AAV8 locoregional delivery induces long-term expression of an immunogenic transgene in macaques despite persisting local inflammation.

Adeno-associated virus (AAV) vectors are considered efficient vectors for gene transfer, as illustrated by recent successful clinical trials targeting retinal or neurodegenerative disorders. However, limitations as host immune responses to AAV capsid or transduction of limited regions must still be overcome. Here, we focused on locoregional (LR) intravenous perfusion vector delivery that allows transduction of large muscular areas and is considered to be less immunogenic than intramuscular (IM) injection. To confirm this hypothesis, we injected 6 cynomolgus monkeys with an AAV serotype 8 (AAV8) vector encoding for the highly immunogenic GFP driven by either a muscle-specific promoter (n = 3) or a cytomegalovirus (CMV) promoter (n = 3). We report that LR delivery allows long-term GFP expression in the perfused limb (up to 1 year) despite the initiation of a peripheral transgene-specific immune response. The analysis of the immune status of the perfused limb shows that LR delivery induces persisting inflammation. However, this inflammation is not sufficient to result in transgene clearance and is balanced by resident regulatory T cells. Overall, our results suggest that LR delivery promotes persisting transgene expression by induction of Treg cells in situ and might be a safe alternative to IM route to target large muscle territories for the expression of secreted therapeutic factors.

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation.

BACKGROUND: Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation. RESULTS: In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells. CONCLUSIONS: Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy.

Transgene regulation using the tetracycline-inducible TetR-KRAB system after AAV-mediated gene transfer in rodents and nonhuman primates.

Numerous studies have demonstrated the efficacy of the Adeno-Associated Virus (AAV)-based gene delivery platform in vivo. The control of transgene expression in many protocols is highly desirable for therapeutic applications and/or safety reasons. To date, the tetracycline and the rapamycin dependent regulatory systems have been the most widely evaluated. While the long-term regulation of the transgene has been obtained in rodent models, the translation of these studies to larger animals, especially to nonhuman primates (NHP), has often resulted in an immune response against the recombinant regulator protein involved in transgene expression regulation. These immune responses were dependent on the target tissue and vector delivery route. Here, using AAV vectors, we evaluated a doxycyclin-inducible system in rodents and macaques in which the TetR protein is fused to the human Krüppel associated box (KRAB) protein. We demonstrated long term gene regulation efficiency in rodents after subretinal and intramuscular administration of AAV5 and AAV1 vectors, respectively. However, as previously described for other chimeric transactivators, the TetR-KRAB-based system failed to achieve long term regulation in the macaque after intramuscular vector delivery because of the development of an immune response. Thus, immunity against the chimeric transactivator TetR-KRAB emerged as the primary limitation for the clinical translation of the system when targeting the skeletal muscle, as previously described for other regulatory proteins. New developments in the field of chimeric drug-sensitive transactivators with the potential to not trigger the host immune system are still needed.

Forelimb treatment in a large cohort of dystrophic dogs supports delivery of a recombinant AAV for exon skipping in Duchenne patients.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disorder caused by mutations in the dystrophin gene, without curative treatment yet available. Our study provides, for the first time, the overall safety profile and therapeutic dose of a recombinant adeno-associated virus vector, serotype 8 (rAAV8) carrying a modified U7snRNA sequence promoting exon skipping to restore a functional in-frame dystrophin transcript, and injected by locoregional transvenous perfusion of the forelimb. Eighteen Golden Retriever Muscular Dystrophy (GRMD) dogs were exposed to increasing doses of GMP-manufactured vector. Treatment was well tolerated in all, and no acute nor delayed adverse effect, including systemic and immune toxicity was detected. There was a dose relationship for the amount of exon skipping with up to 80% of myofibers expressing dystrophin at the highest dose. Similarly, histological, nuclear magnetic resonance pathological indices and strength improvement responded in a dose-dependent manner. The systematic comparison of effects using different independent methods, allowed to define a minimum threshold of dystrophin expressing fibers (>33% for structural measures and >40% for strength) under which there was no clear-cut therapeutic effect. Altogether, these results support the concept of a phase 1/2 trial of locoregional delivery into upper limbs of nonambulatory DMD patients.

hCTLA4-Ig transgene expression in keratocytes modulates rejection of corneal xenografts in a pig to non-human primate anterior lamellar keratoplasty model.

