Transient Immunomodulation Allows Repeated Injections of AAV1 and Correction of Muscular Dystrophy in Multiple Muscles
Authors: Stéphanie Lorain, David-Alexandre Gross, Aurélie Goyenvalle, Olivier Danos, Jean Davoust, Luis Garcia
Journal: Molecular Therapy (2008)

Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (μKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in μKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge. Exon-skipping AAV1-U7-associated therapy is a promising treatment for Duchenne muscular dystrophy (DMD). We have shown earlier that the newly rescued dystrophin protein is stably expressed for months in mice and dogs, and does not induce immune rejection of transduced fibers. In this study, we used the dystrophic mdx mouse as a preclinical model to characterize the immune response to the adeno-associated virus 1 (AAV1) vector, and tested the feasibility of administering multiple AAV1 injections to extend the treatment to several muscles. We found that re-injections of AAV1 vector are compromised as early as 3 days after the first injection, coincident with a rapid increase in AAV1-specific immunoglobulin M (IgM) and IgG in the serum. Adoptive transfer of immune sera confirmed the rapid appearance of an AAV1 neutralization activity, and experiments with immunoglobulin-deficient (μKO) mice proved that antibodies (Abs) are the only effectors responsible for AAV1-U7 elimination. It is important to note, however, that the AAV2 vector still generated an adverse immune response in μKO mice. By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, we found that a mere 5 days of immunomodulation treatment was sufficient to totally abrogate the formation of anti-AAV1 Abs and to allow for the correction of muscular dystrophy in multiple muscles, provided the treatment was administered during each challenge. IntroductionMutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), the most common severe childhood muscular pathology. There is currently no effective treatment for the condition. With antisense sequences linked to a modified U7 small nuclear RNA, we achieved persistent exon skipping that removes the mutated exon and re-establishes the open reading frame on the dystrophin messenger RNA of the animal models for DMD, the mdx mouse, and golden retriever muscular dystrophy dog.1Goyenvalle A Vulin A Fougerousse F Leturcq F Kaplan JC Garcia L et al.Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping.Science. 2004; 306: 1796-1799Crossref PubMed Scopus (405) Google Scholar,2Vulin A Goyenvalle A Barthélémy I Leturcq F Kaplan JC Chelly J et al.Rescue of dystrophin in the GRMD dog by multi-exon skipping using engineered U7 snRNAs.Neuromuscul Disord. 2006; 16: 722-723Abstract Full Text Full Text PDF Google Scholar In the treated muscles, dystrophin expression, muscle morphology amelioration, and significant force recovery were obtained. This successful dystrophin rescue by AAV1-U7 therapy is partly explained by the high tropism of AAV1 (adeno-associated virus 1) for muscle.3Chao H Liu Y Rabinowitz J Li C Samulski RJ Walsh CE Several log increase in therapeutic transgene delivery by distinct adeno-associated viral serotype vectors.Mol Ther. 2000; 2: 619-623Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar Yet most clinical trials so far have been performed using AAV2-derived vectors (for review see ref.4Warrington Jr, KH Herzog RW Treatment of human disease by adeno-associated viral gene transfer.Hum Genet. 2006; 119: 571-603Crossref PubMed Scopus (116) Google Scholar).AAV1-U7 treatment aimed at restoring muscle function body-wide will likely require multiple re-injections. There are at least two reasons for this. First, although AAV vector genomes clearly persist in muscles for several years,5Rivera VM Gao GP Grant RL Schnell MA Zoltick PW Rozamus LW et al.Long-term pharmacologically regulated expression of erythropoietin in primates following AAV-mediated gene transfer.Blood. 2005; 105: 1424-1430Crossref PubMed Scopus (234) Google Scholar the beneficial expression of the antisense may disappear with time, given that there is no evidence of AAV vector genome integration into host chromosomes in either fibers or myogenic stem cells.6Schnepp BC Clark KR Klemanski DL Pacak CA Johnson PR Genetic fate of recombinant adeno-associated virus vector genomes in muscle.J Virol. 2003; 77: 3495-3504Crossref PubMed Scopus (200) Google Scholar Second, despite the significant progress in recent times in body-wide vector delivery to striated muscles and hearts of mice,7Wang Z Zhu T Qiao C Zhou L Wang B Zhang J et al.Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart.Nat Biotechnol. 2005; 23: 321-328Crossref PubMed Scopus (495) Google Scholar,8Gregorevic P Allen JM Minami E Blankinship MJ Haraguchi M et al.rAAV6-microdystrophin preserves muscle function and extends lifespan in severely dystrophic mice.Nat Med. 2006; 12: 787-789Crossref PubMed Scopus (247) Google Scholar,9Denti MA Rosa A D'Antona G Sthandier O De Angelis FG Nicoletti C et al.Body-wide gene therapy of Duchenne muscular dystrophy in the mdx mouse model.Proc Natl Acad Sci USA. 2006; 103: 3758-3763Crossref PubMed Scopus (116) Google Scholar the translation of this approach to large animals and humans is challenging because of the high doses of vector required.When the commonly used AAV2 vector was employed in preclinical models, little or no transduction was detected after the second injection. The presence of neutralizing antibodies (Abs) to AAV2 capsid proteins resulting from the first vector injection (as detected in in vitro studies) appears to be the cause of the poor transduction after the second injection.10Fisher KJ Jooss K Alston J Yang Y Haecker SE High K et al.Recombinant adeno-associated virus for muscle directed gene therapy.Nat Med. 1997; 3: 306-312Crossref PubMed Scopus (574) Google Scholar,11Halbert CL Standaert TA Aitken ML Alexander IE Russell DW Miller AD Transduction by adeno-associated virus vectors in the rabbit airway: efficiency, persistence, and readministration.J Virol. 1997; 71: 5932-5941Crossref PubMed Google Scholar,12Manning WC Zhou S Bland MP Escobedo JA Dwarki V Transient immunosuppression allows transgene expression following readministration of adeno-associated viral vectors.Hum Gene Ther. 1998; 9: 477-485Crossref PubMed Scopus (125) Google Scholar Furthermore, the results of a completed hemophilia B clinical trial have revealed that specific cytotoxic T-lymphocyte (CTL) responses targeting epitopes of the AAV2 capsid protein can limit the duration of therapeutically relevant transgene expression.13Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1536) Google Scholar These CTL responses to AAV2 capsids were further analyzed in healthy donors, and can be attributed to the reactivation of memory T cells.14Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8+ T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar Similar observations in mice have been reported,15Chen J Wu Q Yang P Hsu HC Mountz JD Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.Mol Ther. 2006; 13: 260-269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,16Sabatino DE Mingozzi F Hui DJ Chen H Colosi P Ertl HC et al.Identification of mouse AAV capsid-specific CD8+ T cell epitopes.Mol Ther. 2005; 12: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar and it was shown, in particular, that the AAV2 VP3 protein contains a heparan sulfate proteoglycan RXXR–binding motif which directs T-cell activation.17Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar This VP3 heparan sulfate proteoglycan–binding motif is not present in other AAV serotypes such as AAV8 (ref.17Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar) and AAV1 (ref.18Hauck B Xiao W Characterization of tissue tropism determinants of adeno-associated virus type 1.J Virol. 2003; 77: 2768-2774Crossref PubMed Scopus (96) Google Scholar).Because of its ribonucleotidic nature U7 small nuclear RNA is not immunogenic per se, and its expression induces long-term expression of a truncated but functional dystrophin in mice and dogs.1Goyenvalle A Vulin A Fougerousse F Leturcq F Kaplan JC Garcia L et al.Rescue of dystrophic muscle through U7 snRNA-mediated exon skipping.Science. 2004; 306: 1796-1799Crossref PubMed Scopus (405) Google Scholar,2Vulin A Goyenvalle A Barthélémy I Leturcq F Kaplan JC Chelly J et al.Rescue of dystrophin in the GRMD dog by multi-exon skipping using engineered U7 snRNAs.Neuromuscul Disord. 2006; 16: 722-723Abstract Full Text Full Text PDF Google Scholar This shortened dystrophin is similar to the one observed in revertant dystrophin-positive fibers in the muscles of human DMD patients andmdx mice in an otherwise dystrophin-negative background.19Hoffman EP Morgan JE Watkins SC Partridge TA Somatic reversion/suppression of the mouse mdx phenotype in vivo.J Neurol Sci. 1990; 99: 9-25Abstract Full Text PDF PubMed Scopus (229) Google Scholar,20Nicholson LV The "rescue" of dystrophin synthesis in boys with Duchenne muscular dystrophy.Neuromuscul Disord. 