Time Of Vector Administration Research Articles

Systemic delivery of AAVrh74.tMCK.hCAPN3 rescues the phenotype in a mouse model for LGMD2A/R1

Limb girdle muscular dystrophy (LGMD) 2A/R1, caused by mutations in the CAPN3 gene and CAPN3 loss of function, is known to play a role in disease pathogenicity. In this study, AAVrh74.tMCK.CAPN3 was delivered systemically to two different age groups of CAPN3 knockout (KO) mice; each group included two treatment cohorts receiving low (1.17 × 1014 vg/kg) and high (2.35 × 1014 vg/kg) doses of the vector and untreated controls. Treatment efficacy was tested 20 weeks after gene delivery using functional (treadmill), physiological (in vivo muscle contractility assay), and histopathological outcomes. AAV.CAPN3 gene therapy resulted in significant, robust improvements in functional outcomes and muscle physiology at low and high doses in both age groups. Histological analyses of skeletal muscle showed remodeling of muscle, a switch to fatigue-resistant oxidative fibers in females, and fiber size increases in both sexes. Safety studies revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. These results show that CAPN3 gene replacement therapy improved the phenotype in the CAPN3 KO mouse model at both doses independent of age at the time of vector administration. The improvements were supported by an absence of cardiotoxicity, showing the efficacy and safety of the AAV.CAPN3 vector as a potential gene therapy for LGMDR1.

MUSCLE FUNCTION & HOMEOSTASIS / MOLECULAR THERAPEUTIC APPROACHES: P.63 AAV.CAPN3 gene therapy for Limb-girdle muscular dystrophy type 2A (LGMD2A): Phenotypic improvements in the calpain3 (CAPN3)-null mouse

LGMD2A is caused by mutations in CAPN3 gene. CAPN3 loss of function is shown to play a role in disease pathogenicity, which might be improved by CAPN3 gene therapy. rAAVrh74.tMCK.CAPN3 vector was systemically delivered to young (YC, 6-10 week-old) and old cohorts (OC, 21-23 week-old) of CAPN3-KO mice. Each cohort included two treatment groups receiving low dose (LD; 3E12 vg, n=12) and high dose (HD; 6E12 vg, n=12) of the vector and age-matched untreated (UT) control groups (UT-YC, n=13;UT-OC, n=15). Outcome measures were performed at 20 weeks post-injection. AAV.CAPN3 improved treadmill performance in both cohorts, at both LD and HD subgroups (p<0.0001). There was no significant age-related effect on treadmill activity and the average performance of the treatment groups, HD vs. LD did not differ significantly. In the OC however, females (n=5) from the HD group run longer distance than males from same group (n=7; p<0.01). In the LD and HD treatment groups from OC, in vivo muscle contractility assay showed an improvement in both Twitch (p=0.001) and Tetanic Max responses (p=0.047) at the endpoint. The average of percent change between baseline and endpoint tetanic responses increased in the treated-YC while there was a decline in the UT cohort (p=0.002). Histological analyses of the skeletal muscle (gastroc, tibialis anterior, quadriceps and triceps) showed remodeling of muscle, a switch to fatigue resistant oxidative fibers in both cohorts along with fiber size increase. In-house toxicology studies in the HD cohort revealed no organ tissue abnormalities; specifically, there was no histopathological evidence of cardiotoxicity. AAV.CAPN3 gene therapy improved the LGMD2A-related phenotype in the CAPN3-KO mouse model in both low and high doses tested, independent of age at the time of vector-administration. Improvements shown were supported with the absence of cardiotoxicity, enhancing the efficacy and safety of AAV.CAPN3 vector as a potential gene therapy for LGMD2A.

