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Neutralizing antibodies directed against adeno-associated virus (AAV) are commonly found in humans. In seropositive subjects, vector administration is not feasible as antibodies neutralize AAV vectors even at low titers. Consequently, a relatively large proportion of humans is excluded from enrollment in clinical trials and, similarly, vector redosing is not feasible because of development of high-titer antibodies following AAV vector administration. Plasmapheresis has been proposed as strategy to remove anti-AAV antibodies from the bloodstream. Although safe and relatively effective, the technology has some limitations mainly related to the nonspecific removal of all circulating IgG. Here we developed an AAV-specific plasmapheresis column which was shown to efficiently and selectively deplete anti-AAV antibodies without depleting the total immunoglobulin pool from plasma. We showed the nearly complete removal of anti-AAV antibodies from high titer purified human IgG pools and plasma samples, decreasing titers to levels that allow AAV vector administration in mice. These results provide proof-of-concept of a method for the AAV-specific depletion of neutralizing antibodies in the setting of in vivo gene transfer.

International audience; Adeno-associated virus (AAV) vectors are promising candidates for gene therapy and have been explored as gene delivery vehicles in the treatment of Duchenne Muscular Dystrophy (DMD). Recent studies showed compelling evidence of therapeutic efficacy in large animal models following the intravenous delivery of AAV vectors expressing truncated forms of dystrophin. However, to translate these results to humans, careful assessment of the prevalence of anti-AAV neutralizing antibodies (NAbs) is needed, as presence of preexisting NABs to AAV in serum have been associated with a drastic diminution of vector transduction. Here we measured binding and neutralizing antibodies against AAV serotype 1, 2, and 8 in serum from children and young adults with DMD (n = 130). Results were compared with to age-matched healthy donors (HD, n = 113). Overall, approximately 54% of all subjects included in the study presented IgG to AAV2, 49% to AAV1, and 41% to AAV8. A mean of around 80% of IgG positive sera showed neutralizing activity with no statistical difference between DMD and HD. NAb titers for AAV2 were higher than AAV1, and AAV8 in both populations studied. Older DMD patients (13-24 years old) presented significantly lower anti-AAV8 IgG4 subclass. Anti-AAV antibodies were found to be decreased in DMD patients subjected to a 6-month course of corticosteroids and in subjects receiving a variety of immunosuppressive drugs including B cell targeting drugs. Longitudinal follow up of humoral responses to AAV over up to 6 years showed no change in antibody titers, suggesting that in this patient population, seroconversion is a rare event in humans.

Duchenne muscular dystrophy (DMD) is an incurable X-linked muscle-wasting disease caused by mutations in the dystrophin gene. Gene therapy using highly functional microdystrophin genes and recombinant adeno-associated virus (rAAV) vectors is an attractive strategy to treat DMD. Here we show that locoregional and systemic delivery of a rAAV2/8 vector expressing a canine microdystrophin (cMD1) is effective in restoring dystrophin expression and stabilizing clinical symptoms in studies performed on a total of 12 treated golden retriever muscular dystrophy (GRMD) dogs. Locoregional delivery induces high levels of microdystrophin expression in limb musculature and significant amelioration of histological and functional parameters. Systemic intravenous administration without immunosuppression results in significant and sustained levels of microdystrophin in skeletal muscles and reduces dystrophic symptoms for over 2 years. No toxicity or adverse immune consequences of vector administration are observed. These studies indicate safety and efficacy of systemic rAAV-cMD1 delivery in a large animal model of DMD, and pave the way towards clinical trials of rAAV–microdystrophin gene therapy in DMD patients., Duchenne muscular dystrophy is a progressive degenerative disease of muscles caused by mutations in the dystrophin gene. Here the authors use AAV vectors to deliver microdystrophin to dogs with muscular dystrophy, and show restoration of dystrophin expression and reduction of symptoms up to 26 months of age.