1993; 3: 525-531Abstract Full Text PDF PubMed Scopus (29) Google Scholar This would explain the lack of immune response against the rescued dystrophin, in contrast to restoration of full-length dystrophin.21Ginhoux F Turbant T Leboeuf M Gross DA Lemonnier F Garcia L et al.Evaluation of immune responses to dystrophin in humanized mdx HLA-A*0201 dystrophic mice: application in Duchenne muscular dystrophy gene therapy after U7 snRNA-mediated exon skipping.Molr Ther. 2005; 11: 307Abstract Full Text Full Text PDF Google Scholar Moreover, although the nature of immune response to AAV1 vectors has not been investigated, it has been shown in mice that, when re-injected into muscle, AAV1 vectors encounter an adaptative immune response that blocks subsequent gene delivery with the same brand of vector.22Xiao W Chirmule N Berta SC McCullough B Gao G Wilson JM Gene therapy vectors based on adeno-associated virus type 1.J Virol. 1999; 73: 3994-4003Crossref PubMed Google Scholar,23Riviere C Danos O Douar AM Long-term expression and repeated administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice.Gene Ther. 2006; 13: 1300-1308Crossref PubMed Scopus (140) Google ScholarA thorough understanding of this adaptive response following the use of AAV1-U7-based treatment in diseased muscles of dystrophic mice is crucial for the design of future gene therapy clinical trials, given that the first exposure to the vector could preclude further treatment with the same vector, particularly in the microenvironment of dystrophic muscle which undergoes constant regeneration of fragile fibers.Although we know that targeting of the whole skeletal musculature would require systemic delivery of AAV vectors, early clinical trials most often use the intramuscular route. This is unlikely to be broadly and repetitively applicable for any wide-spread benefit in patients. In this study, using dystrophic mice, we evaluated the immunological consequences of intramuscular injections of AAV1 vectors administered for the purpose of rescuing a tolerated quasi-dystrophin by exon skipping. We found that neutralizing Abs against the vector itself were the only effectors responsible for the failure of efficacy of AAV1 re-injection in mice. We showed that transient inhibition of co-stimulatory molecules during the period of AAV1 delivery, by using anti-CD40 Abs and CTLA4/Fc fusion protein, overcame this immune response and allowed sequential treatments and the consequent rescue of dystrophin in multiple muscles.ResultsImpairment of dystrophin rescue after secondary AAV1-U7 challengeAAV1-U7 treatment requires several injections in different sites to rescue the maximum possible number of muscles. We initially evaluated the effect of sequential challenges in mdx mice by injecting AAV1-U7 first in the tibialis anterior (TA) muscle and 15 days later in the contralateral TA muscle. Whereas the first injected TA exhibited a strong and sustained dystrophin expression, underlining the lack of immune response against the rescued dystrophin, the contralateral injected muscle exhibited the characteristics of an untreated muscle (Figure 1aandb), thereby demonstrating that the second AAV1-U7 injection was ineffective. In view of the finding that the first injected muscle exhibited a territory of dystrophin expression as extensive as would be expected after a single AAV1-U7 injection, we hypothesized that the ineffectiveness of subsequent AAV1-U7 injections resulted from an anti-AAV1 specific immune response.In order to test the immunogenicity of the AAV1 capsid itself, we set up an experimental system in which only the AAV1 capsid was shared between two successive injections, using the murine secreted alkaline phosphatase (mSeAP) (a non-immunogenic self-protein that can be detected in serum) as a reporter transgene for the second challenge. Given that spontaneous self renewal of dystrophic muscle drastically reduces mSeAP activity until it almost completely disappears,24Bartoli M Poupiot J Goyenvalle A Perez N Garcia L Danos O et al.Noninvasive monitoring of therapeutic gene transfer in animal models of muscular dystrophies.Gene Ther. 2006; 13: 20-28Crossref PubMed Scopus (28) Google Scholar we carried out these experiments in normal C57BL/6 mice (B6). This allowed for an appraisal of the kinetics of rejection of the second challenge independent of the expected loss of mSeAP activity connected with the ongoing dystrophic process. B6 mice were intramuscularly