hemophilia B. We sought to investigate whether we could detect T cell responses to AAV capsid in animal models and whether the host would generate different responses to capsids of different serotypes. We first identified the immune dominant H2b- and H2d-restricted CD8 epitopes in the capsids of AAV2 and AAV2/8 in C57BL/6 and BALB/c mice, respectively. Using the dominant epitopes in an Enzyme-linked ImmunoSPOT (ELISPOT) assay, we detected T cell response to AAV2 capsid in both strains of mice 7 days after intramuscular injection of 1x1011 genome copies (GC) of AAV2 vector, such response decreased over the time. However, we could not detect any T cell response to AAV2/8 capsid in either mouse strain, even at a 10-fold higher dose. Intracellular INF-|[gamma]| staining (ICS) was also performed to examine the magnitude of the AAV2-specific CD8 T cell response in C57BL/6 and BALB/c mice. At day 7 after i.m. of 1|[times]|1012 GC of AAV2 vector in C57BL/6 mice, 0.36% of CD8+ cells were detected producing INF-|[gamma]| following in vitro stimulation with the dominant epitope. About 3-fold higher response (1.09%) was detected in BALB/c mice injected with the same amount of vector. ICS was not sensitive enough to detect CD8+ T-cell response to the capsid in mice received 1|[times]|1011 GC of AAV2 vector, suggesting the T cell response to AAV2 capsid is vector dose-related. Such dramatic differential host T cell responses to AAV2 and AAV2/8 capsid were also observed in nonhuman primates administered AAV2, AAV2/7 and AAV2/8 vectors IM. High level T cell responses to capsid were detected by ELISPOT assays on PBMCs from monkeys who received i.m. injection of AAV2 vectors up to 8 weeks but not on PBMCs from monkeys injected with AAV2/7 or 2/8 vectors.

Systemic protein delivery by muscle-gene transfer is limited by a local immune response

AbstractAdeno-associated viral (AAV) vectors have been successfully used for therapeutic expression of systemic transgene products (such as factor IX or erythropoietin) following in vivo administration to skeletal muscle of animal models of inherited hematologic disorders. However, an immune response may be initiated if the transgene product represents a neoantigen. Here, we use ovalbumin (OVA) as a model antigen and demonstrate immune-mediated elimination of expression on muscle-directed AAV-2 gene transfer. Administration to immune competent mice resulted in transient systemic OVA expression. Within 10 days, OVA-specific T-helper cells had been activated in draining lymph nodes, an inflammatory immune response ensued, and OVA-expressing muscle fibers were destroyed by a cytotoxic CD8+ T-cell response. Use of a muscle-specific promoter did not prevent this immune response. Adoptively transferred CD4+ cells transgenic for a T-cell receptor specific to OVA peptide-major histocompatibility complex class II showed antigen-specific, vector dose-dependent proliferation confined to the draining lymph nodes of AAV-OVA–transduced muscle within 5 days after gene transfer and subsequently participated in lymphocytic infiltration of transduced muscle. This study documents that a local immune response limits sustained expression of a secreted protein in muscle gene transfer, a finding that may have consequences for design of clinical protocols.

609. Long Term Persistence of Canine Factor IX-Specific Th1 Lymphocytes in Regional Lymph Nodes after AAV1-Mediated Intramuscular Gene Transfer in a Hemophilia B Dog

Previous studies on AAV-mediated gene therapy for hemophilia B showed that underlying factor IX (F.IX) mutation, vector dose, serotype, and route of administration all play a role in the risk of anti-F.IX antibody formation. Intramuscular (IM) administration of an AAV2 vector in normal and hemophilia B (HB) mice invariably activates F.IX-specific Th2 lymphocytes with production of mainly IgG1 and IgG2b antibody subclasses. Similarly, HB dogs with a missense mutation usually do not develop an immune response against canineF.IX when injected IM with low doses of an AAV2 vector. At high vector doses, a neutralizing antibody response has been observed, characterized by the production of both IgG1 and IgG2 subclasses, with levels of IgG2>IgG1. Although previous observations ruled out the involvement of CD8+ T cells, a recent study on the ovalbumin mouse model of AAV-mediated muscle-directed gene transfer showed a CTL response against the transgene protein at high vector doses (Wang et al., Blood, in press). An HB dog from the Chapel Hill colony (E57) received by IM injection an AAV1 vector expressing canine F.IX under the control of the CMV promoter at a dose of 2.4 |[times]| 10^11vg/kg, vector was co-administered with immunosuppression (IS) (Arruda et al., Blood 2004). One week after vector administration the whole blood clotting time (WBCT) fell to the normal range (18min) and circulating F.IX levels reached 104 ng/ml. However, after discontinuing IS a long-lasting neutralizing antibody response was observed (6 BU >3 years after injection) with production of both anti-cF.IX IgG1 and IgG2 and levels of IgG1>IgG2. In order to study in detail T cell responses against the cF.IX, a peptide library was produced spanning the F.IX protein sequence. PBMCs were isolated from whole blood of E57 and, from two HB dogs that received an AAV2-cF.IX vector delivered to the muscle. Lymphocytes were tested by ELISpot for secretion of IL-4, IL-10 and IFN-|[gamma]| and, interestingly, dogs injected with the AAV2 vector showed a positive IL-10 response while the AAV1 injected dog did not. Next, E57 and a normal dog were sacrificed and lymphocytes from spleen, draining lymph nodes (LN) and non-draining LN were isolated. Lymphocytes from E57 draining LN showed positive results on ELISpot for IFN-|[gamma]| for 2 cF.IX peptide pools. No response was detected for the other tissues and for the control dog. IF staining for CD8+ cells and H&E staining of muscle sections from E57 were negative. These results suggest a long-lived immune response detectable locally in the draining LN. Cytokine and antibody subclass profiles are typical of a Th1 response vs. the Th2 response seen in AAV2 injected dogs. Whether the shift from a Th2 to a Th1 response is driven by higher levels of antigen expression of by the switch in serotypes is currently under study. We conclude that in AAV-mediated, muscle-directed gene transfer, there may be long-term persistence of F.IX-specific Th1 lymphocytes in the regional LN.