International audience; Adeno-associated viruses (AAV) are small, nonenveloped single-stranded DNA viruses which require helper viruses to facilitate efficient replication. These recombinant viruses are some of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Nevertheless, the presence of humoral responses to the wild-type AAV common among humans is one of the limitations of in vivo transduction efficacy in humans using cognate recombinant vector. In this study, based on the serum samples that we were able to collect from various clinical situations, we studied the impact of one to five plasmapheresis (PP), at 1-5 day intervals on neutralizing factor (NAF) titers specific for AAV types 1, 2, 6, and 8 in seropositive patients with diverse pathologies and immunosuppressor treatments. We show that frequent sessions of PP result in drastic reduction of NAF specific for AAV1, 2, 6, and 8 to undetectable levels or titers

Adeno-associated viruses (AAVs) are small, nonenveloped single-stranded DNA viruses that require helper viruses to facilitate efficient replication. Despite the presence of humoral responses to the wild-type AAV in humans, AAV remains one of the most promising candidates for therapeutic gene transfer to treat many genetic and acquired diseases. Characterization of the IgG subclass responses to AAV and study of the prevalence of both IgG and neutralizing factors to AAV types 1, 2, 5, 6, 8, and 9 in the human population are of importance for the development of new strategies to overcome these immune responses. Natural exposure to AAV types 1, 2, 5, 6, 8, and 9 can result in the production of antibodies from all four IgG subclasses, with a predominant IgG1 response and low IgG2, IgG3, and IgG4 responses. Prevalences of anti-AAV1 and -AAV2 total IgG determined by enzyme-linked immunosorbent assay were higher (67 and 72%) than those of anti-AAV5 (40%), anti-AAV6 (46%), anti-AAV8 (38%), and anti-AAV9 (47%). Furthermore, data showed that cross-reactions are important. The two highest neutralizing factor seroprevalences were observed for AAV2 (59%) and AAV1 (50.5%) and the lowest were observed for AAV8 (19%) and AAV5 (3.2%). Vectors based on AAV5, AAV8, and AAV9 may have an advantage for gene therapy in humans. Furthermore, among individuals seropositive for AAV5, AAV8, and AAV9, about 70-100% present low titers. Better characterization of the preexisting humoral responses to the AAV capsid and cross-reactivity will allow development of new strategies to circumvent AAV acquired immune responses.

International audience; Duchenne muscular dystrophy (DMD) is an X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterised by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno Associated Virus vectors (rAAV) hold great promise and result in particular in very efficient transduction of skeletal and cardiac muscles.

BCR/ABL fusion gene, encoding a paradigmatic tyrosine kinase involved in chronic myelogenous leukemia (CML), can modulate the expression of genes involved in natural killer (NK) cell target recognition. Recent reports outline the role of allogeneic antileukemic NK effectors in the graft-versus-leukemia effect but the regulation of NK cell activation in the setting of graft-versus-leukemia effect remains unknown. Here we show that dendritic cells derived from monocytes of CML patients are selectively endowed with NK cell stimulatory capacity in vitro. We further show, using a gene transfer approach in mouse bone marrow progenitors, that ABL/ABL is necessary to promote dendritic cell–mediated NK cell activation. The dendritic cell/NK cell cross-talk in ABL/ABL-induced CML seems unique because JunB or IFN consensus sequence binding protein loss of functions, associated with other myeloproliferative disorders, do not promote dendritic cell–mediated NK cell activation. NK cell activation by leukemic dendritic cells involves NKG2D activating receptors and is blocked by imatinib mesylate. Indeed, ABL/ABL translocation enhances the expression levels of the NKG2D ligands on dendritic cells, which is counteracted by imatinib mesylate. Altogether, the clonal ABL/ABL dendritic cells display the unique and selective ability to activate NK cells and may participate in the NK cell control of CML. This study also highlights the deleterious role of imatinib mesylate at the dendritic cell level for NK cell activation.

