Direct Intramuscular Injection Research Articles

Sustained AAV-mediated Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy with a Brief Course of Immunosuppression

Adeno-associated virus-based vector (AAV)-mediated gene delivery has been successful in some animal models of human disease such as the mdx mouse model of human Duchenne muscular dystrophy (DMD). However, recent evidence of immune-mediated loss of vector persistence in dogs and humans suggests that immune modulation might be necessary to achieve successful long-term transgene expression in these species. We have previously demonstrated that direct intramuscular injection of AAV2 or AAV6 in wild-type random-bred dogs resulted in a robust immune response to capsid or capsid-associated proteins. We now demonstrate that a brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF) is sufficient to permit long-term and robust expression of a canine micro-dystrophin (c-micro-dys) transgene in the skeletal muscle of a dog model for DMD (canine X-linked muscular dystrophy, or cxmd dog) and that its expression restored localization of components of the dystrophin-associated protein complex at the muscle membrane. This protocol has potential applications to human clinical trials to enhance AAV-mediated therapies.

260. Tolerance Induction to FIX in Mice with Pre-Existing Anti-FIX Immunity Following Direct Intramuscular Injection of AAV

260. Tolerance Induction to FIX in Mice with Pre-Existing Anti-FIX Immunity Following Direct Intramuscular Injection of AAV

Hydrodynamic Limb Vein Delivery of a Xenogeneic DNA Cancer Vaccine Effectively Induces Antitumor Immunity

Tumor-associated antigens (TAA) are typically poorly immunogenic "self" antigens. An effective strategy to break tolerance and induce antitumor immunity is by genetic vaccination, employing the orthologous TAA-sequence from a different species. We recently developed a clinically relevant approach for intravascular hydrodynamic limb vein (HLV) delivery of nucleic acids to skeletal muscle. Using the human gp100 xenogeneic TAA in the murine B16 melanoma model, we show that genetic vaccination of mice by HLV plasmid DNA delivery was highly effective at breaking tolerance against the homologous murine gp100 (mgp100) TAA and induced prophylactic antitumor protection. HLV vaccination resulted in an anti-hgp100 humoral and cellular response, with 4-5% of CD8(+) T cells being gp100(25-33)-epitope-specific. Vaccinated animals demonstrated in vivo cytolytic activity against human and mgp100(25-33) peptide-pulsed targets. Antitumor immunity could be adoptively transferred by splenocytes from human gp100-vaccinated animals. Furthermore, a durable antitumor memory response was established as approximately 3% of CD8(+) T cells were gp100(25-33) antigen-specific in mice 6 months after vaccination. Following a single HLV human gp100 DNA boost, this level increased to approximately 17% and protected animals from subsequent B16 tumor rechallenge. Our results warrant further consideration of HLV as a clinically relevant method for cancer gene therapy.

Immunity to Adeno-Associated Virus-Mediated Gene Transfer in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Recombinant adeno-associated virus (rAAV)-mediated gene transfer has shown promise for treating diseases in various animal models including the mdx mouse model of Duchenne muscular dystrophy (DMD). In many cases, however, preclinical studies in inbred mice have not successfully predicted human clinical responses. To assess the potential clinical utility of treating human DMD patients by AAV-mediated gene delivery, we performed a series of direct intramuscular injections in random-bred wild-type dogs. AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for murine studies and administered intramuscularly. The injection sites were biopsied at various time points and analyzed for transgene expression and immunohistochemical analysis. In contrast to the generally nonimmunogenic nature of these vectors in murine studies, both AAV2 and AAV6 vectors elicited robust cellular immune responses regardless of the transgene expressed, the cellular specificity of the promoter, and the muscle type injected. Viral purification by various methods did not diminish T cell-mediated infiltration. Our data indicate that AAV2 and AAV6 capsid proteins can elicit primary cellular immune responses when injected into the skeletal muscle of random-bred dogs, and suggest the possibility of cellular immunity to AAV vectors in humans.

Protease−Activated Receptor 2 (PAR−2) as a Novel Target To Prevent Inhibitor Formation to FIX.