111. AAV7, 8, and 9 Are More Efficient and Less Immunogenic Vectors for Muscle-Directed Gene Therapy for Hemophilia B

Vectors based on AAV serotype 2 have been tested in a phase I clinical trials for hemophilia B. Problems of pre-existing immunity to AAV2 in human population could limit its broad application. Another concern is the anti-FIX inhibitor formation which has been observed in the preclinical animal studies using AAV2 and AAV1 vectors. Pseudotyped AAV vectors with different tissue tropisms and improved gene transfer efficiencies provide potential alternatives to overcome these problems. In this study, we compared the long-term expression of canine factor IX (cFIX) levels and anti-cFIX antibody responses following muscle-directed gene transfer of vectors pseudotyped with AAV1, 2, 5, 7, 8, and 9 in immunocompetent hemophilia B mice. We demonstrated that AAV8, 9, and 7 are superior vectors for muscle-directed gene therapy for hemophilia B, compared to AAV2/1, 2/2, and 2/5 vectors. Our data indicate that AAV2/8 is about 25-fold more efficient than AAV2/1 vector for muscle-directed gene transfer in immunocompetent hemophilia B mice. These novel vectors are not only more efficient in transducing muscle fibers, they are also less immunogenic in eliciting B cell responses to the cFIX transgene product. The absence of antibody response to transgene by AAV8 is independent of the dose of vector and level of transgene expression. Besides, pre-existing immunity to these vectors in human is also less problematic. Therefore, these new AAV serotypes provide unique advantages for their future applications in treating hemophilia and other genetics diseases.

Regional intravascular delivery of AAV-2-F.IX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model

AbstractIn earlier work, we showed that adeno-associated virus–mediated delivery of a Factor IX gene to skeletal muscle by direct intramuscular injection resulted in therapeutic levels of circulating Factor IX in mice. However, achievement of target doses in humans proved impractical because of the large number of injections required. We used a novel intravascular delivery technique to achieve successful transduction of extensive areas of skeletal muscle in a large animal with hemophilia. We provide here the first report of long-term (> 3 years, with observation ongoing), robust Factor IX expression (circulating levels of 4%-14%) by muscle-directed gene transfer in a large animal, resulting in essentially complete correction of the bleeding disorder in hemophilic dogs. The results of this translational study establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B. These findings also have immediate relevance for gene transfer in patients with muscular dystrophy.

603. Correction of Hemophilia B Phenotype by Novel Method of Regional Intravenous Delivery of AAV Vector to Skeletal Muscle of Hemophilia B Dogs

