Next-generation muscle-directed gene therapy by in silico vector design

S Sarcar,M Sampaolesi,M K Chuah,P De Bleser,P In’T Veld,G Dickson,W Tulalamba,S Jarmin,M Loperfido,J Tipanee,M Keyaerts,E Berardi,M Y Rincon,H Evens,E Samara-Kuko,T Lahoutte,D Boon,H Q Pham,T Vandendriessche

doi:10.1038/s41467-018-08283-7

Abstract

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. These vectors should express substantially higher levels of the therapeutic transgene, enabling the use of lower and safer vector doses. In the current study, we identify potent muscle-specific transcriptional cis-regulatory modules (CRMs), containing clusters of transcription factor binding sites, using a genome-wide data-mining strategy. These novel muscle-specific CRMs result in a substantial increase in muscle-specific gene transcription (up to 400-fold) when delivered using adeno-associated viral vectors in mice. Significantly higher and sustained human micro-dystrophin and follistatin expression levels are attained than when conventional promoters are used. This results in robust phenotypic correction in dystrophic mice, without triggering apoptosis or evoking an immune response. This multidisciplinary approach has potentially broad implications for augmenting the efficacy and safety of muscle-directed gene therapy.

Highlights

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials
To design robust muscle-specific gene therapy vectors, we relied on a multistep computational approach (Fig. 1a) that allowed us to identify novel evolutionary conserved skeletal muscle-specific cis-regulatory modules associated with genes that are highly expressed in the skeletal muscle (Table 1 and Supplementary Table 1)
The increased therapeutic efficacy with the next-generation adenoassociated viral vectors (AAVs) vectors expressing the micro-dystrophin 1 (MD1) and FST therapeutic genes from the Sk-CRM4/Des chimeric promoter was consistent with the increase in MD1 and FST expression. These results suggested that the use of the Sk-CRM4/Des chimeric promoter provided an effective and sustained therapeutic effect that is significantly more robust compared to conventional promoters typically used for Duchenne muscular dystrophy (DMD) gene therapy

Summary

Introduction

There is an urgent need to develop the next-generation vectors for gene therapy of muscle disorders, given the relatively modest advances in clinical trials. This multidisciplinary approach provides new insights into regulatory motifs and their relative strength in conferring muscle-specific transcriptional control These novel muscle CRMs increase expression of micro-dystrophin (MD1)[16,17] and follistatin (FST344), a known myostatin inhibitor[32], after gene therapy with serotype 9 adeno-associated viral vectors (AAV9)[33,34]. This results in sustained phenotypic correction in dystrophic mice without any discernable immune complications

Methods

Results

Conclusion