Direct intramuscular injection (IM) of adeno−associated viral (AAV) serotype 2 in humans with hemophilia B (HB) is a promising therapeutic strategy since muscle biopsies obtained >3 years after vector injection demonstrated stable local gene expression. However to achieve therapeutic FIX levels using AAV−2 would required hundreds IM injections. The use of alternate AAV serotypes is an attractive strategy since AAV−1 or AAV−6, resulted in a >10−fold increase in transgene levels compared to AAV−2 in large animals, but the immune response to the transgene product has been consistently demonstrated as a major limitation of this strategy. There is growing evidence that blood proteases play an important role in modulating inflammatory and immune responses through activation of PARs. Mice lacking PAR−1(−/−) or PAR−2(−/−) alleles presented amelioration of immune− or infection−mediated diseases. Here we sought to determine whether inhibition of PARs could be used as a strategy to prevent immune responses to the FIX following AAV−mediated gene transfer to skeletal muscle. We used PAR−1 and PAR−2 knockout mice on C57Bl/6 background and littermate mice received IM injection of AAV1−CMV−hFIX. At dose 5x1011vg/kg, PAR−2 (−/−) mice (n=5) exhibited circulating FIX levels of 500± 99ng/ml (8–10%) which remained stable for the duration of the experiment (10 weeks), and no antibodies for FIX were detected (n=5). In contrast, all PAR−2 (+/+) mice (n=4) developed antibodies to FIX which inhibits FIX clotting activity, as determined by Bethesda assay (2.1± 0.6 BU). However, when similar vector doses were delivered to PAR−1(−/−) or PAR−1(+/+) (n=4/genotype) mice, antibodies to FIX developed in all animals. We next tested a higher vector dose in PAR−2 models. At dose 1x1012vg/kg, PAR−2(−/−) mice(n=7) resulted in FIX levels of 1,500±353ng/ml, and again no antibodies for FIX were detected. At the same dose, 6 out of 10 mice of PAR−2 (+/+)/(+/−) developed inhibitory antibodies (1.8± 0.7 BU). Further increase in the vector dose to 5 x 1012 vg/kg resulted in the development of inhibitor to FIX in both PAR−2 (−/−) (4/11 mice, 36%) and PAR−2(+/+)/(+/−) (10/17 mice, 60%). This suggests a threshold value in the protective effect in the PAR−2 (−/−) model. We sought to assay for FIX−specific T−cell by ELISPOT assay to quantify IFN−γ secretion from splenocytes of PAR−2 (−/−) and PAR−2 (+/+) mice injected at 5x1011 or 1x1012vg/kg. No difference in IFN−γ secretion was observed between PAR−2 (−/−) and their controls. Moreover, upon repeated challenges with FIX protein following vector injection antibody to FIX was detected in only 1/4 PAR−2 (+/+) mouse and none of 5 PAR−2 (−/−). Thus, PAR−2 inhibition does not compromise the tolerance to FIX.In a different model, intravenous injection of FIX protein into normal mice upon simultaneous activation of PAR−2 by using specific agonist peptide the rates of FIX antibody formation were comparable with those of a control peptide group. Thus, PAR−2−mediating antibody formation to FIX may differ among distinct immunologic challenges. Together, these data suggest that PARs play a role in the immune response to FIX and that inhibition of PAR−2 (but not PAR−1) could be a novel target in preventing inhibitor formation in hemophilia gene therapy and potentially for protein−based therapy.

Efficient Induction of Tolerance to FIX by Direct Intramuscular Delivery of AAV1.