injected at day 0 with 1011 AAV1-U7 vg as previously described, and subsequently received 1010 AAV1-mSeAP vg in contralateral muscles alternately from day 1 to day 7. This dosage of AAV1-mSeAP allowed robust and reproducible levels of alkaline phosphatase activity in sera and/or tissue sections. In groups of mice re-injected on day 3, the mSeAP serum level dipped to 10% of the AAV1-mSeAP expression after a single injection, and was undetectable after a week (Figure 1c). These experiments suggest that mice challenged with AAV1 in TA raise an immune response directed against the AAV1 capsid within 1 week, preventing successful AAV1 re-injection. The transduction efficiency of the second challenge using AAV1-mSeAP seemed, therefore, to be limited by the magnitude of the anti-AAV1 immune response. Remarkably, no signs of cell-death or muscle-tissue renewal were detected at the secondary sites, thereby suggesting the absence of muscle-fiber destruction by either complement or cytotoxic activity.Humoral response against AAV1 capsidIt is well known that the lack of dystrophin triggers muscle-fiber micro-damage spread over the whole skeletal musculature. Scavenger cells potentially involved in the immune system, such as macrophages, home in toward these sites of necrosis, provoking the inflammation-like reactions that are essential for local muscle repair. Yet, there is no evidence that these circumstances lead to immunological problems or hypersensitivity in dystrophic individuals (mdx mice, golden retriever muscular dystrophy dogs, and human DMD patients). In order to confirm this, we first compared the humoral responses against AAV1 in mdx and normal B6 mice after intramuscular injection of 1011 AAV1-U7 vg (Figure 2a). As expected, the immunoglobulin G (IgG) and IgM titers, as evaluated by enzyme-linked immunosorbent assay (ELISA) tests, were identical in both cases.Figure 2Neutralizing antibodies curtail adeno-associated virus 1 (AAV1) muscle transduction efficiency, in vivo. (a) Tibialis anterior (TA) muscles from B6 (black) and mdx (grey) mice were injected with 1011 AAV1-U7 viral genomes (vg), and the anti-AAV1 immunoglobulin G (IgG) (black or grey triangles) and IgM (black or grey squares) titers were evaluated every week by enzyme-linked immunosorbent assay (ELISA). The data shown represent the mean values from three mice per time point ± SEM. (b) Anti-AAV1 IgM, and (c) IgG titers were evaluated after AAV1-U7 injections of 1011, 1010, and 109 vg into the TAs of B6 mice. The data shown represent the mean values from four mice per time point ± SEM. One of two representative experiments is shown. (d) From the mice in the experiment described in b and c, that had received 1011 AAV1-U7 vg injections into their TAs, sera were drawn at days 7, 14, and 28 (d7, d14, and d28) after the injections, and these sera were transferred at different doses (600, 150, 30, and 6 μl) by intravenous injection into naïve mice. One day after serum transfer, these naive mice were injected in their TAs with 1010 vg of AAV1-mSeAP. The data shown represent the mean values of murine secreted alkaline phosphatase (mSeAP) serum levels of three mice per time point ± SEM. The AAV1-mSeAP expression level of a single infection (lane 1) corresponds to 100% of transduction efficiency. One of two representative experiments is shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also performed a dose–response experiment by injecting B6 mice with 109, 1010, and 1011 AAV1-U7 vg. Not surprisingly, AAV1-specific IgM were detected earlier than IgG, peaking at day 7 and then decreasing to a level slightly higher than the basal titer observed in pre-immune sera (Figure 2b). At day 7 after injection, AAV1-specific IgG Abs started to be detected in the sera of mice infused with 1011 vg, and rapidly increased thereafter to reach a plateau at week 4 (Figure 2c). Interestingly, mice challenged with 10- or 100-fold less AAV1 particles exhibited the same Ab titer progression, although the final levels were different and were maintained until the end of the experiment 6 months later.The neutralizing properties of the Abs formed were assayed by transferring serum from injected mice [1011 AAV1 vg intramuscularly (IM)] into naïve recipient mice 1 day before the IM AAV1-mSeAP challenge. Figure 2d shows that 600 μl of immune serum drawn on day 7 (d7) was needed to inhibit 50% of the AAV1-mSeAP transduction, whereas as little as 150 μl of a d14 immune serum was sufficient to totally block transduction. A partial inhibition was also observed with as little as 30 μl of a d28 immune serum, certainly because of the higher Ab concentration, and probably also because of affinity maturation of the neutralizing anti-AAV1 Abs. In this experiment, antigen specificity was verified in terms of the inefficiency of sera from naïve mice or animals immunized twice with AAV2 (data not shown).Absence of cytotoxic immune response against AAV1 vectorsCTL responses directed against AAV2 capsid epitopes have been recently described in humans13Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko JJ et al.Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1536) Google Scholar,14Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8+ T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar and mice.15Chen J Wu Q Yang P Hsu HC Mountz JD Determination of specific CD4 and CD8 T cell epitopes after AAV2- and AAV8-hF.IX gene therapy.Mol Ther. 2006; 13: 260-269Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar,16Sabatino DE Mingozzi F Hui DJ Chen H Colosi P Ertl HC et al.Identification of mouse AAV capsid-specific CD8+ T cell epitopes.Mol Ther. 2005; 12: 1023-1033Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar,17Vandenberghe LH Wang L Somanathan S Zhi Y Figueredo J Calcedo R et al.Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid.Nat Med. 2006; 12: 967-971Crossref PubMed Scopus (173) Google Scholar However, there is no direct evidence of an impairment in gene delivery on account of AAV-specific cytotoxic attack in vivo. Two factors may contribute toward masking CTL responses directed against the AAV capsid: (i) the very efficient Ab neutralization of AAV particles before their entry into target cells, and (ii) the occurrence of cellular immune responses directed against the transgene itself in some conditions. Using our experimental system, in which mSeAP transgene is not immunogenic per se, we monitored CTL responses with the ability to inhibit AAV-based gene transfer in muscle by performing sequential AAV challenges in μKO mice. In these mice the heavy chain of IgM (μ chain) is invalidated, leading to the absence of mature B-cells.25Kitamura D Roes J Kuhn R Rajewsky K A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene.Nature. 1991; 350: 423-426Crossref PubMed Scopus (1528) Google Scholar Even in the absence of Ab response, we found no evidence of cellular immunity against AAV1 in vivo; a second injection of AAV1-mSeAP in μKO mice was as efficient as the first one using a single-injection protocol (Figure 3a). The absence of cytotoxic activity against AAV1 did not depend upon a delay in CTL induction. This is clear from the fact that a third injection of AAV1-mSeAP 1 month later in μKO mice was as efficient as a first injection (data not shown). In sharp contrast, we found that when AAV2 vectors were used, the success of gene transfer after secondary challenge was drastically reduced in μKO mice, showing a 30% reduction in transduction efficiency compared to a single injection (Figure 3b).Figure 3Adeno-associated virus 2 (AAV2)-injected, but not AAV1-injected, muscles present features of cytotoxic attack. (a) B6 (black) and μKO (grey) mice were either injected in their tibialis anteriors (TAs) or not injected with 1011 AAV1-U7 viral genomes (vg), and 15 days later they were injected in the contralateral TA with AAV1-mSeAP (1010 vg). Serum levels of murine secreted alkaline phosphatase (mSeAP) were measured 15 days after AAV1-mSeAP injection. (b) This experiment was similar to the one described in a except that AAV2 vectors were used. The data shown represent the mean values of mSeAP serum levels of four mice per group ± SEM. One of two representative experiments is shown. (c) Three days after the last injection, B6 TA sections were immunostained for CD8+ and CD11b+ cell surface markers. Left panel, results after AAV1 injections; right panel, results after AAV2 injections. Blue, nucleus staining with 4',6-diamidino-2-phenylindole (DAPI); green, CD8+ and CD11b staining; 1st, after one injection; 2nd, after the second injection. One representative immunostained section of six different muscles is shown for each condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)By immunostaining whole B6 muscle sections, we observed that each injection provoked a cellular infiltration, probably caused by needle intrusions, but the cellular composition of the infiltrate varied with the number of injections and the AAV serotype (Figure 3c). The