International audience; Loss-of-function mutations in the myotubularin gene (MTM1) cause X-linked myotubular myopathy (XLMTM), a fatal, congenital pediatric disease that affects the entire skeletal musculature. Systemic administration of a single dose of a recombinant serotype 8 adeno-associated virus (AAV8) vector expressing murine myotubularin to Mtm1-deficient knockout mice at the onset or at late stages of the disease resulted in robust improvement in motor activity and contractile force, corrected muscle pathology, and prolonged survival throughout a 6-month study. Similarly, single-dose intravascular delivery of a canine AAV8-MTM1 vector in XLMTM dogs markedly improved severe muscle weakness and respiratory impairment, and prolonged life span to more than 1 year in the absence of toxicity or a humoral or cell-mediated immune response. These results demonstrate the therapeutic efficacy of AAV-mediated gene therapy for myotubular myopathy in small- and large-animal models, and provide proof of concept for future clinical trials in XLMTM patients.

Dendritic cells (DC) are professional antigen-presenting cells that can be used as immune adjuvant for anti-tumoural therapies. This approach requires the generation of large quantities of DC that are fully characterized on the immunophenotypical and functional levels. In a murine model, we analysed the in vitro effects of granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined with interleukin-4 (IL-4) or Flt3 ligand (Flt3-L) on the number, immunophenotype and functions of bone marrow-derived DC. In GM-CSF cultures, we have identified two populations based on their level of expression of major histocompatibility complex (MHC) class II molecules: MHC-IIhi cells, exhibiting the typical morphology and immunophenotype of myeloid DC (CD11c+ 33D1+ DEC-205+ F4/80+), and MHC-IIlo cells, heterogeneous for DC markers (30% CD11c+; 50% 33D1+; DEC-205-; F4/80+). The addition of Flt3-L to GM-CSF induced a twofold increase in MHC-IIhi DC number; besides, the MHC-IIlo cells lost all DC markers. In contrast, after addition of IL-4 to GM-CSF, the two populations displayed a very similar phenotype (CD11c+ 33D1- DEC-205+ F4/80-), differing only in their expression levels of MHC class II and costimulatory molecules, and showed similar stimulatory activity in mixed leucocyte reaction. We next analysed the migration of these cultured cells after fluorescent labelling. Twenty-four hours after injection into the footpads of mice, fluorescent cells were detected in the draining popliteal lymph nodes, with an enhanced migration when cells were cultured with GM-CSF+Flt3-L. Finally, we showed that MHC-IIhi were more efficient than MHC-IIlo cells in an anti-tumoral vaccination protocol. Altogether, our data highlight the importance of characterizing in vitro-generated DC before use in immunotherapy.

Mutations in the myotubularin gene (MTM1) result in X-linked myotubular myopathy (XLMTM), a fatal pediatric disease of skeletal muscle characterized by small myofibers with frequent central nuclei and abnormal mitochondrial accumulations. Patients with XLMTM typically present with severe hypotonia, muscle weakness and respiratory failure. Previous local studies in Mtm1-mutant mice demonstrated potential efficacy of gene therapy to treat the disease. Here we report long-term survival data in mice and dogs following intravenous delivery of an adeno-associated virus serotype 8 (AAV8) vector expressing myotubularin under the muscle-specific desmin promoter. XLMTM dogs (n = 3) were treated at 8 weeks of age with full-length canine MTM1 cDNA using an AAV8 vector. Affected XLMTM dogs were infused with AAV8–MTM1 (2.5 × 10 13 vg/kg), IV under high pressure into the saphenous vein while a tourniquet was applied around the upper thigh. While AAV8–MTM1 was administered by hindlimb infusion, effects were observed systemically in dogs. Mice were given intravenous AAV8–MTM1 by tail vein injection. In both murine and canine models, myotubularin transgene was expressed in all limb muscles and this translated into marked improvement of contractile force in mice and dogs. All mutant mice, including those injected at a late stage of the disease, survived until the end of the 6-month study. The lifespan of MTM1 mutant dogs that normally survive to 4 months was prolonged beyond 1 year in the first of three treated animals. Two additional treated dogs remain robust and continue to thrive beyond 8 months-of-age. Our findings in these two species provide proof-of-principle that intravenous AAV-mediated myotubularin replacement can rescue the severe phenotype of both small and large animal models of myotubular myopathy.