Hemophilia B is an X-linked recessive genetic disease resulting from deficiency in coagulation factor IX (FIX). The current therapy for hemophilia B is life-long replacement of FIX through recombinant FIX or purified blood products in response to bleeding events. However, this replacement therapy is non-prophylactic, costly, and can be complicated by formation of inhibitory anti-FIX antibodies in up to 5% of patients. While somatic gene therapy is expected to provide a final cure for hemophilia B, it may also cause high incidence of FIX antibodies formation and other adverse immune responses following gene delivery. Direct intramuscular injection of adeno-associated virus (AAV) is a safe and promising procedure for hemophilia B gene therapy. This treatment, however, elicits anti-FIX antibodies in immune competent animal models. We have previously reported that intramuscular injection of AAV1 expressed high levels of canine FIX and induced FIX tolerance in a mouse model of hemophilia B, but AAV2 elicited anti-FIX antibodies. Here, we report efficient induction of human FIX (hFIX) tolerance in naive as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1×1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected close to 1000ng/ml of hFIX antigen by ELISA 8 weeks post AAV injection (n=5). No significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. In addition, subsequent challenge with recombinant hFIX in complete Freund's adjuvant did not cause anti-FIX antibodies to be produced and the level of hFIX in the blood remained constant. However, anti-FIX antibodies, but not hFIX antigen, were measured in the mice injected with the same dose of AAV2 (n=7). Subsequent injection of AAV1 vector into the skeletal muscle of these AAV2-injected mice resulted in the disappearance of anti-FIX antibodies and emergence of FIX antigen at similar levels to AAV1-injected naive mice in the circulation of these mice. In addition, direct intramuscular injection of AAV1 also induced FIX tolerance in mice that developed anti-FIX antibodies after exposure to recombinant FIX proteins (n=6). Similar experiments in mice with different genetic and MHC backgrounds have also demonstrated efficient induction of tolerance to FIX, implying that AAV1-hFIX can induce tolerance regardless of MHC haplotype. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. To elucidate the mechanism of different immune responses to FIX following intramuscular injection of AAV1 and AAV2, we are examining variations in antigen presentation, interaction between antigen presenting cells and antigen-specific T cells, and fate of antigen-specific T cells following intramuscular injection of AAV1 and AAV2 vectors. In summary, our results demonstrate efficient induction of FIX following direct intramuscular injection of AAV1 vectors. Investigations to elucidate the underlying mechanism are ongoing in our lab.

Glucose transporter proteins GLUT1 and GLUT3 like immunoreactivities in the fetal sheep brain are not reduced by maternal betamethasone treatment

Synthetic glucocorticoids administered to accelerate fetal lung maturation in threatened preterm delivery change electrocortical brain activity in the human and sheep fetus and alter structural neuronal proteins in fetal baboon and sheep. We hypothesized that these changes are due to a decreased amount of glucose transporter proteins (GLUT). Glucose uptake into cerebral neurons is selectively facilitated by glucose transporter protein GLUT1 in the blood brain barrier and GLUT3 in neuronal membranes. GLUT1 and GLUT3 immunoreactivity was examined in fetal sheep brain sections of the frontal neocortex, caudate putamen and hippocampus at 0.73 gestation after fetal exposure to betamethasone by direct fetal intravenous infusion or maternal intramuscular injections at the clinically relevant dosage. Betamethasone did not alter GLUT1 and GLUT3 immunoreactivity in any of the brain regions investigated, independently of the dose and route of administration. These data indicate that alteration of GLUT expression is unlikely to explain the cerebral functional effects of antenatal glucocorticoids.

567. Adeno-Associated Virus Vector 8 (AAV8) Provides Efficient Gene Delivery to Skeletal Muscles In Utero by Systemic Adminstration

Top of pageAbstract Adenoviral-associated viral vectors (AAV) have developed as excellent gene delivery vehicles to various tissues. Recently newer AAV serotypes have been characterized and they demonstrate varying transduction efficiencies in different tissues. Our laboratory is specifically interested in exploiting AAV serotypes that transduce skeletal muscle from a systemic gene delivery. We have previously shown that in utero gene delivery with AAV1 and AAV2 only transduce skeletal muscle when delivered as a direct intramuscular injection in the fetal pup. Recent studies in post-natal mice demonstrate efficient transduction of skeletal muscle after systemic delivery of AAV8. We were therefore interested whether intraperitoneal administration of AAV8 in embryonic day 16 CD1 mice could provide transgene expression in striated muscle. We pursued a biodistribution study of AAV8 in mice at 9 weeks of age that have received AAV8 by intraperitoneal administration at embryonic day 16 in utero. Gene expression was observed by histology and quantitative |[beta]|-galactosidase assay in skeletal muscles, intercostal muscles and diaphragm of intraperitoneally injected pups. Our results support the potential of AAV8 as an efficient vector for crossing the blood vessel barrier to attain systemic gene transfer to skeletal muscle in utero. Furthermore, our data encourage us to pursue studies of AAV8 gene delivery of microdystrophin into the mdx (dystrophin-deficient) mouse model in utero.

