Stance in statistical analysis, Dr Alvin Koh, and Ting Yixin for their comments on the original manuscript.Author ContributionsConceived and designed the experiments: WLH YPL. Analyzed the data: WLH CHL BHT MPC WHLL TS YPL. Wrote the paper: WLH.
Recombinant adeno-associated viral vectors (rAAV vectors) have been extensively developed as a means of delivering gene expression cassettes in vivo to a variety of post-mitotic cell types with the ultimate purpose of ameliorating disease symptoms [1]. The capacity of rAAV vectors to achieve strong and long-lasting transduction of non-dividing cells without significant pathogenicity or genomic integration has also made them valuable tools for manipulating and elucidating gene function in animal models. To this end, rAAV vectors have shown promise as prospective interventions for understanding and treating a variety of conditions IQ-1 site affecting the neuromuscular, cardiac, respiratory, hepatic, circulatory and sensory systems [2].In experiments using rAAV vectors to manipulate gene function, reporter genes such as b-galactosidase [3,4,5], human placental alkaline phosphatase (hPLAP) [4,6,7], luciferase [8,9] and green fluorescent protein (GFP) [10,11] are commonly used as experimental controls. Vectors carrying reporter genes not normally expressed in muscle offer a measure of transduction efficiency, and dose and time dependent SC-66 chemical information effects of transgene expression, while controlling for the influence of administering an equivalent dose of recombinant viral vectors as used in the experimental condition. However, the expression of such nonnative genes in skeletal muscle may alter cellular function and therefore complicate the interpretation of effects attributed toReporter Genes Can Promote Inflammation in Muscledelivery of an experimental vector, if used as an experimental control. Previous studies have observed inflammatory responses in mammalian skeletal muscle following administration of rAAV vectors carrying expression cassettes encoding non-native genes such as bacterial b-galactosidase [3,12,13,14] and coagulation factor IX [15]. However, effects appear to vary by gene, as we, and others have successfully employed rAAV vectors to transduce mammalian skeletal muscle with genes encoding for proteins not normally expressed in the host 1317923 species [16,17]. Other groups have reported that over-expression of native proteins can cause toxic effects in skeletal muscle, suggesting that the level of transgene expression may be determine whether cellular breakdown and local inflammation is caused by perturbation of functions within the target cell, as an alternative to activation of immunogenic responses [18]. Given that recombinant AAV vectors are capable of achieving highly effective delivery of gene expression cassettes, and that reporter gene-based vectors are routinely used as “nonfunctional” controls when experimentally manipulating muscle, it is important to ascertain whether commonly used reporter transgenes can elicit effects of their own when expressed in skeletal muscle. To answer this question, we examined murine hind limb muscles following administration of pseudotyope-6 rAAV vectors carrying expression cassettes encoding for hPLAP and GFP reporter genes. We report herein that local administration of rAAV6:hPLAP vector to skeletal muscle causes dose- and timedependent pro-inflammatory macrophage recruitment as well as significant skeletal muscle damage. These effects were independent of promoter type,.Stance in statistical analysis, Dr Alvin Koh, and Ting Yixin for their comments on the original manuscript.Author ContributionsConceived and designed the experiments: WLH YPL. Analyzed the data: WLH CHL BHT MPC WHLL TS YPL. Wrote the paper: WLH.
Recombinant adeno-associated viral vectors (rAAV vectors) have been extensively developed as a means of delivering gene expression cassettes in vivo to a variety of post-mitotic cell types with the ultimate purpose of ameliorating disease symptoms [1]. The capacity of rAAV vectors to achieve strong and long-lasting transduction of non-dividing cells without significant pathogenicity or genomic integration has also made them valuable tools for manipulating and elucidating gene function in animal models. To this end, rAAV vectors have shown promise as prospective interventions for understanding and treating a variety of conditions affecting the neuromuscular, cardiac, respiratory, hepatic, circulatory and sensory systems [2].In experiments using rAAV vectors to manipulate gene function, reporter genes such as b-galactosidase [3,4,5], human placental alkaline phosphatase (hPLAP) [4,6,7], luciferase [8,9] and green fluorescent protein (GFP) [10,11] are commonly used as experimental controls. Vectors carrying reporter genes not normally expressed in muscle offer a measure of transduction efficiency, and dose and time dependent effects of transgene expression, while controlling for the influence of administering an equivalent dose of recombinant viral vectors as used in the experimental condition. However, the expression of such nonnative genes in skeletal muscle may alter cellular function and therefore complicate the interpretation of effects attributed toReporter Genes Can Promote Inflammation in Muscledelivery of an experimental vector, if used as an experimental control. Previous studies have observed inflammatory responses in mammalian skeletal muscle following administration of rAAV vectors carrying expression cassettes encoding non-native genes such as bacterial b-galactosidase [3,12,13,14] and coagulation factor IX [15]. However, effects appear to vary by gene, as we, and others have successfully employed rAAV vectors to transduce mammalian skeletal muscle with genes encoding for proteins not normally expressed in the host 1317923 species [16,17]. Other groups have reported that over-expression of native proteins can cause toxic effects in skeletal muscle, suggesting that the level of transgene expression may be determine whether cellular breakdown and local inflammation is caused by perturbation of functions within the target cell, as an alternative to activation of immunogenic responses [18]. Given that recombinant AAV vectors are capable of achieving highly effective delivery of gene expression cassettes, and that reporter gene-based vectors are routinely used as “nonfunctional” controls when experimentally manipulating muscle, it is important to ascertain whether commonly used reporter transgenes can elicit effects of their own when expressed in skeletal muscle. To answer this question, we examined murine hind limb muscles following administration of pseudotyope-6 rAAV vectors carrying expression cassettes encoding for hPLAP and GFP reporter genes. We report herein that local administration of rAAV6:hPLAP vector to skeletal muscle causes dose- and timedependent pro-inflammatory macrophage recruitment as well as significant skeletal muscle damage. These effects were independent of promoter type,.