Comparisons of Infant and Adult Mice Reveal Age Effects for Liver Depot Gene Therapy in Pompe Disease

Pompe disease is caused by the deficiency of lysosomal acid α-glucosidase (GAA). It is expected that gene therapy to replace GAA with adeno-associated virus (AAV) vectors will be less effective early in life because of the rapid loss of vector genomes. AAV2/8-LSPhGAA (3 × 1010 vector genomes [vg]/mouse) was administered to infant (2-week-old) or adult (2-month-old) GAA knockout mice. AAV vector transduction in adult mice significantly corrected GAA deficiency in the heart (p < 0.0001), diaphragm (p < 0.01), and quadriceps (p < 0.001) for >50 weeks. However, in infant mice, the same treatment only partially corrected GAA deficiency in the heart (p < 0.05), diaphragm (p < 0.05), and quadriceps (p < 0.05). The clearance of glycogen was much more efficient in adult mice compared with infant mice. Improved wire hang test latency was observed for treated adults (p < 0.05), but not for infant mice. Abnormal ventilation was corrected in both infant and adult mice. Vector-treated female mice demonstrated functional improvement, despite a lower degree of biochemical correction compared with male mice. The relative vector dose for infants was approximately 3-fold higher than adults, when normalized to body weight at the time of vector administration. Given these data, the dose requirement to achieve similar efficacy will be higher for the treatment of young patients.

77. Antigen-Specific Modulation of Capsid Immunogenicity with Tolerogenic Nanoparticles Results in Successful AAV Vector Readministration

Gene transfer approaches based on the adeno-associated virus (AAV) vector platform have shown great therapeutic potential in both preclinical studies and clinical trials. Neutralizing immune responses to AAV, however, are an important limitation to the use of AAV vectors as therapeutic tools, as even low-titer anti-capsid neutralizing antibodies (NAb) can lead to vector clearance and lack of efficacy. In particular, anti-AAV NAbs develop at high titers following vector administration and persist for several years after AAV vector administration, making vector re-administration hard if not impossible. Here we tested a novel strategy to modulate immune responses directed against AAV vectors based on the co-administration of biodegradable tolerogenic poly(lactic acid co-glycolytic acid) (PLGA) nanoparticles (tNP) containing rapamycin at the time of vector administration. C57BL/6 mice (n=5/group) received an AAV8 vector encoding for luciferase (AAV8-Luc) at a dose of 4×1012 vg/kg injected intravenously alone, or formulated with empty PLGA nanoparticles (NP), or formulated with tNP containing 100 µg of rapamycin. Three weeks after treatment, anti-AAV8 antibodies were measured and animals received a second intravenous infusion with an AAV8 vector encoding for human coagulation factor IX (AAV8-hFIX) at a dose of 4×1012 vg/kg formulated with NP or tNP. An anti-AAV8 antibody ELISA and an in vitro neutralization assay was used to follow humoral immune responses to the vector. While no development of anti-AAV8 antibodies was observed in the tNP-treated animals after the first and second vector administration, control groups developed robust humoral immune responses to the AAV8 capsid, which prevented vector readministration. Consequently, efficient hFIX transgene expression deriving from the second AAV8 vector administration was observed only when tNP were used, at levels identical to animals that received only a single administration of AAV8-hFIX. Lack of antibody formation against the AAV8 capsid in animals treated with tNP was also accompanied by a downregulation of both CD4+ and CD8+ T cell responses in the liver. A mild decrease in the frequency of CD4+ T cells in spleen, with no change in frequency of regulatory T cells, were also noted. In a separate set of experiments, we tested the antigen-specificity of the treatment of tNP with AAV8 administration. Mice (n=5/group) received 4×1012 vg/kg of an AAV8-luc vector with tNP followed by either challenge with hFIX protein in complete Freund's adjuvant, AAV5-hFIX vector intravenous injection, or AAV8-hFIX vector intravenous injection. All animals pretreated with AAV8-luc and tNP developed antibodies against the hFIX and the AAV5 antigens, while they anti-AAV8 antibody titers were significantly decreased. In conclusion, tNP administration together withAAV vector prevents anti-capsid immune responses in an antigen-specific manner and allows for AAV vector readministration, addressing one of the most important challenges of the in vivo gene transfer field.

Tolerance Induction To FIX Padua With AAV Liver Gene Transfer In Inhibitor-Prone Hemophilia B Dogs

Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX [FIX] deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB.We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies.Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months.An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections).The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases.Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity. Disclosures:High:Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity's Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.

