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

Abstract

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.

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells

“…As a global messenger, H 2 O 2 affects many biological processes beyond oxidative stress response, such as cell metabolism, cellular fate, immunity and homeostasis [31], [32]. The mechanism shown here for the generation of O 2 •– and H 2 O 2 by fluorescent proteins explains the cytotoxicity and tissue abnormalities seen in cells and animals expressing fluorescent proteins [10], [11], [12], [15], [16]. Given the extensive use of fluorescent proteins, especially eGFP, in studies of different human diseases (Supplemental Data Table S1) and the known role of O 2 •– and H 2 O 2 in the pathophysiology of those conditions, we call for a careful evaluation of experimental design, validation and interpretation of results in investigations using fluorescent proteins.…”

EGFP reporter protein: its immunogenicity in Leishmania-infected BALB/c mice

“…Although there are some reporters about the toxic effect of EGFP expression in some cells (such as muscle (Wallace et al 2013), Ku80-deficient hamster cells (Koike et al 2013), retina (Rex et al 2004), mouse fibroblast, hamster kidney cell and Huh-7 cells from hepatoma cancer (Liu et al 1999), adult stem cells from rat hepatic (Taghizadeh and Sherley 2008), but recombinant parasites encoding reporters as EGFP have received great attention and are routinely used (Breton et al 2005;Costa Sdos et al 2011;Kamau et al 2001;Mehta et al 2008;Singh et al 2009;Varela M et al 2009) without adverse effects on parasite.…”

Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

“…To test vector potency, we packaged the D4Z4.V5.pLAM provirus into AAV serotype 6 (AAV6) capsids [ 22 ], and injected 3 x 10 10 particles (reported as DNAse resistant particles, or DRP) into tibialis anterior (TA) muscles of wild-type C57BL/6 mice. We also delivered 3 x 10 10 particles of AAV.CMV.GFP vectors [ 23 ], or saline, in separate animals to serve as injection controls. In contrast to the massive muscle lesions and subsequent widespread regeneration induced by CMV-based DUX4 vectors ( Fig.…”

“…All were packaged with AAV6 capsids as previously described [ 8 ]. Vectors carrying CMV.eGFP, CMV.DUX4.V5 and CMV.DUX4.HOX.V5 expression cassettes were previously described [ 8 , 23 ]. The D4Z4.V5 and D4Z4.V5.2 vectors were newly constructed for this work.…”