infiltrates were composed mainly of CD11b+ cells, macrophages, and neutrophils, whose number increased after the second injection. Importantly, we found no CD8+ cells in the muscles after one or multiple AAV1 injections, whereas AAV2 transfer was associated with the presence of CD8+ cells in the infiltrate of only the secondary injected muscle. CD8+ cells were equally recruited after IM infusions of AAV2 in μKO mice, but never with AAV1 (data not shown).Taken together, these results demonstrate that the ineffectiveness of AAV1-U7 in the context of rechallenge by multiple infusions is caused only by pre-existing humoral immune response and neutralizing Abs against AAV1, whereas cellular immunity may also play a role in the process of rejection of AAV2 and transduced fibers.Transient immunomodulation therapy with MR1 and CTLA4/FcIn order to overcome the humoral immune response to AAV1, we chose to administer an immunomodulation treatment along with the injection of AAV1-U7 in mdx mice. The use of classical immunosuppressive treatments such as FK506, mycophenolate mofetil, and cyclosporin have failed to inhibit the formation of anti-AAV1 neutralizing Abs (data not shown). We then explored blocking T/B-cell interactions by interfering with the co-stimulatory pathways B7/CD28 (CTLA4) and CD40L/CD40, using two specific agents, CTLA4/Fc fusion protein and MR1 Abs. We found that transient co-stimulation blockade, performed at day 0 at the time of AAV1-U7 injection and on days 2 and 4, was sufficient to inhibit the formation of anti-AAV1 IgG Abs up to the end of the experiment 6 months later (Figure 4a). The formation of anti-AAV1 IgM Abs was also considerably curtailed by this treatment (Figure 4b). Most importantly, this co-stimulation blockade resulted in the recovery of transduction efficiency after a second AAV1-U7 challenge in the contralateral TA at week 2, as evidenced by the presence of a specific reverse transcriptase polymerase chain reaction (RT-PCR) product from the skipped dystrophin messenger RNA and the detection of rescued dystrophin protein in muscle sections (Figure 5b).Figure 4Immunomodulation by co-stimulation blockage inhibits anti-AAV1 immunoglobulin G (IgG) formation. (a) Anti-AAV1 IgG and (b) anti-AAV1 IgM formations in mdx mice were evaluated every week by enzyme-linked immunosorbent assay after intramuscular treatment with AAV1-U7 (1011 viral genomes) on day 0 (d0). Transient immunomodulation with MR1 and CTLA4/Fc was either carried out intraperitoneally at d0, d2, and d4 (open triangles or open squares) or not carried out (closed triangles or closed squares). The data shown represent the mean values of four mice per time point ± SEM.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Immunomodulation by co-stimulation blockade restores the efficiency of multiple AAV1-U7 injections. Tibialis anteriors (TAs), contralateral TA, and gastrocnemius muscles of mdx mice were injected with AAV1-U7 (1011 viral genomes) at d0 (1st), d14 (2nd) and d28 (3rd). (a) Control without immunosuppression. (b) The mice were immunosuppressed as described for Figure 4, at d0, d2, and d4 (IS, immunosuppression). (c) The mice were immunosuppressed at d0, d2, d4, d14, d16, and d18 (IS X2). Left panels: the mean values of immunoglobulin G (IgG) (closed triangles or open triangles) and IgM (closed squares or open squares) titers from two mice are represented. Middle panels: 6 weeks after the latest injection, dystrophin rescue was monitored by NCL-DYS2 immunostaining on whole transverse sections of muscles. Right panels: RNA samples were analyzed using reverse transcriptase polymerase chain reaction (RT-PCR) for detection of the normal dystrophin (open arrowheads, 901 base pair (bp) fragment) and exon 23-skipped messenger RNAs (closed arrowheads, 688 bp). One representative immunostained section, and RT-PCR results from two different muscles is shown for each condition.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In order to test the feasibility of repeated injections, we performed a third injection in the gastrocnemius muscle at day 28. When the immune response in the mice was suppressed during the first AAV1-U7 injection, dystrophin rescue in the third injected muscle was only partial, and anti-AAV1 IgGs appeared 2 weeks later at week 6 (Figure 5b). However, when the mice were immunosuppressed during the second AAV1-U7 injection also, the efficiency of gene transfer after the third injection of AAV1-U7 rose to a high level (Figure 5c). Impo