Suppression of Inhibitor Formation in Protein and Gene Therapy for Hemophilia Using Ex Vivo Expanded Treg

Abstract 13Hemophilia A and B result from deficiency in clotting factor VIII (FVIII) or IX (FIX), respectively. In a subset of patients, treatment by factor replacement therapy is limited by formation of inhibitory antibodies to the clotting factors, representing a serious complication that increases risks of morbidity and mortality. Immune responses to the therapeutic coagulation factors are also a concern in newly emerging gene therapies. Regulatory T cells (Treg) offer to be a novel alternative pathway toward immune tolerance. Treg has been identified as a major component of immune tolerance to coagulation factors in pre-clinical studies. Therefore, we hypothesize that ex vivo expanded autologous Treg can suppress inhibitor formation. Our study seeks to test this approach in hemophilic mice. Initially, we optimized in vitro expansion of murine BALB/c-derived Treg. Using flow sorting, GFP+ cells were purified (>98% purity) from spleens of BALB/c knock-in mice containing a GFP reporter linked to FoxP3 expression with an IRES sequence. Sorted cells were stimulated in culture using anti-CD28/anti-CD3 beads in the presence of high-level IL-2 (1000 U/ml). IL-2 was replenished every second day in culture. After ∼1 week, cells were freshly stimulated. At the end of 2 weeks, viability, purity, and FoxP3/GFP expression was confirmed. Greater than 30-fold expansion was repeatedly accomplished. Assumming a dose of 1×106 Treg/mouse, expansion is sufficiently robust to treat >30 mice starting with Treg from 2–3 donor mice. Ex vivo expanded Tregs were adoptively transferred to male hemophilia A mice (BALB/c F8e16 −/−), which were then treated with F.VIII (1 IU human B domain-deleted F.VIII, IV, once per week) for two months. Bethesda assays demonstrated that Treg transplant had effectively suppressed inhibitor formation. Inhibitor titers in control mice were 15–20 BU at 1 month and 30–40 BU at two months. In contrast, Treg treated mice (n=5 per group) formed at most low-titer inhibitors (2–3 BU for both time points). By 2 months, peripheral Treg frequencies had returned to near baseline. To further demonstrate presence of a Treg population capable of suppressing antibody formation against F.VIII, a secondary transfer of sorted CD4+CD25+ splenocytes was performed. Recipient hemophilia A mice were immunized against F.VIII in adjuvant. Compared to mice receiving control Treg, there was significant (P<0.05) suppression of inhibitor formation against F.VIII. In other experiments, Treg therapy was also able to significantly reduce inhibitor titers in hemophilia A mice with pre-existing F.VIII inhibitors. We chose hemophilia A mice (n=6) that developed on average 25 BU from F.VIII replacement therapy. Half of these received Treg transplant, and all mice received 8 more weeks of F.VIII treatment. Inhibitor titers in the control group increased to ∼100 BU. Treg therapy substantially reduced this response (to 15–20 BU, P<0.001). In order to evaluate Treg therapy for hemophilia B, BALB/c F9 −/− × FoxP3-GFP mice were generated. Treg were isolated from these mice as described above, expanded in vitro, and transferred to (BALB/c F9 −/−) hemophilia B mice. Two days later, to test their effectiveness in a gene therapy setting, these mice were treated by intramuscular injection of AAV1 vector expressing human F.IX. By 6 weeks after gene transfer, control mice had formed high-titer antibody against hF.IX (>20 mg IgG/ml plasma, ∼ 10 BU). In contrast, anti-hF.IX formation was undetectable in mice that had received Treg prior to vector administration (n=4/group). While perhaps not as potent as antigen-specific Treg, our data demonstrate the ability of highly purified and ex vivo expanded bulk Treg to control inhibitor formation and thus support their utility for tolerance induction in hemophilia. Their effectiveness may involve emergence of a more specific Treg population after repeated in vivo exposure to antigen. In gene therapy, Treg transplant may be a more desirable alternative to use of immune suppressive drugs. Providing additional immune regulation around the time of vector administration, i.e. when activation signals are provided to the immune system, could be sufficient to prevent immune rejection long-term while inducing antigen-experienced Treg for durable tolerance. Disclosures:Herzog:Genzyme Corp.: Royalties, AAV-FIX technology, Royalties, AAV-FIX technology Patents & Royalties.