Top of pageAbstract Intramuscular (IM) injection of AAV-F.IX in humans was safe and resulted in gene transfer and expression. However, at the doses administered, it did not result in sustained circulating levels >1% of normal. Although access to the skeletal muscle can be easily obtained by direct IM injection. However, achievement of target doses in humans proved impractical because of the large number of injections required. We sought to exploit techniques that allowed widespread transduction of muscle fibers. The major obstacle to vector distribution to large areas of skeletal muscle is the vascular endothelial barrier. However, the rich network of capillaries can be chemically and/or mechanically modified to ensure vascular leakage of vectors. In hemophilia B dogs, we showed (Blood, 2005) that infusion of AAV2-canineF.IX through femoral artery in the presence of histamine to increase capillary permeability, results in long-term expression of robust therapeutic F.IX levels (4-14% normal). Here we report the application of a novel approach, afferent transvenular extravasation (ATVE), in which the vector is injected through a superficial saphenous vein in the distal part of the limb under elevated hydrostatic pressure. Briefly, a tourniquet was placed at the level of the proximal thigh. AAV2-F.IX was diluted in 20 ml of PBS/kg and infused rapidly under the pressure of 300 mmHg. The vector-solution dwells for 15 min before tourniquet release. Hemophilia B dogs treated with AAV2 at doses of 1|[times]|1012vg/kg (n=2) expressed F.IX levels of 2% (100-125ng/ml). Increasing the vector dose to 3|[times]|1012vg/kg (n=3) resulted in circulating F.IX plateau levels of 6%-23% (300-1160 ng/ml). F.IX is biologically active as judged by excellent correlation between clotting times/antigen levels and near normalization of the whole blood clotting time. A sixth dog received an AAV dose of 8.5|[times]|1012vg/kg; this dose seems to be excessive since after F.IX levels increased to 713 ng/ml (day 21) we documented a slow decrease to <100ng/ml due to low-level of a non-neutralizing antibody to F.IX. Muscle enzymes levels increased transiently in 2 of 6 dogs up to day 2 post-injection. Endothelial cell damage markers such as vWF and IL-6 assayed at several time points following vector delivery did not differ from baseline levels. Thus, ATV provides an efficient, non-invasive, and safe strategy for widespread delivery of vector to skeletal muscle in the absence of vasoactive drugs. The doses required to achieve therapeutic F.IX levels (6-23%) are similar to doses already prove to be safe in earlier clinical trials. Furthermore, ATVE abolishes the dose advantage in gene transfer of liver-directed over muscle-directed gene transfer for hemophilia B. These results establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B.

A Novel Method of Regional Intravenous Delivery of AAV Vector to Skeletal Muscle Results in Correction of Canine Hemophilia B Phenotype.

Previously we demonstrated that intramuscular (IM) injection of AAV-F.IX in humans was safe and resulted in long-term >gene transfer and expression of injected muscle biopsied at time points up to 10 months post injection. However, at the doses administered, it did not result in sustained circulating levels >1%. Although access to the skeletal muscle can be easily obtained by direct IM injection, achievement of target doses in humans has proved impractical because of hundreds of injections required. We sought to exploit techniques that allowed transduction of large numbers of muscle fibers by injecting AAV vectors via intravascular. The major obstacle to vector distribution to large areas of skeletal muscle is the vascular endothelial barrier. However, the network of capillaries can be chemically and/or mechanically modified to ensure vascular leakage of fluid containing vectors. Earlier we showed that intravascular delivery of AAV2-CMV-canine F.IX, at doses in the range of 2–4 x 1012 vg/kg, by infusion vector through isolated femoral artery in the presence of histamine to increase capillary permeability (isolated limb perfusion), results in long-term expression (> 3 years) of therapeutic levels (4–14% normal) of F.IX, and phenotypic correction of severe canine hemophilia B. Here we report a novel approach, anterograde limb perfusion (ALP), in which the vector is injected through a superficial saphenous vein in the distal part of the limb under elevated hydrostatic pressure without vasoactive drug. Briefly, a tourniquet was placed at the level of the proximal thigh. Access to the target vessel was achieved by a small incision of the skin overlying the saphenous vein and an intravascular catheter was placed. The vector was diluted in 20 ml of PBS/kg and infused rapidly using a pressure of 300 mmHg. The vector-containing solution dwells for 15 minutes before tourniquet release. The procedure was well tolerated and muscle enzyme levels increased transiently (up to day 2 post-infusion) in 1 of 3 dogs. We treated two dogs (15 and 16 kg) with AAV2-CMV-cF.IXvector at dose of 3 x1012 vg/kg. Circulating F.IX antigen levels slowly increased to 292 and 299 ng/ml at day 14, reaching levels of 674 and 876 ng/ml day 21 (13 and 17 % of normal, ongoing observation). The shortening of the aPTT and the whole blood clotting time to nearly normal values confirmed that F.IX is biologically active. A third ALP-treated dog received 8 x1012 vg/kg of the AAV vector; F.IX levels rose to 577 ng/ml (>10% of normal), at days 13. This dose seems to be excessive since we documented the development of low-level of a non-neutralizing antibody to F.IX that likely increased the clearance of the transgene product. Thus, ALP provides an efficient, non-invasive strategy for widespread delivery of vector to skeletal muscle in the absence of vasoactive drugs. The doses required to achieve therapeutic F.IX levels are similar to those proved to be safe in earlier clinical trials. Furthermore, ALP abolishes the dose advantage in gene transfer of liver-directed over muscle-directed gene transfer for hemophilia B. These results establish an experimental basis for clinical studies of this delivery method in humans with hemophilia B.