598. Effective Humoral and Cellular Smallpox-Immune Responses Using a Combination of Plasmids Delivered by Intramuscular Injection Followed by Constant-Current Electroporation in Rabbits

Previous studies have examined smallpox antigen-expressing plasmids in mice and non-human primates with limited efficacy. Recent studies showed that the electroporation technique can be used to dramatically increase plasmid uptake into the target tissues (e.g., skeletal muscle) when compared to simple injection. Consequently, the expression levels for the encoded transgene are orders of magnitude higher, likely improving potency. In a pilot experiment, uptake and distribution of plasmids with and without electroporation have been analyzed in the rabbit. Electroporated muscle showed a better distribution and uptake. We evaluated immune responses to smallpox antigens, after direct intramuscular plasmid injection followed by constant-current electroporation in groups of rabbits (n=3/group). The plasmids were administered either individually or in combination and their relative responses analyzed. Group 1 rabbits were immunized with a combination of eight plasmids expressing varying smallpox antigens (A13L, A14L, A27L, A33L, B5R, D8L, H3L, L1R). Group 2 rabbits were immunized with a combination of four different antigens (A27L, B5R, D8L, L1R). The final group received an immunization with an individual plasmid expressing a single antigen (B5R). All plasmids were administered via a single IM injection of 800 |[mu]|g (100 |[mu]|g each antigen and/or empty vector up to 800 |[mu]|g per injection) into semimembranosus muscle followed by electroporation using the ADViSYS constant-current EKD system, at 0.6 Amps, 3 pulses, 52 ms/pulse, 1 sec between pulses. Rabbits were immunized twice, at Study d 0 and at Study d 64. Sera were collected from rabbits at different time points, including on Day 85 and used to determine antibody responses by protein ELISA. Antibody responses were improved by electroporation in both immunized groups irrespective of the number of antigens delivered. A specific ELISpot assay for TNF is being developed to determine T-cell response rates in rabbits following DNA immunization. Rabbit PBMCs collected 2 weeks after first immunization were stimulated for 24h with 15mer peptides that span the entire length of the A27L gene. Immunization followed by electroporation induced improved antigen specific T-cell responses against A27L. Thus, the combination immunization strategy using smallpox antigens and constant-current electroporation appears important for driving better humoral and cellular-based responses in a relevant animal model.