Duchenne Muscular Dystrophy (DMD) is a X-linked inherited muscle-wasting disease primarily affecting young boys with a prevalence of 1:5,000. The disease is caused by loss-of-function mutations in the gene encoding for the Dystrophin protein and is characterized by systemic, progressive, irreversible and severe loss of muscle function. Among vector systems that allow efficient in vivo gene transfer, recombinant Adeno-Associated Virus vectors (rAAV) hold great promise and allow very efficient transduction of skeletal and cardiac muscles. However, full-length dystrophin cDNA exceeds the packaging capacity for a single rAAV gene-delivery cassette. Therefore, truncated versions namely micro-dystrophins have been designed and optimized to contain few clinically important regions of the dystrophin protein. We have tested a rAAV2/8 vector encoding a sequence optimised canine micro-dystrophin transgene, driven by a muscle-synthetic Spc512 promoter (rAAV2/8-Spc512-µDys) in a total of 12 Golden Retriever Muscular Dystrophy (GRMD) dogs, the canine model of DMD. Isolated limb perfusion studies using a single administration of vector induced high levels of micro-dystrophin expression in the treated limb (up to 90% dystrophin positive fibres) with significant normalisation of histological, NMR imaging and spectroscopy parameters and muscle strength, without deleterious immune responses. Similarly, single-dose intravascular delivery of the same rAAV2/8-Spc512-µDys, in absence of immunosuppression, led to long-term transduction of distant muscle groups and extended lifespan (up to 2 years). Profound improvement of multiple clinical features was observed, including gait and respiratory parameters and no toxicity or deleterious humoral and/or cell-mediated immune responses were observed. This study demonstrates the safety and long term efficacy of rAAV2/8-Spc5.12-µDys gene therapy in a relevant large-animal models of DMD and paves the way towards human clinical gene therapy using systemic peripheral vein administration of vector, and applicable to all DMD patients regardless of their genotype.

MTM1 gene mutations cause X-linked myotubular myopathy (XLMTM), a fatal congenital myopathy characterized by small myofibers with frequent central nuclei. XLMTM patients typically present with severe hypotonia, muscle weakness and respiratory failure. Previous intramuscular (IM) injection studies in Mtm1 KO mice demonstrated potential efficacy of gene therapy to treat the disease. We extended these results to a large animal model by testing IM delivery of an AAV8-canine MTM1 vector into the cranial tibialis muscle of dogs with XLMTM. We observed dramatic improvement in strength of treated limb muscles to levels approaching that of age-matched normal littermates. This response was detectable at 1 week post-injection and improvement continued until a terminal measurement at 6 weeks post-injection. Concomitantly, the AAV-injected XLMTM muscles showed substantial increase in mass and myofiber size, and decreases in pathological features. We also utilized the Mtm1 KO mouse to determine the response to systemic intravenous delivery of an AAV serotype 9 vector expressing myotubularin under the muscle-specific desmin promoter. Myotubularin was rapidly and persistently expressed in muscles scattered throughout the body and this translated into robust improvement of skeletal muscle pathology and contractile force, and normalized motor activity of treated mice. Importantly, the lifespans of treated mice, which normally survive ∼7–8 weeks, were prolonged to >6 months. We then evaluated the effects of a targeted protein-replacement agent (3E10Fv-MTM1) injected into the tibialis anterior muscle of Mtm1 KO mice. Injection of 3E10Fv-MTM1 over 2 weeks increased contractile function (p

