Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation

Abstract

Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

Ex Vivo Gene Editing of the Dystrophin Gene in Muscle Stem Cells Mediated by Peptide Nucleic Acid Single Stranded Oligodeoxynucleotides Induces Stable Expression of Dystrophin in a Mouse Model for Duchenne Muscular Dystrophy

“…Most of the research aimed at determining the efficacy of SCs in restoring dystrophin expression for the treatment of DMD following engraftment has implemented the use of reporter systems to identify donor cells into transplanted muscles . Although these systems have proven to be valuable tools, expression of proteins like β‐galactosidase (β‐gal) or green fluorescent protein as experimental controls has been associated with a strong proinflammatory response toward the reporter protein and increased muscle damage rendering the assessment of the long‐term therapeutic efficacy of SC‐mediated regenerative approaches to DMD difficult to perform . Our experimental settings overcome these limitations by ensuring that the expression of dystrophin detected following engraftment of mdx 5cv SCs treated with the targeting PNA‐ssODN is the result of correction that has occurred at the genomic level and not the result of expansion of revertant fibers that may have occurred over time.…”

Dose-dependent Toxicity of Humanized Renilla reniformis GFP (hrGFP) Limits Its Utility as a Reporter Gene in Mouse Muscle

“…28 Potential toxicity is another issue, as some fluorescent proteins have proven toxic to various cells and tissues. 22,23,25,26 , 28,29,30 In this study, we were concerned about some published reports suggesting the prominently used Aequorea eGFP gene could be myopathic, and we therefore developed an AAV6 vector utilizing the hrGFP, based on the hypothesis that it was potentially less deleterious to adult mouse muscle. 13,14,22,23,25,26,29 Contrary to expectations, we found that hrGFP caused dose-dependent muscle toxicity.…”

“…2 , 14,15,16,17,18,19,20,21 Because some reports suggested Aequorea -derived eGFP could be toxic in striated muscle, and Renilla hrGFP was billed as a potentially safer alternative for which no obvious toxicity was previously noted, we used hrGFP as a reporter in our first generation AAV6 miRNA shuttle vectors. 2,13,15 , 22,23,24,25,26 In our original proof-of-concept study using this vector system, we used constitutively active promoters (U6 and cytomegalovirus (CMV)) to co-deliver therapeutic or control miRNAs and hrGFP to muscles of newborn mice. 2 We found no overt evidence of vector toxicity in diseased or wt mouse muscles 4 months after injection of 1-day-old mice.…”

Skeletal muscle‐specific overexpression of heat shock protein 72 improves skeletal muscle insulin‐stimulated glucose uptake but does not alter whole body metabolism

“…A recombinant AAV vector plasmid containing a skeletal muscle‐specific creatine kinase promoter (pCK6), a gift of S. D. Hauschka to P. Gregorevic, and the cDNA construct for mouse Hspa1a (Figure C) was designed and generated using standard cloning techniques, and was used subsequently to prepare recombinant AAV6:Hsp72 viral vectors . Animals received either this vector (AAV:Hsp72) or a vector containing a multiple cloning site (MCS) as control (AAV:CON).…”