Rabbit Anti-Thymocyte Globulin (rATG) Administrated Concomitantly with Liver Delivery of AAV2-hFIX Can Promote Inhibitor Formation In Rhesus Macaques.

AbstractAbstract 3765Adeno-associated viral (AAV) vectors are one of the most extensively studied vector platforms for gene therapy applications. Our group is currently developing AAV vectors for the therapeutic treatment of hemophilia B (HB) in humans. The first clinical trial using an AAV2 vector to express human Factor IX (hFIX) (AAV2-hFIX16) from the liver of HB patients revealed a cytotoxic T lymphocyte (CTL) response directed against AAV capsid that occurred 4–6 weeks following treatment that was associated with a decline in transgene expression. Thus, immunosuppressive (IS) therapies may be required during AAV2 vector administration at high doses to prevent or to halt the immune mediated destruction of transduced hepatocytes. Previous work in murine and non-human primate (NHP) models has shown that sustained AAV-mediated expression of transgenes can induce tolerance, and that this is in part, dependent on CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Here we investigate the safety of a Treg sparing anti-T cell IS regimen in the context of liver mediated AAV2 gene transfer. Rabbit anti-thymocyte globulin (rATG) is an immune suppressive drug that is used in solid organ transplant and autoimmune disease. rATG has been shown to dramatically deplete the majority of T-cells, however some studies have shown that rATG spares Tregs and can induce tolerance in human T cells. rATG was administered to rhesus macaques (along with an 8-week course of Mycophenolate Mofetil (MMF) and sirolimus) either at the time of AAV vector administration (AAV2-hFIX16), or 5 weeks post-vector administration (rescue therapy). The administration of ATG at week 5 had no detrimental effect on hFIX expression and was not associated with inhibitor formation (n=3) indicating that rATG might be safe to use as an IS ‘rescue’ agent, after the detection of an ongoing immune response against transduced cells. Interestingly we observed that early administration of rATG prevented tolerance induction and resulted in inhibitor formation in 2 of 3 animals upon withdrawal of IS. The inhibitor formation was associated with transient elevations in circulating levels of IL-2, IL-4, IL-10 and IFN-g. These results are comparable to previous findings in NHP using an anti-CD25 IS regimen (Daclizumab) at the time of vector administration (Blood 2007, 110(7):2334-41). We conclude that the timing of IS regimens is critical, and that IS regimens that alter the numbers, frequency, and/or function of T-cells at the time of vector administration can result in neutralizing antibodies (inhibitors) to the transgene product (hFIX). These data suggest that there might be multiple mechanisms responsible for maintaining tolerance in this model, and that Tregs alone might not be sufficient. This study highlights the critical need for safety studies in large animal models of potential immune suppressive regimens in the context of gene transfer before translating to the clinic. Disclosures:High:Genzyme, Inc: Consultancy, Patents & Royalties; Third Rock Ventures: Consultancy; Novo-Nordisk: Consultancy; Shire, Inc.: Consultancy.

Timing of Therapeutic Intervention Determines Functional and Survival Outcomes in a Mouse Model of Late Infantile Batten Disease

Classical late infantile neuronal ceroid lipofuscinosis (cLINCL) is a monogenic disorder caused by the loss of tripeptidyl peptidase 1 (TPP1) activity as a result of mutations in CLN2. Absence of TPP1 results in lysosomal storage with an accompanying axonal degeneration throughout the central nervous system (CNS), which leads to progressive neurodegeneration and early death. In this study, we compared the efficacies of pre- and post-symptomatic injections of recombinant adeno-associated virus (AAV) for treating the cellular and functional abnormalities of CLN2 mutant mice. Intracranial injection of AAV1-hCLN2 resulted in widespread human TPP1 (hTPP1) activity in the brain that was 10-100-fold above wild-type levels. Injections before disease onset prevented storage and spared neurons from axonal degeneration, reflected by the preservation of motor function. Furthermore, the majority of CLN2 mutant mice treated pre-symptomatically lived for at least 330 days, compared with a median survival of 151 days in untreated CLN2 mutant controls. In contrast, although injection after disease onset ameliorated lysosomal storage, there was evidence of axonal degeneration, motor function showed limited recovery, and the animals had a median lifespan of 216 days. These data illustrate the importance of early intervention for enhanced therapeutic benefit, which may provide guidance in designing novel treatment strategies for cLINCL patients.