985. Efficient Induction of Tolerance To FIX by Direct Intramuscular Delivery of AAV1

Hemophilia B is an X-linked recessive genetic disease resulting from deficiency in coagulation factor IX (FIX). The current therapy for hemophilia B is life-long replacement of FIX through recombinant FIX or blood products in response to bleeding events. However, this replacement therapy is non-prophylactic, costly, and can be complicated by formation of inhibitory anti-FIX antibodies in up to 5% of patients. While somatic gene therapy is expected to provide a final |[ldquo]|cure|[rdquo]| for hemophilia B, it may also cause high incidence of FIX antibodies formation and other adverse immune responses following gene delivery. Direct intramuscular injection of adeno- associated virus (AAV) is a safe and promising procedure for hemophilia B gene therapy. This treatment, however, elicits anti-FIX antibodies in immune competent animal models. We have previously reported that intramuscular injection of AAV1 expressed high levels of canine FIX and induced FIX tolerance in a mouse model of hemophilia B, but AAV2 elicited anti-FIX antibodies. Here, we report efficient induction of human FIX (hFIX) tolerance in na|[iuml]|ve as well as FIX-pre-immunized animals by direct intramuscular injection of AAV1 vectors. Following injection of 1x1011 of AAV1 expressing hFIX per mouse in hemostatically-normal and FIX knock out mice, we detected higher than 500ng/ml of hFIX antigen by ELISA as early as 2 weeks post AAV injection (n=5), while no significant level of anti-FIX antibodies could be detected in these mice, by either ELISA or modified Bethesda inhibitor assay. However, anti-FIX antibodies, but not hFIX antigen, could be measured in the mice injected with the same doses of AAV2 (n=5). Subsequent injection of AAV1 vector into the skeletal muscle of these AAV2-injected mice resulted in the disappearance of anti-FIX antibodies and emergence of FIX antigen at similar levels to AAV1- injected na|[iuml]|ve mice in the circulation of these mice as early as 2 weeks post AAV1 injection. In addition, direct intramuscular injection of AAV1 also induced FIX tolerance in mice that developed anti-FIX antibodies after exposure to recombinant FIX proteins (n=5). Investigation of FIX tolerance by AAV1 in mice with different genetic and MHC backgrounds is ongoing. We hypothesize that the immediate expression of high levels of FIX from the non-pathogenic AAV1 induces FIX tolerance. We previously showed a linear relation between canine FIX antigen levels and the AAV1 doses injected to skeletal muscle of mice. We are currently conducting a viral dose curve to determine the lowest titer of AAV1 (as well as the minimal levels of hFIX) necessary to induce tolerance to FIX. To elucidate the mechanism of the different immune responses to FIX following intramuscular injection of AAV1 and AAV2, we are examining variations in antigen presentation, interaction between antigen presenting cells and antigen-specific T cells, and fate of antigen-specific T cells following intramuscular injection of AAV1 and AAV2 vectors. In summary, our results demonstrate efficient induction of FIX following direct intramuscular injection of AAV1 vectors. Investigations to elucidate the underlying mechanism are ongoing in our lab.

Intramuscular gene transfer of fibroblast growth factor-1 using improved pCOR plasmid design stimulates collateral formation in a rabbit ischemic hindlimb model

Fibroblast growth factor 1 (FGF1) is an angiogenic factor known to play a role in the growth of arteries. The purpose of this study was to evaluate the usefulness of direct intramuscular injection of an optimized expression plasmid encoding FGF1 to augment collateral formation and tissue perfusion in a rabbit ischemic hindlimb model. Truncated FGF1 fused to the human fibroblast interferon (FIN) signal peptide was expressed from a newly designed plasmid backbone with an improved safety profile for gene therapy applications. In vitro, optimization of plasmid design yielded in a dramatic increase in expression efficiency for FGF1, independent of the presence of a signal peptide, as analyzed by Western Blotting. In vivo, successful transgene expression could be demonstrated by FGF1 immunostaining after gene application. FGF1 plasmid containing FIN signal peptide (100, 500, and 1,000 mug), when injected into ischemic muscle areas of rabbits 10 days after ligation of the external iliac artery, exhibited a pronounced therapeutic effect on collateral formation to the ischemic hindlimb in a dose-depending manner, as assessed by physiological (blood pressure ratio, maximal intra-arterial Doppler flow) and anatomical (angiographic score, histologic evaluation of capillary density) measurements 30 days after therapy, compared to saline or lacZ control plasmid. FGF1 plasmid without a signal peptide sequence resulted in a comparable therapeutic effect on collateral formation at comparable doses (500 and 1,000 mug). Our results indicate that intramuscular FGF1 gene application could be useful to stimulate collateral formation in a situation of chronic peripheral ischemia. The presence of a signal peptide does not seem to be obligatory to achieve bioactivity of intramuscular transfected FGF1. An optimized vector design improved both biosafety of gene transfer and expression efficiency of the transgene, rendering this vector highly suitable for human gene therapy. Therefore, this new generation vector encoding FGF1 might be useful as an alternative treatment for patients with chronic ischemic disorders not amenable to conventional therapy.

Characterization of the Immune Response to Canine Factor IX Following AAV-Mediated Intravascular Gene Delivery to Skeletal Muscle in Hemophilia B Dogs.

