30 research outputs found
Polymers for Improving the In Vivo Transduction Efficiency of AAV2 Vectors
Background: Adeno-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. Methodology: Here, 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. Conclusions: Our 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
Approches de thérapies géniques pour des maladies neuromusculaires
Gene therapy of myopathies such as Duchenne muscular dystrophy requires a systemic approach in order to treat the whole musculature. The AAV vector is currently the most efficient delivery system for muscle transduction. We show that the biodistribution of AAV administered intravenously can be modified using different adjuvant strategies in healthy mice. In particular, the pre-injection of polymers enables an improvement of muscle transduction by AAV, and can also decrease the neutralizing immune response induced by the intravenous injection of this vector. We also explored in this work the impact of exogenous and endogenous modulating factors â such as the administration procedure or some blood factors â on the AAV transduction capacity. In a second approach, we evaluated gene transfer in dystrophic muscles in order to secrete in the blood circulation a transgenic protein associating the soluble TNF-α receptor I and the Fc fragment of an immunoglobulin (TNFR-Is/mIgG1). The comparison of the kinetic of secretion after muscle gene transfer in healthy and dystrophic mice indicates that the inflammatory context of dystrophic muscle increases the immune response against the transgene. We also show that while the expression and secretion of a low immunogenic murine variant of TNFR-Is/mIgG1 improves the mdx muscle function, it does not confer a selective advantage to muscle fibers which still undergo cycles of necrosis and regeneration.La thĂ©rapie gĂ©nique de myopathies telles que la dystrophie musculaire de Duchenne nĂ©cessite une approche systĂ©mique afin de traiter l'ensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administrĂ© par voie veineuse peut ĂȘtre modifiĂ©e en utilisant diverses stratĂ©gies adjuvantes chez la souris saine. La prĂ©-injection de polymĂšres permet ainsi d'amĂ©liorer la transduction des muscles par le vecteur AAV, ou encore de baisser la rĂ©ponse immune neutralisante induite par l'injection intraveineuse du vecteur. Nous abordons Ă©galement l'impact de facteurs modulateurs exogĂšnes ou endogĂšnes â tels que la procĂ©dure d'administration ou certains facteurs sanguins â sur la transduction systĂ©mique de l'AAV. Dans une seconde approche, nous avons Ă©valuĂ© le transfert de gĂšnes dans le muscle dystrophique afin de sĂ©crĂ©ter dans la circulation sanguine une protĂ©ine transgĂ©nique fusionnant le rĂ©cepteur soluble I du TNF-α avec le fragment constant d'une immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinĂ©tiques de sĂ©crĂ©tion obtenu aprĂšs le transfert de gĂšne dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une rĂ©ponse immune contre le transgĂšne. Nous montrons que l'expression et la sĂ©crĂ©tion d'un variant murin peu immunogĂšne du TNFR-Is/mIgG1 amĂ©liore la fonction musculaire de la souris mdx sans toutefois confĂ©rer un avantage sĂ©lectif aux fibres musculaires dystrophiques qui continuent leur cycle de nĂ©crose et de rĂ©gĂ©nĂ©ration
Gene therapy approaches for neuromuscular disorders
La thĂ©rapie gĂ©nique de myopathies telles que la dystrophie musculaire de Duchenne nĂ©cessite une approche systĂ©mique afin de traiter lâensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administrĂ© par voie veineuse peut ĂȘtre modifiĂ©e en utilisant diverses stratĂ©gies adjuvantes chez la souris saine. La prĂ©-injection de polymĂšres permet ainsi dâamĂ©liorer la transduction des muscles par le vecteur AAV, ou encore de baisser la rĂ©ponse immune neutralisante induite par lâinjection intraveineuse du vecteur. Nous abordons Ă©galement lâimpact de facteurs modulateurs exogĂšnes ou endogĂšnes â tels que la procĂ©dure dâadministration ou certains facteurs sanguins â sur la transduction systĂ©mique de lâAAV. Dans une seconde approche, nous avons Ă©valuĂ© le transfert de gĂšnes dans le muscle dystrophique afin de sĂ©crĂ©ter dans la circulation sanguine une protĂ©ine transgĂ©nique fusionnant le rĂ©cepteur soluble I du TNF-α avec le fragment constant dâune immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinĂ©tiques de sĂ©crĂ©tion obtenu aprĂšs le transfert de gĂšne dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une rĂ©ponse immune contre le transgĂšne. Nous montrons que lâexpression et la sĂ©crĂ©tion dâun variant murin peu immunogĂšne du TNFR-Is/mIgG1 amĂ©liore la fonction musculaire de la souris mdx sans toutefois confĂ©rer un avantage sĂ©lectif aux fibres musculaires dystrophiques qui continuent leur cycle de nĂ©crose et de rĂ©gĂ©nĂ©ration.Gene therapy of myopathies such as Duchenne muscular dystrophy requires a systemic approach in order to treat the whole musculature. The AAV vector is currently the most efficient delivery system for muscle transduction. We show that the biodistribution of AAV administered