A major impediment to the use of adeno-associated virus (AAV)-mediated gene delivery to muscle in clinical applications is the pre-existing immune responses against the vector. Pre-existing humoral response to different AAV serotypes is now well documented. In contrast, cellular responses to AAV capsid have not been analyzed in a systematic manner, despite the risk of T cell reactivation upon gene transfer. AAV1 has been widely used in humans to target muscle. In this study, we analyzed PBMCs and sera of healthy donors for the presence of AAV1 capsid-specific T cell responses and AAV1 neutralizing factors. Approximately 30% of donors presented AAV1 capsid-specific T cells, mainly effector memory CD8+ cells. IFN-γ–producing cells were also observed among effector memory CD4+ cells for two of these donors. Moreover, to our knowledge, this study shows for the first time on a large cohort that there was no correlation between AAV1-specific T cell and humoral responses. Indeed, most donors presenting specific Ig and neutralizing factors were negative for cellular response (and vice versa). These new data raise the question of prescreening patients not only for the humoral response, but also for the cellular response. Clearly, a better understanding of the natural immunology of AAV serotypes will allow us to improve AAV gene therapy and make it an efficient treatment for genetic disease.

Background Gene modified dendritic cells (DC) are able to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific manner. Among the different DC subsets, plasmacytoid DC (pDC) are well known for their ability to recognize and respond to a variety of viruses by secreting high levels of type I interferon. Methods We analyzed here, the transduction efficiency of a pDC cell line, GEN2.2, and of pDC derived from CD34+ progenitors, using lentiviral vectors (LV) pseudotyped with different envelope glycoproteins such as the vesicular stomatitis virus envelope (VSVG), the gibbon ape leukaemia virus envelope (GaLV) or the feline endogenous virus envelope (RD114). At the same time, we evaluated transgene expression (E-GFP reporter gene) under the control of different promoters. Results We found that efficient gene transfer into pDC can be achieved with VSVG-pseudotyped lentiviral vectors (LV) under the control of phoshoglycerate kinase (PGK) and elongation factor-1 (EF1α) promoters (28% to 90% of E-GFP+ cells, respectively) in the absence of phenotypic and functional maturation. Surprisingly, promoters (desmin or synthetic C5–12) described as muscle-specific and which drive gene expression in single strand AAV vectors in gene therapy protocols were very highly active in pDC using VSVG-LV. Conclusion Taken together, our results indicate that LV vectors can serve to design pDC-based vaccines in humans, and they are also useful in vitro to evaluate the immunogenicity of the vector preparations, and the specificity and safety of given promoters used in gene therapy protocols.

Dendritic cells (DC) are antigen-presenting cells pivotal for inducing immunity or tolerance. Gene transfer into DC is an important strategy for developing immunotherapeutic approaches against infectious pathogens and cancers. One of the vectors previously described for the transduction of human monocytes or DC is the recombinant adeno-associated virus (rAAV), with a genome conventionally packaged as a single-stranded (ss) molecule. Nevertheless, its use is limited by the poor and variable transduction efficiency of DC. In this study, AAV type 1 (AAV1) and AAV2 vectors, which expressed the enhanced green fluorescent protein and were packaged as ss or self-complementary (sc) duplex strands, were used to transduce different DC subsets generated ex vivo and the immunophenotypes, states of differentiation, and functions of the subsets were carefully examined. We show here for the first time that a single exposure of monocytes (Mo) or CD34+progenitors (CD34) to sc rAAV1 or sc rAAV2 leads to high transduction levels (5 to 59%) of differentiated Mo-DC, Mo-Langerhans cells (LC), CD34-LC, or CD34-plasmacytoid DC (pDC), with no impact on their phenotypes and functional maturation of these cells, compared to those of exposure to ss rAAV. Moreover, we show that all these DC subpopulations can also be efficiently transduced after commitment to their differentiation pathways. Furthermore, these DC subsets transduced with sc rAAV1 expressing a tumor antigen were potent activators of a CD8+-T-cell clone. Altogether, these results show the high potential of sc AAV1 and sc AAV2 vectors to transduce ex vivo conventional DC, LC, or pDC or to directly target them in vivo for the design of new DC-based immunotherapies.