Progress towards an effective gene therapy for hemophilia B (HB) has been facilitated by large animal studies. Previous work has shown intravascular delivery of an adeno-associated viral (AAV) serotype 2 vector expressing canine factor IX (cF.IX) to skeletal muscle in HB dogs resulted in long-term expression of cF.IX at levels of 4–20% of normal, which nearly corrected the disease phenotype. However, occurrence of inhibitors to F.IX in some animals raises concerns of a potential immune response to the transgene product, prompting a more thorough examination of T cell responses in this setting. Early work revealed that transient immunosuppression (IS) with cyclophosphamide was required to avoid inadvertent antibody formation to F.IX. Here we report in detail the nature and the duration of T cell responses against the transgene product. Six HB dogs from the Chapel Hill colony received AAV2 vector at three different doses (1x1012 vg/kg, n=3; 3 x 1012 vg/kg, n=2; 8 x 1012 vg/kg, n=1) in addition to weekly infusion with cyclophosphamide (6 doses total). PBMCs were isolated from whole blood prior to vector infusion, during IS and after removal from IS, and used to measure the T cell response to F.IX by ELISpot assay for IL-10 and IFN-γ secretion using a cF.IX peptide library composed of 15-mers overlapping by 10 amino acids, spanning the entire protein sequence. Peptides were arranged into a matrix of pools, such that each peptide was contained in two orthogonal pools. Interestingly, in the IL-10 assay, one common T cell epitope corresponding to peptide 68 in the cF.IX library was found in all intravascularly-administered dogs from each of the three dose groups. The same epitope was also detectable in naïve HB dogs. Another epitope, corresponding to peptide 84, was found in a dog injected with the highest dose of vector after it developed a non-neutralizing antibody response against the cF.IX transgene product. Peptide 84 spans the region of the protein that contains the missense mutation responsible for HB (Chapel Hill mutation, Glu379 → Gly), which is a key difference in the newly introduced transgene product. Furthermore, the lack of any IFN-γ secretion coupled with the marked IL-10 response gives a cytokine profile that is characteristic of a Th2 response. This is in contrast with the Th1 response seen in previous studies with direct intramuscular injection of an AAV serotype 1 expressing cF.IX in dogs from the same colony. IS successfully reduced T cell responses to undetectable levels, while IL-10 secretion was detectable in PBMCs before vector delivery and one month after IS was discontinued. Overall circulating cF.IX levels did not seem to be affected by late restoration of T cells responses. No T cell responses against AAV capsid were detectable by ELISpot on PBMCs in any of the dogs studied. Interestingly, the relatively mild IS regimen also appeared to reduce the formation of neutralizing antibodies against AAV capsid, regardless of the route of administration. In summary, the nature of T cell responses (Th2 vs. Th1) suggests that route of administration, and/or AAV serotype, may play a role in the determination of the immune response elicited. We conclude that IS may provide a means to decrease T cell responses to the transgene following intravascular delivery of AAV-F.IX to skeletal muscle.

791. Immunity to AAV-Mediated Gene Therapy in a Random-Bred Canine Model of Duchenne Muscular Dystrophy

Top of pageAbstract Introduction: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. Studies in the mdx mouse model of DMD have shown that muscle membrane integrity and function can be improved by AAV-mediated delivery of a functional dystrophin protein. To assess the potential clinical utility of treating human DMD patients with AAV-mediated gene delivery, we performed a series of direct intra-muscular injections in random-bred normal dogs and in dogs with muscular dystrophy caused by a dystrophin mutation (xmd dogs). Methods: AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for the murine studies and purified either on a heparin column or with a combination of heparin column and cesium chloride gradient. Direct intramuscular injections of virus (1012, 1010, or 108 viral genomes per site) in a total volume of 250 ul were performed. The injection sites were biopsied under anesthesia between 2 and 12 weeks after injection. Results: Injection of either AAV6 or AAV2 expressing CMV-b-galactosidase (b-gal) induced a strong inflammatory response containing both CD8+ and CD4+ T-lymphocytes with peak tissue destruction 4 weeks following viral injection. A similar robust immune response was seen with injection of AAV6-RSV-alkaline-phosphatase, AAV6-CMV-canine-factor-IX (cFIX) and with empty AAV6 capsid alone. Additional purification of the AAV6-CMV-cFIX by centrifugation through a cesium chloride gradient did not diminish the immune response. Continuous immunosuppression with cyclosporine (CSP) and mycophenolate mofetil (MMF) largely prevented the immune response to AAV6-CMV-b-gal in a normal dog for up to four weeks and permitted robust transgene expression. The same immunosuppressive regimen did not prevent an immune response to AAV6-CMV-cFIX or AAV6-CMV-human-micro-dystrophin in an xmd dog, suggesting that a more aggressive immunosuppressive regimen might be necessary in the xmd dog. Conclusions: Taken together, our results suggest that AAV capsid proteins, or proteins associated with the capsid, elicit significant immune responses when directly injected into skeletal muscle of normal random-bred dogs. The combination of CSP and MMF effectively prevents the immune responses in a normal dog, but not in an xmd dog, possibly due to the pre-existing inflammatory nature of the DMD muscle disease or due to genetic variation in a random-bred animal population. The robust immune responses to AAV2 and AAV6 in random-bred dogs contrast with the lack of an immune response in studies by others in inbred mice and dogs. Further studies will be necessary to determine the nature of the immune responses and to develop appropriate immunosuppressive regimen for AAV gene delivery to xmd muscle.