BACKGROUND: Human corneal allografting is an established procedure to cure corneal blindness. However, a shortage of human donor corneas as well as compounding economic, cultural, and organizational reasons in many countries limit its widespread use. Artificial corneas as well as porcine corneal xenografts have been considered as possible alternatives. To date, all preclinical studies using de-cellularized pig corneas have shown encouraging graft survival results; however, relatively few studies have been conducted in pig to non-human primate (NHP) models, and particularly using genetically engineered donors. METHODS: In this study, we assessed the potential benefit of using either hCTLA4-Ig transgenic or α1,3-Galactosyl Transferase (GT) Knock-Out (KO) plus transgenic hCD39/hCD55/hCD59/fucosyl-transferase pig lines in an anterior lamellar keratoplasty pig to NHP model. RESULTS: Corneas from transgenic animals expressing hCTLA4-Ig under the transcriptional control of a neuron-specific enolase promoter showed transgene expression in corneal keratocytes of the stroma and expression was maintained after transplantation. Although a first acute rejection episode occurred in all animals during the second week post-keratoplasty, the median final rejection time was 70 days in the hCTLA4-Ig group vs. 21 days in the wild-type (WT) control group. In contrast, no benefit for corneal xenograft survival from the GTKO/transgenic pig line was found. At rejection, cell infiltration in hCTLA4Ig transgenic grafts was mainly composed of macrophages with fewer CD3+ CD4+ and CD79+ cells than in other types of grafts. Anti-donor xenoantibodies increased dramatically between days 9 and 14 post-surgery in all animals. CONCLUSIONS: Local expression of the hCTLA4-Ig transgene dampens rejection of xenogeneic corneal grafts in this pig-to-NHP lamellar keratoplasty model. The hCTLA4-Ig transgene seems to target T-cell responses without impacting humoral responses, the control of which would presumably require additional peripheral immunosuppression.

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain.

Severe deficiency in lysosomal ß-glucuronidase (ß-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in ß-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the >200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, ß-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.

Generation and in vivo evaluation of IL10-treated dendritic cells in a nonhuman primate model of AAV-based gene transfer.

Preventing untoward immune responses against a specific antigen is a major challenge in different clinical settings such as gene therapy, transplantation, or autoimmunity. Following intramuscular delivery of recombinant adeno-associated virus (rAAV)-derived vectors, transgene rejection can be a roadblock to successful clinical translation. Specific immunomodulation strategies potentially leading to sustained transgene expression while minimizing pharmacological immunosuppression are desirable. Tolerogenic dendritic cells (TolDC) are potential candidates but have not yet been evaluated in the context of gene therapy, to our knowledge. Following intramuscular delivery of rAAV-derived vectors expressing an immunogenic protein in the nonhuman primate model, we assessed the immunomodulating potential of autologous bone marrow-derived TolDC generated in the presence of IL10 and pulsed with the transgene product. TolDC administered either intradermally or intravenously were safe and well tolerated. While the intravenous route showed a modest ability to modulate host immunity against the transgene product, intradermally delivery resulted in a robust vaccination of the macaques when associated to intramuscular rAAV-derived vectors-based gene transfer. These findings demonstrate the critical role of TolDC mode of injection in modulating host immunity. This study also provides the first evidence of the potential of TolDC-based immunomodulation in gene therapy.

scAAV9 intracisternal delivery results in efficient gene transfer to the central nervous system of a feline model of motor neuron disease.