Long-term correction of the disease phenotype following gene therapy in vivo with adeno-associated virus (AAV) vectors has been demonstrated in several preclinical and clinical studies. With clinical development, however, it has become obvious that the host immune system represents an important obstacle to safe and effective gene transfer with AAV vectors. Indeed T cell responses directed against the viral capsid can have a detrimental effect on the duration of transgene expression and can result in tissue-specific toxicity due to the clearance of vector infected cells. ELISpot and flow cytometry analysis of immune responses to the vector capsid in peripheral blood mononuclear cells (PBMCs) show that both CD4+ and CD8+ T cells reactive to the AAV capsid are detectable in peripheral blood following gene transfer in humans, however little is known about T cell clonality following gene transfer in PBMC vs. locally in the tissue in which the vector is delivered.Here we used TCRbeta deep sequencing of T cells infiltrating muscle injected with AAV vectors and correlated results with a time course of sequencing of TCRbeta performed in fractionated CD8+ and CD8- PBMC from subjects injected with an AAV1 vector expressing γ-sarcoglycan (AAV1-γ-sarco). PBMC and muscle samples from three subjects who received an intramuscular injection of 4.5×1010 vector genomes of AAV1-γ-sarco (Brain 2012; 135:483) were analyzed. In one of these subjects, AAV capsid IFN-γ ELISpot on PBMCs showed activation of T cells responses peaking at day 30 post gene delivery. Consistent with this finding, TCRbeta sequencing of sorted CD8+ T cells showed the de novo expansion of T cell clones in PBMC after gene transfer, which were detectable at day 30 and 180 but not at baseline before vector administration. Additional CD8+ T cell clones present at baselines also expanded between day 0 and day 180. Interestingly, CD8+ T cell clones that expanded in PBMC following gene transfer were not found in biopsies of injected muscle collected at day 30. Conversely, several T cell clones shared between muscle and more in PBMC CD8+ than CD8- population did not expand upon AAV vector injection, possibly representing the underlying inflammation of muscle characteristic of the disease. Finally, we found T cell clones over-represented in muscle following AAV injection compared to PBMC, suggesting specific differences in homing of T cells in different body tissues. For example in one subject, we observed a clear expansion of T cell clones in muscle at day 30 post vector administration compared to baseline; these clones were not detected in the CD8+ T cell compartment in peripheral blood.In conclusion, this work addresses the crucial issue of correlation of T cell responses to AAV in peripheral blood vs. the transduced tissue. Our results suggest that T cell reactivity in peripheral blood does not necessarily represent the state of inflammation in the injected tissue, at the site of antigen presentation.

Gene and cell therapies are promising for long-term reconstitution of diseased tissues, and for restoration of therapeutic levels of circulating proteins, correcting inherited genetic disorders. Among the fundamental biological questions raised, the immune response to gene therapy is of great importance as it can hinder the success of gene correction. The onset of an immune response depends on the mutational genotype in each patient; the strength of antitransgene responses may also vary depending on gene delivery methods, based on plasmids, viral vectors or cell vectors, and on the route of injection. As a result, in most gene therapy protocols, the immune system will detect the packaging viral proteins and the transgene itself, causing humoral and cellular responses, which may lead to complete eradication of the corrected cells whenever the transgene protein behaves as a truly foreign antigen, that is, its sequence is partially or completely absent from the host. In this review, we will consider three main goals of immunology research in the field of gene therapy, namely, (1) deciphering dendritic cell/viral vector interactions, (2) inducing antigen-specific tolerance for long-term reconstitution of diseased tissues, and (3) monitoring immune responses to gene transfer in humanized animal models carrying HLA transgenes. Keywords: gene transfer; muscular dystrophy; immune responses; viral vectors; dendritic cells; regulatory T cells; tolerance induction; HLA molecules; animal models; immunomonitoring