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.

The Effects of Intradermal Vaccination with DNA Encoding for the T-cell Receptor on the Induction of Experimental Autoimmune Encephalomyelitis in B10.PL Mice

Intradermal gene administration was found to induce a more profound immune response than direct intramusclular gene injection. We performed intradermal vaccination of B10.PL mice with DNA encoding for the Vβ8.2 region of the T-cell receptors (TCR). Three weeks later, these mice were immunized with rat myelin basic protein (MBP). Daily mean clinical scores and mortality rate were lower in this group compared with controls. The proliferative responses of lymph node cells to rat MBP were slightly less in the vaccination groups than in the control groups (p<0.05). However, we detected no differences between the two groups with regard to the production of MBP-specific IgG, IgG1, & IgG2a antibodies. The levels of cytokine mRNA expression in the vaccination groups were observed higher than in the control groups without antigen-specific stimulation, but all of cytokine expressions between the vaccination and control groups after antigen-specific stimulation were identical. These results demonstrate that intradermal DNA vaccines encoding for TCR might prove to be useful in the control of autoimmune disease.

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.

Importance of Transplantation Variables in Marrow to Muscle Conversion.

We have previously shown that conversion of marrow to skeletal muscle is increased by cardiotoxin injury and we have demonstrated levels of up to 12% conversion. We have now used the same model of cardiotoxin injury to determine variables that affects this conversion. Muscle fibers were defined by their morphology and presence of desmin and dystrophin staining and absence of CD45 staining. In a GFP to B6 transplant model, there was a significant increase in the incidence of muscle chimersim with increasing the dose of marrow donor cells unrelated to the level of hematopoietic chimersim. Radiation was a critical factor and a combination of 500cGy of whole body and 500cGy of lower extremity radiation was found as the optimal model. However radiation impact was mostly due to its effect on hematopoietic chimerism and there was very little local effect on muscle. Higher doses of radiation were detrimental and resulted in significant muscle atrophy. Transplantation of marrow cells to NOD/Scid mice without radiation resulted in muscle engraftment although this was a rare event. Furthermore, timing of the cardiotoxin injury relative to the transplantation was an important factor since injection of cardiotoxin before or right after transplant resulted in minimal conversion while injury seven days or more after transplant resulted in significant conversion events. When sublethal doses of cardiotoxin were injected systemically after marrow transplant GFP positive muscle fibers were seen but the incidence was much lower than direct intramuscular injections of cardiotoxin. Multiple cycles of mobilization and injury also improved the incidence of GFP positive fibers in the regenerated muscle. We conclude that in addition to injury, cell mobilization and cell selection, improvements in the efficiency of this process can be achieved by applying specific doses and distribution of radiation (i.e. local and systemic), by giving the appropriate doses of marrow cells and by selecting the appropriate time for injury. Combining these models gives quantitatively significant fiber conversions approaching levels that would be therapeutically effective in the treatment of muscular dystrophies.