490. Successful Readministration of AAV8 Following Administration of Nondepleting CD4 Antibody at the Time of Vector Administration

Transgene expression from the liver following recombinant adeno-associated viral vector (rAAV) gene transfer is mediated mainly by episomally maintained genomes. For chronic disorders such as haemophilia this raises concerns that transgene expression will decline to subtherapeutic levels over time with the natural turnover of hepatocytes in humans. Neutralising antibodies that develop at the time of primary vector administration will however prevent readministration of the same serotype. Therefore strategies that allow repeated administration of the optimal vector system may be required to maintain transgene expression at therapeutic levels. We have used a non-depleting CD4 antibody (NDCD4) in conjunction with other immunosuppressive agents in an attempt to attenuate the primary immune response to AAV. Cohorts of male C57BL/6 mice (n= 7-8) were treated with NDCD4 (0.5mg/mouse) alone, or in combination with cyclosporine (CyA) 25mg/kg or mycophenolate mofetil (MMF) 20mg/kg on days |[minus]|1, 0, +1, 3, 6, 8 after tail vein administration of a pseudotype 8 vector encoding human factor IX. Additional cohorts received CyA or MMF alone and one group received no treatment. CD4 T lymphocyte analysis by FACs with an antibody against CD4 epitope B over 56 days, showed a moderate decline in the NDCD4 treated cohorts (34%) compared to na|[iuml]|ve mice (sham treated). At day 98 anti-AAV8 antibody titers were reduced in cohorts receiving NDCD4 alone (11.3|[plusmn]|3.5) or in combination with MMF (12.3|[plusmn]|5.5). However, the humoral response in CyA+NDCD4 was undetectable. Secondary challenge with a low titer (5|[times]|1010) of AAV8 encoding human |[beta]| interferon (h|[beta]|IFN) resulted in serum h|[beta]|IFN levels of 7.3|[plusmn]|3.9 ng/mL in the CyA+NDCD4 cohort. These levels were comparable (9.0|[plusmn]|5.2 ng/mL) to those in animals receiving only AAV8 h|[beta]|IFN. Hence treatment with NDCD4 antibody and cyclosporine at the time of vector administration can substantially attenuate the immune response to primary immunisation with AAV, enabling repeat administration and efficient transduction with vectors based on the same serotype. Unlike previously used strategies our approach offers significant safety advances and will have widespread appeal.

904. The Systemic Human Immune Response to Intraparenchymal Administration of AAV

BACKGROUND Recombinant AAV-ASPA has been developed for gene delivery to the CNS for Canavan Disease, a rare leukodystrophy with no treatment. This NINDS funded project is the first clinical study of a viral vector used for gene delivery in a neurodegenerative disorder. METHODS Subjects were administered AAV serotype 2 at a maximum dose of 1 × 10E12 vector genomes per subject, via intraparenchymal infusions to the subcortex . The immune response and safety profiles were monitored in the follow- up of 13 subjects who ranged from 3–82 months of age at the time of vector administration.

37. Duration of Immunologically Detectable AAV-2 Capsid in an Animal Model

In a Phase I trial for Hemophilia B six subjects underwent infusion of an adeno-associated viral (AAV) vector expressing human Factor IX (F.IX) under the control of a liver specific promoter. One subject in the high dose cohort (5×10e12 vg/kg) attained therapeutic F.IX levels in the range of 5-12% for 5 wks after vector infusion, but these declined to baseline ( 1 week but <4 wks after rAAV-2 vector injection. This mouse model will allow further investigation of the effectiveness of transient immune suppression at the time of vector administration and for the period surrounding immunologic persistence of capsid proteins.

Immune response to full-length dystrophin delivered to Dmd muscle by a high-capacity adenoviral vector.