intravenously can be modified using different adjuvant strategies in healthy mice. In particular, the pre-injection of polymers enables an improvement of muscle transduction by AAV, and can also decrease the neutralizing immune response induced by the intravenous injection of this vector. We also explored in this work the impact of exogenous and endogenous modulating factors â such as the administration procedure or some blood factors â on the AAV transduction capacity. In a second approach, we evaluated gene transfer in dystrophic muscles in order to secrete in the blood circulation a transgenic protein associating the soluble TNF-α receptor I and the Fc fragment of an immunoglobulin (TNFR-Is/mIgG1). The comparison of the kinetic of secretion after muscle gene transfer in healthy and dystrophic mice indicates that the inflammatory context of dystrophic muscle increases the immune response against the transgene. We also show that while the expression and secretion of a low immunogenic murine variant of TNFR-Is/mIgG1 improves the mdx muscle function, it does not confer a selective advantage to muscle fibers which still undergo cycles of necrosis and regeneration
Approches de thérapies géniques pour des maladies neuromusculaires
Gene therapy of myopathies such as Duchenne muscular dystrophy requires a systemic approach in order to treat the whole musculature. The AAV vector is currently the most efficient delivery system for muscle transduction. We show that the biodistribution of AAV administered intravenously can be modified using different adjuvant strategies in healthy mice. In particular, the pre-injection of polymers enables an improvement of muscle transduction by AAV, and can also decrease the neutralizing immune response induced by the intravenous injection of this vector. We also explored in this work the impact of exogenous and endogenous modulating factors â such as the administration procedure or some blood factors â on the AAV transduction capacity. In a second approach, we evaluated gene transfer in dystrophic muscles in order to secrete in the blood circulation a transgenic protein associating the soluble TNF-α receptor I and the Fc fragment of an immunoglobulin (TNFR-Is/mIgG1). The comparison of the kinetic of secretion after muscle gene transfer in healthy and dystrophic mice indicates that the inflammatory context of dystrophic muscle increases the immune response against the transgene. We also show that while the expression and secretion of a low immunogenic murine variant of TNFR-Is/mIgG1 improves the mdx muscle function, it does not confer a selective advantage to muscle fibers which still undergo cycles of necrosis and regeneration.La thĂ©rapie gĂ©nique de myopathies telles que la dystrophie musculaire de Duchenne nĂ©cessite une approche systĂ©mique afin de traiter l'ensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administrĂ© par voie veineuse peut ĂȘtre modifiĂ©e en utilisant diverses stratĂ©gies adjuvantes chez la souris saine. La prĂ©-injection de polymĂšres permet ainsi d'amĂ©liorer la transduction des muscles par le vecteur AAV, ou encore de baisser la rĂ©ponse immune neutralisante induite par l'injection intraveineuse du vecteur. Nous abordons Ă©galement l'impact de facteurs modulateurs exogĂšnes ou endogĂšnes â tels que la procĂ©dure d'administration ou certains facteurs sanguins â sur la transduction systĂ©mique de l'AAV. Dans une seconde approche, nous avons Ă©valuĂ© le transfert de gĂšnes dans le muscle dystrophique afin de sĂ©crĂ©ter dans la circulation sanguine une protĂ©ine transgĂ©nique fusionnant le rĂ©cepteur soluble I du TNF-α avec le fragment constant d'une immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinĂ©tiques de sĂ©crĂ©tion obtenu aprĂšs le transfert de gĂšne dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une rĂ©ponse immune contre le transgĂšne. Nous montrons que l'expression et la sĂ©crĂ©tion d'un variant murin peu immunogĂšne du TNFR-Is/mIgG1 amĂ©liore la fonction musculaire de la souris mdx sans toutefois confĂ©rer un avantage sĂ©lectif aux fibres musculaires dystrophiques qui continuent leur cycle de nĂ©crose et de rĂ©gĂ©nĂ©ration
Approches de thérapies géniques pour des maladies neuromusculaires
La thĂ©rapie gĂ©nique de myopathies telles que la dystrophie musculaire de Duchenne nĂ©cessite une approche systĂ©mique afin de traiter l ensemble de la musculature. Le vecteur AAV est actuellement le plus efficace pour transduire le muscle. Nous montrons que la biodistribution du vecteur AAV administrĂ© par voie veineuse peut ĂȘtre modifiĂ©e en utilisant diverses stratĂ©gies adjuvantes chez la souris saine. La prĂ©-injection de polymĂšres permet ainsi d amĂ©liorer la transduction des muscles par le vecteur AAV, ou encore de baisser la rĂ©ponse immune neutralisante induite par l injection intraveineuse du vecteur. Nous abordons Ă©galement l impact de facteurs modulateurs exogĂšnes ou endogĂšnes tels que la procĂ©dure d administration ou certains facteurs sanguins sur la transduction systĂ©mique de l AAV. Dans une seconde approche, nous avons Ă©valuĂ© le transfert