Role of interleukin-1beta in acute inflammation and graft death after cell transplantation to the heart.

Poor survival of grafted cells is a major factor hindering the therapeutic effect of cell transplantation; however, the causes of cell death remain unclear. We hypothesized that interleukin-1beta (IL-1beta) might play a role in the acute inflammatory response and graft death after cell transplantation and that inhibition of IL-1beta might improve graft survival. 14C-labeled male skeletal muscle precursor cells were implanted into female mouse hearts by direct intramuscular injection. The amount of 14C-label provides an estimate of the surviving cell number, whereas the amount of male-specific Smcy gene measured by polymerase chain reaction indicates the total (surviving+proliferated) number of donor-derived cells. At 10 minutes after implantation, 44.8+/-2.4% of the grafted cells survived and this steadily decreased to 14.6+/-1.1% by 24 hours, and to 7.9+/-0.6% by 72 hours (n=6 in each point). Proliferation of the surviving cells, which began after 24 hours, resulted in an increase in the total cell number from 15.5+/-0.8% at 24 hours to 24.4+/-1.6% at 72 hours. Acute inflammation was prominent at 24 hours and was reduced by 72 hours, in parallel with IL-1beta expression. Administration of anti-IL-1beta antibody improved graft survival at both 24 (25.6+/-1.6%) and 72 hours (14.8+/-1.1%) and resulted in a 2-fold increase in the total cell number at 72 hours (45.8+/-2.4%). The effects of IL-1beta inhibition corresponded with a reduced inflammatory response. IL-1beta is involved in acute inflammation and graft death after direct intramyocardial cell transplantation. Targeted inhibition of IL-1beta may be a useful strategy to improve graft survival.

Capillary regression in vascular endothelial growth factor-deficient skeletal muscle.

Skeletal muscle angiogenesis is an important physiological adaptation to increased metabolic demand, possibly dependent on vascular endothelial growth factor (VEGF), the increased expression of which is a known early response to exercise. To test the hypothesis that VEGF is essential to muscle capillary maintenance, we evaluated the consequences of targeted skeletal muscle inhibition of VEGF expression in postnatal, cage-confined VEGFloxP(+/+) mice. To delete VEGF, cre recombinase expression was accomplished using direct intramuscular injection of a recombinant adeno-associated cre recombinase expressing viral vector. Four weeks postinfection, VEGF-inactivated regions revealed 64% decreases in capillary density and capillary-to-fiber ratio. Substantial apoptosis was also observed in VEGF-depleted regions. There was no evidence of rescue at 8 wk, with a persistent 67% reduction in capillary-to-fiber ratio and a 69% decrease in capillary density. These data implicate VEGF as an essential survival factor for muscle capillarity and also demonstrate insufficient VEGF-dependent signaling leads to apoptosis in mouse skeletal muscle.

Muscle stem cells can act as antigen-presenting cells: implication for gene therapy.

Research has shown that the use of a muscle-specific promoter can reduce immune response and improve gene transfer to muscle fibers. We investigated the efficiency of direct and ex vivo gene transfer to the skeletal muscles of 6- to 8-week-old mdx mice by using two adenoviral vectors: adenovirus (AD) encoding the luciferase gene under the cytomegalovirus (CMV) promoter (ADCMV) and AD encoding the same gene under the muscle creatine kinase (MCK) promoter (ADMCK). Direct intramuscular injection of ADMCK triggered a lower immune response that enabled more efficient delivery and more persistent expression of the transgene than did ADCMV injection. Similarly, ex vivo gene transfer using ADCMV-transduced muscle-derived stem cells (MDSCs) induced a stronger immune response and led to shorter transgene expression than did ex vivo gene transfer using ADMCK-transduced MDSCs. This immune response was due to the release of the antigen after MDSC death or to the ADCMV-transduced MDSCs acting as antigen-presenting cells (APCs) by expressing the transgene and rapidly initiating an immune response against subsequent viral inoculation. The use of a muscle-specific promoter that restricts transgene expression to differentiated muscle cells could prevent MDSCs from becoming APCs, and thereby could improve the efficiency of ex vivo gene transfer to skeletal muscle.