TY - JOUR
T1 - Dose-dependent toxicity of humanized Renilla reniformis GFP (hrGFP) limits its utility as a reporter gene in mouse muscle
AU - Wallace, Lindsay M.
AU - Moreo, Andrew
AU - Clark, K. Reed
AU - Harper, Scott Q.
N1 - Funding Information:
We thank Louise Rodino-Klapac for assistance with the Bioquant software package. Funding for the Harper Lab that enabled this study came from the National Institutes of Health (National Institute of Arthritis and Musculoskeletal and Skin Diseases, 1R01AR062123 to S.Q.H.; National Institute of Neurological Disorders and Stroke R21NS072260 and 1R21NS078327 to S.Q.H.; National Institutes of Health KL2 Clinical and Translational Scholar Award KL2 RR025754 to S.Q.H.); the FSHD Global Foundation (to S.Q.H.); The FSH Society (to S.Q.H.); and the Muscular Dystrophy Association (grant no. 4358 to S.Q.H.). L.M.W. is a fellow on the Muscle Disease and Biology National Institutes of Health T32 Training Grant at Ohio State University/Nationwide Children's Hospital. The authors declared no conflict of interest.
PY - 2013
Y1 - 2013
N2 - Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×1010 -1 × 1011 particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 109 AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.
AB - Gene therapy has historically focused on delivering protein-coding genes to target cells or tissues using a variety of vectors. In recent years, the field has expanded to include gene-silencing strategies involving delivery of noncoding inhibitory RNAs, such as short hairpin RNAs or microRNAs (miRNAs). Often called RNA interference (RNAi) triggers, these small inhibitory RNAs are difficult or impossible to visualize in living cells or tissues. To circumvent this detection problem and ensure efficient delivery in preclinical studies, vectors can be engineered to coexpress a fluorescent reporter gene to serve as a marker of transduction. In this study, we set out to optimize adeno-associated viral (AAV) vectors capable of delivering engineered miRNAs and green fluorescent protein (GFP) reporter genes to skeletal muscle. Although the more broadly utilized enhanced GFP (eGFP) gene derived from the jellyfish, Aequorea victoria was a conventional choice, we were concerned about some previous studies suggesting this protein was myotoxic. We thus opted to test vectors carrying the humanized Renilla reniformis-derived GFP (hrGFP) gene, which has not seen as extensive usage as eGFP but was purported to be a safer and less cytotoxic alternative. Employing AAV6 vector dosages typically used in preclinical gene transfer studies (3×1010 -1 × 1011 particles), we found that hrGFP caused dose-dependent myopathy when delivered to wild-type (wt) mouse muscle, whereas identical titers of AAV6 carrying eGFP were relatively benign. Dose de-escalation at or below 8 × 109 AAV particles effectively reduced or eliminated hrGFP-associated myotoxicity, but also had dampening effects on green fluorescence and miRNA-mediated gene silencing in whole muscles. We conclude that hrGFP is impractical for use as a transduction marker in preclinical, AAV-based RNA interference therapy studies where adult mouse muscle is the target organ. Moreover, our data support that eGFP is superior to hrGFP as a reporter gene in mouse muscle. These results may impact the design of future preclinical gene therapy studies targeting muscles and non-muscle tissues alike.
KW - AAV
KW - EGFP
KW - HrGFP
KW - Muscle toxicity
UR - https://www.scopus.com/pages/publications/84878849131
U2 - 10.1038/mtna.2013.16
DO - 10.1038/mtna.2013.16
M3 - Article
C2 - 23591809
AN - SCOPUS:84878849131
SN - 2162-2531
VL - 2
SP - e86
JO - Molecular Therapy - Nucleic Acids
JF - Molecular Therapy - Nucleic Acids
M1 - e86
ER -