de gĂšnes dans le muscle dystrophique afin de sĂ©crĂ©ter dans la circulation sanguine une protĂ©ine transgĂ©nique fusionnant le rĂ©cepteur soluble I du TNF-a avec le fragment constant d une immunoglobuline (TNFR-Is/mIgG1). La comparaison des cinĂ©tiques de sĂ©crĂ©tion obtenu aprĂšs le transfert de gĂšne dans le muscle de souris saines ou de souris dystrophiques mdx indique que le contexte inflammatoire du muscle dystrophique favorise une rĂ©ponse immune contre le transgĂšne. Nous montrons que l expression et la sĂ©crĂ©tion d un variant murin peu immunogĂšne du TNFR-Is/mIgG1 amĂ©liore la fonction musculaire de la souris mdx sans toutefois confĂ©rer un avantage sĂ©lectif aux fibres musculaires dystrophiques qui continuent leur cycle de nĂ©crose et de rĂ©gĂ©nĂ©ration.Gene therapy of myopathies such as Duchenne muscular dystrophy requires a systemic approach in order to treat the whole musculature. The AAV vector is currently the most efficient delivery system for muscle transduction. We show that the biodistribution of AAV administered intravenously can be modified using different adjuvant strategies in healthy mice. In particular, the pre-injection of polymers enables an improvement of muscle transduction by AAV, and can also decrease the neutralizing immune response induced by the intravenous injection of this vector. We also explored in this work the impact of exogenous and endogenous modulating factors such as the administration procedure or some blood factors on the AAV transduction capacity. In a second approach, we evaluated gene transfer in dystrophic muscles in order to secrete in the blood circulation a transgenic protein associating the soluble TNF-a receptor I and the Fc fragment of an immunoglobulin (TNFR-Is/mIgG1). The comparison of the kinetic of secretion after muscle gene transfer in healthy and dystrophic mice indicates that the inflammatory context of dystrophic muscle increases the immune response against the transgene. We also show that while the expression and secretion of a low immunogenic murine variant of TNFR-Is/mIgG1 improves the mdx muscle function, it does not confer a selective advantage to muscle fibers which still undergo cycles of necrosis and regeneration.EVRY-Bib. Ă©lectronique (912289901) / SudocSudocFranceF
Fasting Increases the In Vivo Gene Delivery of AAV Vectors
International audienceSuccessful gene therapy of many genetic diseases requires efficient delivery of the gene to several tissues of the organism. Adeno-associated virus (AAV) is, to date, the sole vehicle that allows to achieving this result but only at the condition of administering very large amounts of vectors. This, however, raises questions about the feasibility of the large-scale production and about the safety of the approach. One way to overcome both problems would be to develop strategies that increase the in vivo efficiency. Here, we investigated the effect of fasting on the transduction efficiency of AAV serotypes 2, 6, and 9. The transgene expression was followed for several weeks and vector biodistribution was determined by real-time polymerase chain reaction (PCR) . The results show that fasting increases the transduction efficiency of all three serotypes. Altogether, we present here a simple and clinically acceptable approach that may allow to reducing the vector dose
Histidine-rich designer peptides of the LAH4 family promote cell delivery of a multitude of cargo
International audienc
Comment lâĂ©pissage alternatif contribue au contrĂŽle de la plasticitĂ© des structures de clathrine
International audienceNo abstract availabl
Soluble TNF-α receptor secretion from healthy or dystrophic mice after AAV6-mediated muscle gene transfer
International audienceMuscle is an attractive target because it is easily accessible; it also offers a permissive environment for adeno-associated virus (AAV)-mediated gene transfer and has an abundant blood vascular supply providing an efficient transport system for the secretion of proteins. However, gene therapy of dystrophic muscle may be more difficult than that of healthy tissue because of degenerative-regenerative processes, and also because of the inflammatory context. In this study we followed the expression levels of secreted inhibitors of the proinflammatory tumor necrosis factor (TNF) cytokine after intramuscular (i.m.) injection of AAV6 into dystrophic mdx and healthy C57BL/10 mice. We used two chimeric proteins, namely, the human or murine TNF-soluble receptor I fused with the murine heavy immunoglobulin chain. We conducted an AAV6 dose-response study and determined the kinetics of transgene expression. In addition, we followed the antibody response against the transgenes and studied their expression pattern in the muscle. Our results show that transduction efficiency is reduced in dystrophic muscles as compared with healthy ones. Furthermore, we found that the immune response against the secreted protein is stronger in mdx mice. Together, our results underscore that the pathological state of the muscle has to be taken into consideration when designing gene therapy approaches