Adenoviral vector-mediated gene transfer to skeletal muscle is a promising potential treatment for Duchenne muscular dystrophy. However, the immunological response to viral antigens and the therapeutic protein expressed by the delivered gene could prevent effective treatment. In this study, we investigated the immune response induced by adenoviral and dystrophin antigens presented by high-capacity adenoviral vector-mediated dystrophin and beta-galactosidase delivery to skeletal muscle of a mouse model that is both dystrophin-deficient and lacZ transgenic. Direct intramuscular gene delivery of the high-capacity adenoviral vector encoding full-length murine dystrophin resulted in stable expression of recombinant dystrophin for 5 months in mice treated as neonates and for 4 weeks in mice treated as adults. We observed neutralizing antibody to adenoviral antigens only in mice treated as adults and not in mice treated as neonates. This suggested that adenoviral antigens were only presented at the time of vector administration when the neonatal immune system was not yet mature. In contrast, antibodies to dystrophin were observed both in mice treated as neonates and in mice treated as adults. The development of an anti-dystrophin antibody response in mice treated with the high-capacity adenoviral vector as neonates suggested that dystrophin antigens were presented to the immune system at a time remote from the gene delivery, when the immune system was mature. Interestingly, an antibody response against beta-galactosidase developed late in the course of mice treated with the high-capacity adenoviral vector as neonates, suggesting a loss of tolerance to beta-galactosidase, a self-antigen in these transgenic mice. Our results suggest that future human trials of dystrophin gene delivery will need to address the potential for immunity induced by ongoing segmental degeneration of partially treated muscle fibers and presentation of recombinant dystrophin antigens in the context of a Duchenne muscular dystrophy patient.

Efficient lentiviral transduction of liver requires cell cycling in vivo.

Human-immunodeficiency-virus (HIV)-based lentiviral vectors are a promising tool for in vivo gene therapy. Unlike Moloney-murine-leukaemia-based retroviruses (MLV), lentiviruses are believed to stably transduce quiescent (non-cycling) cells in various organs. No previous studies, however, have directly established the cell-cycle status of any transduced cell type at the time of vector administration in vivo. In vitro studies using wild-type HIV or HIV-based vectors have shown that, in some cases, cell-cycle activation is required for infection, even though cellular mitosis is not an absolute requirement for integration. Even if the block in reverse transcription is overcome in quiescent T cells, productive infection by HIV cannot be rescued in the absence of cell-cycle activation. The potential use of these vectors for gene therapy prompted our study, which establishes a cell-cycle requirement for efficient transduction of hepatocytes in vivo.

Transient inhibition of CD28 and CD40 ligand interactions prolongs adenovirus-mediated transgene expression in the lung and facilitates expression after secondary vector administration.

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced beta-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.

Transient subversion of CD40 ligand function diminishes immune responses to adenovirus vectors in mouse liver and lung tissues

First-generation adenovirus vectors will have limited application in gene therapy for chronic diseases because of destructive host immune responses. Important immune effectors include CD8+ T cells, which mediate target cell destruction and ablate transgene expression, and B cells, which produce neutralizing antibodies that block effective readministration of vector. Previous studies indicated that activation of CD4+ T cells by virus capsid proteins is necessary for full realization of effector function of CD8+ T cells and B cells. In this paper, we present a strategy for preventing CD4+ T-cell activation by an adenovirus vector delivered to mouse liver and lung tissues which is based on interfering with T-cell priming via CD40 ligand-CD40 interactions. Adenovirus transgene expression was stabilized in mice genetically deficient in CD40 ligand (CD40L), and neutralizing antibody to adenovirus did not develop, allowing efficient readministration of vector. A transient blockade of T-cell activation with an antibody to CD40L infused into the animal at the time of adenovirus vector-mediated gene transfer led to stabilization of transgene expression and diminished production of neutralizing antibody, allowing readministration of vector. In vitro T-cell assays suggested that a block in the primary activation of CD4+ T cells was responsible for the lack of B-cell- and cytotoxic-T-cell-dependent responses. This suggests a strategy for improving the potential of adenovirus vectors based on administration of an antibody to CD40L at the time of vector administration.