Conceptually, dendritic cells (DCs) take up and process exogenous antigens that are presented on MHC (major histocompatibility complex) class II molecules to stimulate CD4+ T cells, and present endogenously-produced proteins to CD8+ T cells through an MHC class I-dependent pathway. In this study, we compared the antitumor effects generated in vivo after vaccinations with DCs either loaded with exogenous protein (DCs-exo) or presenting antigens derived from endogenous-synthesized protein (DCs-endo). We used the murine MC26SC31 colon carcinoma cell line expressing on the cell surface the human CD4 (hCD4) molecule as a model tumor-associated antigen (TAA). Bone marrow (BM)-derived DCs-endo were obtained from histocompatible transgenic mice constitutively expressing hCD4; BM-derived DCs-exo were obtained after in vitro loading DCs, from syngenic normal mice, with purified soluble hCD4 protein (shCD4). Altogether, our results show that intravenous (i.v.) administration of DCs-exo and DCs-endo can trigger similar cytotoxic and humoral protecting immune responses against a lethal tumor challenge. hCD4 antigen-specific T cell-mediated cytotoxic immune response was not accompanied by elevated INF-gamma serum levels. This suggests, in the case of DCs-exo, a possible in vivo CTL cross-priming triggering a CD8+ T cell-mediated immune response in the absence of de novo antigen synthesis within the DCs.

We have previously demonstrated that a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping injected in GRMD by locoregional transvenous perfusion of the forelimb, restores up to 80% dystrophin expression and improves histological, Nuclear Magnetic Resonance (NMR) pathological indices and strength in a dose-dependent manner at 3 months post injection (PInj). A recent paper suggested that dystrophin-positive fibers and rAAV genome drastically decrease with time, especially between 3 and 6 months PInj. This may compromise the clinical development of an AAV-mediated gene transfer approach. To assess this point, we realized a 6 to 7 months follow-up in a group of 3 GRMD aged 3–4 months when injected in both forelimbs with 2.5E13 vg/kg of a rAAV8 U7snRNA vector promoting exon skipping. Dogs were injected, followed up and evaluated (NMR, strength assessment, Dystrophin expression, vector biodistribution) in the same way as the previously reported large cohort of dogs that were followed for 3 months. Muscle biopsies were performed at 3 months and complete autopsy at 6 to 7 months PInj. Compared to previously reported dogs injected with the same dose, evaluation of dystrophin expression, histological aspect, AAV genome, NMR parameters and strength showed not differences indicating that the treatment effect was fully maintained over 6 months. No loss in AAV genome quantification and dystrophin expression was observed between the 3 months muscle biopsy specimens and 6 months muscle autopsy in the 3 dogs. A decrement in transgene copy numbers at 6 months PInj was observed in non muscular tissues. These data show, for the first time, that AAV-mediated exon skipping fully maintains dystrophin expression and functional recovery over a six months period. We suggest that long-term rAAV genome stability is obtained in dystrophic muscles when a substantial reversion of the muscular pathological pattern is reached.

The initiation of T-cell-mediated antitumor immune responses requires the uptake and processing of tumor antigens by dendritic cells and their presentation on MHC-I molecules. Here we show in a human in vitro model system that exosomes, a population of small membrane vesicles secreted by living tumor cells, contain and transfer tumor antigens to dendritic cells. After mouse tumor exosome uptake, dendritic cells induce potent CD8+ T-cell-dependent antitumor effects on syngeneic and allogeneic established mouse tumors. Therefore, exosomes represent a novel source of tumor-rejection antigens for T-cell cross priming, relevant for immunointerventions.

The initiation of host-protective mechanisms against pathogens (viruses, parasites) involves macrophages, dendritic cells (DCs) and natural killer (NK) cells that may interact either locally at the ports of entry or distally, but most likely through soluble mediators such as IFN, IL-12, and TNFα. NK-cell activation has the potential of contributing to beneficial immunoregulatory mechanisms critical in promoting early defenses against pathogenic agents and tumor metastasis. However, NK-cell overexpression of cytokines may be detrimental to the host therefore their activation must be tightly regulated. Flt3 ligand (FL) expands dramatically the number of DCs and enhances the generation of NK cells in vivo . Mature DCs are known to secrete various cytokines capable of activating NK cells. However, these soluble factors do not play a major role in the activation of resting NK cells by DCs.

