Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression

Anna V Starikova,Maryana V Bardina,Denis A Reshetov,Svetlana G Vassilieva,Vladislav O Soldatov,Irina M Savchenko,Victoria V Skopenkova,Anna V Polikarpova,Oleg A Velyaev,Anna A Shmidt,Tatiana V Egorova

doi:10.1038/s41598-022-04892-x

Anna V Starikova, Maryana V Bardina + Show 9 more

Open Access

https://doi.org/10.1038/s41598-022-04892-x

Copy DOI

Abstract

High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (µDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Recent reports highlighted microutrophin (µUtrn) as a less immunogenic functional dystrophin substitute for gene therapy. In the current study, we created a human codon-optimized µUtrn which was subjected to side-by-side characterization with previously reported mouse and human µUtrn sequences after rAAV9 intramuscular injections in mdx mice. Long-term studies with systemic delivery of rAAV9-µUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to µDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized µUtrn overexpression. Furthermore, we verified that muscle-specific promoters MHCK7 and SPc5-12 drive a sufficient level of rAAV9-µUtrn expression to ameliorate the dystrophic phenotype in mdx mice. Our results provide ground for taking human codon-optimized µUtrn combined with muscle-specific promoters into clinical development as safe and efficient gene therapy for DMD.

Highlights

High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials
Duchenne muscular dystrophy (DMD) is a severe and progressive muscle-wasting disorder affecting one in 5000 boys[1]. It is caused by mutations in the DMD gene leading to the loss of dystrophin, a large structural protein located beside the sarcolemma
Studies utilizing microdystrophin vectors have previously demonstrated that codon optimization can significantly increase the level of transgene p rotein[22,23]

Summary

Introduction

High expectations have been set on gene therapy with an AAV-delivered shortened version of dystrophin (μDys) for Duchenne muscular dystrophy (DMD), with several drug candidates currently undergoing clinical trials. Safety concerns with this therapeutic approach include the immune response to introduced dystrophin antigens observed in some DMD patients. Long-term studies with systemic delivery of rAAV9μUtrn demonstrated robust transgene expression in muscles, with localization to the sarcolemma, functional improvement of muscle performance, decreased creatine kinase levels, and lower immunogenicity as compared to μDys. An extensive toxicity study in wild-type rats did not reveal adverse changes associated with high-dose rAAV9 administration and human codon-optimized μUtrn overexpression. Banks et al.[21] reported that rAAV6-CK8e-μUtrn improved dystrophic pathophysiology, with fiber-type expression preferences

Methods

Results

Conclusion