Hers have extensively demonstrated that recipients not previously exposed typically tolerate intramuscular administration of rAAV vectors without evidence of cellular damage [17]. Recombinant AAV vectors typically exert very little evidence of adverse effects upon target cells, as they lack the coding regions of their wildtype genome, are derived from wildtype viruses that are notReporter Genes Can Promote Inflammation in Muscleassociated with specific human pathologies, and typically do not promote modification of the host cell’s genome. Our data are consistent with previous findings, as we were able to directly administer rAAV vectors lacking a functional gene (rAAV6:CMVMCS) to murine musculature without 3-Bromopyruvic acid KDM5A-IN-1 site chemical information causing ensuing cellular damage and inflammation. The transduction of skeletal muscles with constructs expressing non-native proteins can also promote an immune reaction and associated tissue damage, as this has been demonstrated following intramuscular administration of rAAV vectors [30,31]. However, this response appears to vary depending on the gene being expressed, as many other studies (including work of our own) have employed rAAV vectors to successfully transduce mammalian musculature with constructs encoding for non-native genes without observing ensuing tissue damage and inflammation [4,16,32]. In our studies reported here, we have shown similarly well-tolerated expression of non-native transgenes, by using rAAV vectors to express human follistatin-288 in murine skeletal muscles. We have also achieved robust expression of Renilla-derived green fluorescent protein in murine skeletal muscles without evidence of cellular degeneration and inflammation, depending on the vector dose used. Our findings of a positive correlation between rAAV6:hPLAP vector dose and the incidence of inflammation and cellular damage in murine muscles (and a similar correlation albeit requiring higher doses for rAAV6:GFP) suggest that specific gene products may perturb cellular function if expressed at sufficiently high levels. In support of this idea, it has been reported that dosedependent toxic effects can be observed even after expressing muscle-specific transgenes in skeletal muscle via vector based approaches [18]. Some studies have used tissue-specific promoter/ enhancer elements to reduce toxicity in transduced musculature and minimize the potential for unintentional transgene expression from antigen producing cells [19,33,34], whereas others have reported that the use of muscle-specific promoters does not prevent a deleterious reaction [3,35]. The inflammatory response we observed in muscles transduced with hPLAP expression cassettes was less-pronounced at early time-points when the CMV promoter 22948146 was substituted with a muscle-specific, creatine kinase-derived promoter (CK6) [19]. The reduced inflammation induced by hPLAP when driven by the muscle specific promoter correlated with reduced expression levels of hPLAP within TA muscles at this time point. However, significant damage was still observed in muscles treated with rAAV6:CK6-hPLAP at later time points, concomitant with progressive increases in hPLAP expression. Our findings are consistent with previous research in which the inflammatory response to transduction of mammalian musculature was not eliminated but delayed by substituting in a muscle-specific promoter instead of a CMV promoter [3,35]. The CK6 promoter is considerably less potent in its ability to drive reporter gene expression in.Hers have extensively demonstrated that recipients not previously exposed typically tolerate intramuscular administration of rAAV vectors without evidence of cellular damage [17]. Recombinant AAV vectors typically exert very little evidence of adverse effects upon target cells, as they lack the coding regions of their wildtype genome, are derived from wildtype viruses that are notReporter Genes Can Promote Inflammation in Muscleassociated with specific human pathologies, and typically do not promote modification of the host cell’s genome. Our data are consistent with previous findings, as we were able to directly administer rAAV vectors lacking a functional gene (rAAV6:CMVMCS) to murine musculature without causing ensuing cellular damage and inflammation. The transduction of skeletal muscles with constructs expressing non-native proteins can also promote an immune reaction and associated tissue damage, as this has been demonstrated following intramuscular administration of rAAV vectors [30,31]. However, this response appears to vary depending on the gene being expressed, as many other studies (including work of our own) have employed rAAV vectors to successfully transduce mammalian musculature with constructs encoding for non-native genes without observing ensuing tissue damage and inflammation [4,16,32]. In our studies reported here, we have shown similarly well-tolerated expression of non-native transgenes, by using rAAV vectors to express human follistatin-288 in murine skeletal muscles. We have also achieved robust expression of Renilla-derived green fluorescent protein in murine skeletal muscles without evidence of cellular degeneration and inflammation, depending on the vector dose used. Our findings of a positive correlation between rAAV6:hPLAP vector dose and the incidence of inflammation and cellular damage in murine muscles (and a similar correlation albeit requiring higher doses for rAAV6:GFP) suggest that specific gene products may perturb cellular function if expressed at sufficiently high levels. In support of this idea, it has been reported that dosedependent toxic effects can be observed even after expressing muscle-specific transgenes in skeletal muscle via vector based approaches [18]. Some studies have used tissue-specific promoter/ enhancer elements to reduce toxicity in transduced musculature and minimize the potential for unintentional transgene expression from antigen producing cells [19,33,34], whereas others have reported that the use of muscle-specific promoters does not prevent a deleterious reaction [3,35]. The inflammatory response we observed in muscles transduced with hPLAP expression cassettes was less-pronounced at early time-points when the CMV promoter 22948146 was substituted with a muscle-specific, creatine kinase-derived promoter (CK6) [19]. The reduced inflammation induced by hPLAP when driven by the muscle specific promoter correlated with reduced expression levels of hPLAP within TA muscles at this time point. However, significant damage was still observed in muscles treated with rAAV6:CK6-hPLAP at later time points, concomitant with progressive increases in hPLAP expression. Our findings are consistent with previous research in which the inflammatory response to transduction of mammalian musculature was not eliminated but delayed by substituting in a muscle-specific promoter instead of a CMV promoter [3,35]. The CK6 promoter is considerably less potent in its ability to drive reporter gene expression in.