We have developed a murine model to study the involvement of dendritic cells (DC) in human immunodeficiency virus (HIV) routing from an inoculation site to the lymph nodes (LN). Murine bone marrow-derived DC migrate to the draining LN within 24 h after subcutaneous injection. After incubation of these cells with heat-inactivated (Hi) HIV type 1 (HIV-1), HIV RNA sequences were detected in the draining LN only. Upon injection of DC pulsed with infectious HIV, the virus recovered in the draining LN was still able to productively infect human T cells. After a vaginal challenge with Hi HIV-1, the virus could be detected in the iliac and sacral draining LN at 24 h after injection. After an intravenous challenge, the virus could be detected in peripheral LN as soon as 30 min after injection. The specific depletion of a myeloid-related LN DC population, previously shown to take up blood macromolecules and to translocate them into the LN, prevented HIV transport to LN. Together, our data demonstrate the critical role of DC for HIV routing to LN after either a vaginal or an intravenous challenge, which does not require their infection. Therefore, despite the fact that the mouse is not infectable by HIV, this small animal model might be useful to test preventive strategies against HIV.

Reduction of insulin and triglycerides delays glomerulosclerosis in obese Zucker rats. To evaluate the effect of insulin and/or triglycerides on the pathogenesis of glomerulosclerosis, acarbose (BAYg5421), an inhibitor of intestinal α-glucosidases, was administered as a dietary admix (40mg/100g chow) to Zucker obese rats (ZOA), from 1.5 months until sacrifice at 1.5, 5, 8, 10 and 15 months. Obese (ZO) and lean (ZL) rats served as controls. Despite a similar food intake, ZOA weighed less than ZO at all ages. Acarbose reduced serum triglycerides at all ages, and insulin until 10 months. Glycemia remained normal in all groups. Proteinuria developed with age and to a greater degree in ZO than in ZOA rats. In ZL, a faint proteinuria appeared only in the oldest animals. Glomerulosclerosis, tubular and interstitial lesions rapidly affected ZO kidneys. These lesions were reduced in ZOA until 10 months. Acarbose did not modify the hypertrophy of the glomeruli that developed after three months, but slowed down the expansion of the mesangial domain seen in ZO. Thus, by reducing the amount of ingested glucose, acarbose yielded a normal glycemia with a lesser production of insulin and reduced renal impairment. Therefore, insulin could be a key factor involved in the pathogenesis of glomerulosclerosis, either directly or through a control of triglyceride concentrations.

Muscular dystrophies refer to a group of inherited disorders characterized by progressive muscle weakness, wasting and degeneration. So far, there are no strongly effective treatments but new gene-based therapies are currently being developed with particular advances in using exon skipping and other RNA-based approaches, conventional gene replacement strategies, and cell-based gene therapy. In the case of DMD, a number of groups are testing gene therapy with adeno-associated virus vectors expressing engineered microdystrophins (AAV-MDs). In our hands, highly sequence optimised AAV-MDs are available for use in mouse, dog and humans, expressed using a strong skeletal and cardiac muscle specific synthetic promoter, have been tested in detail in mdx mice, and in the GRMD dog. Here we report the outcome of a study of AAV-canine MD delivery (5× E12 vg/kg) by isolated limb perfusion in GRMD dogs from ages 3 months to 6 months. No immunosuppression was applied. In skeletal muscles of the treated limb at 3-months after vector administration we report widespread vector biodistribution, and sustained high level MD expression (up to 95% of fibres). In addition the treated limb exhibited very significantly improved parameters of muscle degeneration, fibrosis, 1H-MRI and 31P-NMR spectroscopy, and muscle strength. In the context of immune parameters, AAV administration elicited anti-AAV2/8 antibodies and a transient elevation of serum cytokines (wk5–10), but no evidence of cellular immunity to microdystrophin or to the AAV vector was observed. This study strongly supports the hypothesis that the current optimised microdystrophin is highly functional, not only in mice, but also in a large animal model, and that AAV2/8 vector delivery results in sustained expression of microdystrophin without adverse immune responses. The current optimised AAV-microdystrophin configuration thus lays a sound basis for a translation programme towards clinical trials in DMD patients.

BackgroundAdeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency.MethodologyHere, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2.ConclusionsOur results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.