On the basis of previous studies suggesting that vascular endothelial growth factor (VEGF) could protect motor neurons from degeneration, adeno-associated virus vectors (serotypes 1 and 9) encoding VEGF (AAV.vegf) were administered in a limb-expression 1 (LIX1)-deficient cat-a large animal model of lower motor neuron disease-using three different delivery routes to the central nervous system. AAV.vegf vectors were injected into the motor cortex via intracerebral administration, into the cisterna magna, or intravenously in young adult cats. Intracerebral injections resulted in detectable transgene DNA and transcripts throughout the spinal cord, confirming anterograde transport of AAV via the corticospinal pathway. However, such strategy led to low levels of VEGF expression in the spinal cord. Similar AAV doses injected intravenously resulted also in poor spinal cord transduction. In contrast, intracisternal delivery of AAV exhibited long-term transduction and high levels of VEGF expression in the entire spinal cord, yet with no detectable therapeutic clinical benefit in LIX1-deficient animals. Altogether, we demonstrate (i) that intracisternal delivery is an effective AAV delivery route resulting in high transduction of the entire spinal cord, associated with little to no off-target gene expression, and (ii) that in a LIX1-deficient cat model, however, VEGF expressed at high levels in the spinal cord has no beneficial impact on the disease course.

Variation patterns in individual fish responses to chemical stress among estuaries, seasons and genders: the case of the European flounder (Platichthys flesus) in the Bay of Biscay.

The objective was to describe and model variation patterns in individual fish responses to contaminants among estuaries, season and gender. Two hundred twenty-seven adult European flounders were collected in two seasons (winter and summer) in four estuaries along the Bay of Biscay (South West France), focusing on a pristine system (the Ster), vs. three estuaries displaying contrasted levels of contaminants (the Vilaine, Loire and Gironde). Twenty-three variables were measured by fish, considering the load of contaminants (liver metals, liver and muscle persistent organic pollutants, muscle polycyclic aromatic hydrocarbons); the gene expression (Cyt C oxydase, ATPase, BHMT, Cyt P450 1A1, ferritin); the blood genotoxicity (Comet test); and liver histology (foci of cellular alteration-tumour, steatosis, inflammation, abnormal glycogen storage). Canonical redundancy analysis (RDA) was used to model these variables using gender, season and estuary of origin as explanatory variables. The results underlined the homogeneity of fish responses within the pristine site (Ster) and more important seasonal variability within the three contaminated systems. The complete model RDA was significant and explained 35 % of total variance. Estuary and season respectively explained 30 and 5 % of the total independent variation components, whilst gender was not a significant factor. The first axis of the RDA explains nearly 27 % of the total variance and mostly represents a gradient of contamination. The links between the load of contaminants, the expression of several genes and the biomarkers were analysed considering different levels of chemical stress and a possible multi-stress, particularly in the Vilaine estuary.

Histopathological lesions and DNA adducts in the liver of European flounder (Platichthys flesus) collected in the Seine estuary versus two reference estuarine systems on the French Atlantic coast.

An epidemiological survey was conducted in the Seine estuary and in two smaller and relatively preserved estuaries on the French Atlantic coast in order to estimate the occurrence of liver lesions in European flounder, Platichthys flesus, and also to seek putative risk factors for the recorded pathologies. Four hundred and seventy-eight fish of both sexes and of different size ranges were sampled in the three studied areas, 338 of which in the Seine estuary. All fish were examined for histopathological liver lesions, while DNA adducts and otoliths were analyzed on a subsample. Five categories of hepatic lesions were recorded with the following prevalence for the Seine estuary: 36.7 % inflammations, 8 % parasites (mainly encysted nematodes), 6.5 % foci of cellular alteration (FCA), 5.3 % foci of necrosis or regeneration (FNR), and 1.5 % tumors. Inflammation occurrence increased according to age, contrary to parasitic infestations and FCA which were more prevalent in young fish, notably those of <1 year old (group 0). Tumors were only observed in females of more than two winters. Females exhibited a higher prevalence of tumors (3.0 %) and FCA (6.5 %) than males (0 and 2.6 %, respectively). Parasitic and infectious lesions and FNR were equally distributed in males and females. The prevalence of FNR was also shown to vary according to sampling season, with significantly more occurrences of liver necrosis in the fish collected in summer than in spring. Spatial differences were observed with a higher occurrence of encysted parasites in flounders from the upper Seine estuary, while inflammations predominated in flounders living downstream. Temporal trends were also noted, with an increased prevalence of parasitic infestations, inflammations, and FCA in the 2002-2003 period in comparison to the 1996-1997 one. The three flounder populations from the Seine estuary (Normandy), Ster estuary (Brittany), and Bay of Veys (Normandy) showed different spectra of hepatic lesions. Flounders from the Bay of Veys had relatively few liver lesions as compared to flounders from the two other estuaries. Flounders from the Ster estuary exhibited the highest prevalence of parasites (37.2 %) and inflammations (51.1 %). Finally, FCA and liver tumors occurred at very similar levels in both flounder populations from the Seine and the Ster estuaries. Group 0 flounders inhabiting the upper Seine estuary were more prone to parasitic and pre-neoplastic hepatic lesions and had higher levels of liver DNA adducts than the older ones living downstream. It was postulated that group 0 European flounders may serve as valuable bioindicators for assessing the quality of estuarine waters and the health status of euryhaline fish populations.

Gene transfer of human CD40Ig does not prevent rejection in a non-human primate kidney allotransplantation model.

BACKGROUND: Blockade of costimulation signaling required for immune response, such as CD40/CD40L and CD28/B7, is a reasonable strategy to prevent rejection and in defined combinations may allow donor specific tolerance. Indeed, in rodents, costimulation blockade with CD28/B7 antagonists or with CD40Ig was able to induce regulatory T cells and transplant tolerance whereas in primates, anti-CD40 antibodies, anti-CD40L antibodies or CTLA4Ig, used as monotherapy, significantly delayed graft rejection. METHODS: Using an adeno-associated virus (AAV) vector mediated gene transfer of a human CD40Ig fusion protein (hCD40Ig) in primates, we evaluated the capacity of this costimulation blockade molecule interfering with CD40/CD40L signaling in prolonging kidney transplants in cynomolgus monkeys. RESULTS: This gene transfer strategy allowed for maintaining a plateau of hCD40Ig production within two months and avoided a high-scale production phase of this molecule. Although the hCD40Ig was able to bind efficiently to human and macaque CD40L and high (>200 µg/ml) transgene expression was obtained, no effect on graft survival was observed. In addition, there was no inhibition of humoral response to vaccination. In vitro, hCD40Ig strongly increased mixed lymphocyte reaction, and when compared to the anti-CD40L antibody h5C8, was not as potent to induce complement-dependent cytotoxicity. CONCLUSION: These data suggest that CD40/CD40L blockade using a non-depleting CD40Ig fusion protein, a therapeutic strategy that showed efficacy in rodents, is not able to modulate the immune response in primates. These data highlight important biological differences between rodent and primate models to evaluate therapeutic strategies at the preclinical level.

Towards the establishment of a porcine model to study human amebiasis.

BACKGROUND: Entamoeba histolytica is an important parasite of the human intestine. Its life cycle is monoxenous with two stages: (i) the trophozoite, growing in the intestine and (ii) the cyst corresponding to the dissemination stage. The trophozoite in the intestine can live as a commensal leading to asymptomatic infection or as a tissue invasive form producing mucosal ulcers and liver abscesses. There is no animal model mimicking the whole disease cycle. Most of the biological information on E. histolytica has been obtained from trophozoite adapted to axenic culture. The reproduction of intestinal amebiasis in an animal model is difficult while for liver amebiasis there are well-described rodent models. During this study, we worked on the assessment of pigs as a new potential model to study amebiasis. METHODOLOGY/PRINCIPAL FINDINGS: We first co-cultured trophozoites of E. histolytica with porcine colonic fragments and observed a disruption of the mucosal architecture. Then, we showed that outbred pigs can be used to reproduce some lesions associated with human amebiasis. A detailed analysis was performed using a washed closed-jejunal loops model. In loops inoculated with virulent amebas a severe acute ulcerative jejunitis was observed with large hemorrhagic lesions 14 days post-inoculation associated with the presence of the trophozoites in the depth of the mucosa in two out four animals. Furthermore, typical large sized hepatic abscesses were observed in the liver of one animal 7 days post-injection in the portal vein and the liver parenchyma. CONCLUSIONS: The pig model could help with simultaneously studying intestinal and extraintestinal lesion development.

Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinoma.

BACKGROUND: Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). METHODS: Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. RESULTS: The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 10(8) plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. CONCLUSIONS: The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.

Systemic delivery of allogenic muscle stem cells induces long-term muscle repair and clinical efficacy in duchenne muscular dystrophy dogs.

Duchenne muscular dystrophy (DMD) is a genetic progressive muscle disease resulting from the lack of dystrophin and without effective treatment. Adult stem cell populations have given new impetus to cell-based therapy of neuromuscular diseases. One of them, muscle-derived stem cells, isolated based on delayed adhesion properties, contributes to injured muscle repair. However, these data were collected in dystrophic mice that exhibit a relatively mild tissue phenotype and clinical features of DMD patients. Here, we characterized canine delayed adherent stem cells and investigated the efficacy of their systemic delivery in the clinically relevant DMD animal model to assess potential therapeutic application in humans. Delayed adherent stem cells, named MuStem cells (muscle stem cells), were isolated from healthy dog muscle using a preplating technique. In vitro, MuStem cells displayed a large expansion capacity, an ability to proliferate in suspension, and a multilineage differentiation potential. Phenotypically, they corresponded to early myogenic progenitors and uncommitted cells. When injected in immunosuppressed dystrophic dogs, they contributed to myofiber regeneration, satellite cell replenishment, and dystrophin expression. Importantly, their systemic delivery resulted in long-term dystrophin expression, muscle damage course limitation with an increased regeneration activity and an interstitial expansion restriction, and persisting stabilization of the dog's clinical status. These results demonstrate that MuStem cells could provide an attractive therapeutic avenue for DMD patients.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Safe, efficient, and reproducible gene therapy of the brain in the dog models of Sanfilippo and Hurler syndromes.

Recent trials in patients with neurodegenerative diseases documented the safety of gene therapy based on adeno-associated virus (AAV) vectors deposited into the brain. Inborn errors of the metabolism are the most frequent causes of neurodegeneration in pre-adulthood. In Sanfilippo syndrome, a lysosomal storage disease in which heparan sulfate oligosaccharides accumulate, the onset of clinical manifestation is before 5 years. Studies in the mouse model showed that gene therapy providing the missing enzyme α-N-acetyl-glucosaminidase to brain cells prevents neurodegeneration and improves behavior. We now document safety and efficacy in affected dogs. Animals received eight deposits of a serotype 5 AAV vector, including vector prepared in insect Sf9 cells. As shown previously in dogs with the closely related Hurler syndrome, immunosuppression was necessary to prevent neuroinflammation and elimination of transduced cells. In immunosuppressed dogs, vector was efficiently delivered throughout the brain, induced α-N-acetyl-glucosaminidase production, cleared stored compounds and storage lesions. The suitability of the procedure for clinical application was further assessed in Hurler dogs, providing information on reproducibility, tolerance, appropriate vector type and dosage, and optimal age for treatment in a total number of 25 treated dogs. Results strongly support projects of human trials aimed at assessing this treatment in Sanfilippo syndrome.

Chikungunya disease in nonhuman primates involves long-term viral persistence in macrophages.

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that induces in humans a disease characterized by fever, rash, and pain in muscles and joints. The recent emergence or reemergence of CHIKV in the Indian Ocean Islands and India has stressed the need to better understand the pathogenesis of this disease. Previous CHIKV disease models have used young or immunodeficient mice, but these do not recapitulate human disease patterns and are unsuitable for testing immune-based therapies. Herein, we describe what we believe to be a new model for CHIKV infection in adult, immunocompetent cynomolgus macaques. CHIKV infection in these animals recapitulated the viral, clinical, and pathological features observed in human disease. In the macaques, long-term CHIKV infection was observed in joints, muscles, lymphoid organs, and liver, which could explain the long-lasting CHIKV disease symptoms observed in humans. In addition, the study identified macrophages as the main cellular reservoirs during the late stages of CHIKV infection in vivo. This model of CHIKV physiopathology should allow the development of new therapeutic and/or prophylactic strategies.

Lack of immunotoxicity after regional intravenous (RI) delivery of rAAV to nonhuman primate skeletal muscle.

In the absence of an immune response from the host, intramuscular (IM) injection of recombinant adeno-associated virus (rAAV) results in the permanent expression of the transgene from mouse to primate models. However, recent gene transfer studies into animal models and humans indicate that the risk of transgene and/or capsid-specific immune responses occurs and depends on multiple factors. Among these factors, the route of delivery is important, although poorly addressed in large animal models. Here, we compare the IM and the drug-free regional intravenous (RI) deliveries of rAAV in nonhuman primate (NHP) skeletal muscle monitoring the host immune response toward the transgene. We show that IM is consistently associated with immunotoxicity and the destruction of the genetically modified myofibers, whereas RI allows the stable expression of the transgene. This has important implications for the design of clinical trials for gene transfer in skeletal muscle.

Differential effects of hypoxia on osteochondrogenic potential of human adipose-derived stem cells.

Human adipose tissue-derived stem cells (hATSC) have been contemplated as reparative cells for cartilage engineering. Chondrogenic differentiation of hATSC can be induced by an enriched culture medium and a three-dimensional environment. Given that bone is vascularized and cartilage is not, oxygen tension has been suggested as a regulatory factor for osteochondrogenic differentiation. Our work aimed at determining whether hypoxia affects the osteochondrogenic potential of hATSC. hATSC were cultured in chondrogenic or osteogenic medium for 28 days, in pellets or monolayers, and under 5% or 20% oxygen tension. Cell differentiation was monitored by real-time PCR (COL2A1, aggrecan, Runx2, and osteocalcin). The chondrogenic differentiation was further evaluated by Alcian blue and immunohistological staining for glycosaminoglycans (GAGs) and type II collagen, respectively. Osteogenic differentiation was also assessed by the staining of mineralized matrix (Alizarin Red) and measurement of alkaline phosphatase (ALP) activity. The expression of chondrogenic markers was upregulated when hATSC were exposed to hypoxia in chondrogenic medium. Conversely, osteocalcin expression, mineralization, and ALP activity were severely reduced under hypoxic conditions even in the presence of osteogenic medium. Our data strongly suggest that hypoxia favors the chondrogenic differentiation of hATSC as evidenced by the expression of the chondrogenic markers, whereas it could alter their osteogenic potential. Our results highlight the differential regulatory role of hypoxia on the chondrogenic and osteogenic differentiation processes of hATSC. These data could help us exploit the potential of tissue engineering and stem cells to replace or restore the function of osteoarticular tissues.

Efficient intracerebral delivery of AAV5 vector encoding human ARSA in non-human primate.

Metachromatic leukodystrophy (MLD) is a lethal neurodegenerative disease caused by a deficiency in the lysosomal arylsulfatase A (ARSA) enzyme leading to the accumulation of sulfatides in glial and neuronal cells. We previously demonstrated in ARSA-deficient mice that intracerebral injection of a serotype 5 adeno-associated vector (AAV) encoding human ARSA corrects the biochemical, neuropathological and behavioral abnormalities. However, before considering a potential clinical application, scaling-up issues should be addressed in large animals. Therefore, we performed intracerebral injection of the same AAV vector (total dose of 3.8 x 10(11) or 1.9 x 10(12) vector genome, three sites of injection in the right hemisphere, two deposits per site of injection) into three selected areas of the centrum semiovale white matter, or in the deep gray matter nuclei (caudate nucleus, putamen, thalamus) of six non-human primates to evaluate vector distribution, as well as expression and activity of human ARSA. The procedure was perfectly tolerated, without any adverse effect or change in neurobehavioral examination. AAV vector was detected in a brain volume of 12-15 cm(3) that corresponded to 37-46% of the injected hemisphere. ARSA enzyme was expressed in multiple interconnected brain areas over a distance of 22-33 mm. ARSA activity was increased by 12-38% in a brain volume that corresponded to 50-65% of injected hemisphere. These data provide substantial evidence for potential benefits of brain gene therapy in patients with MLD.