Long-Term Functional Correction of Pompe Disease and Increased α-Glucosidase Expression after Gene Therapy with Novel Combinations of Muscle-Targeted Transcriptional <i>Cis</i> -Regulatory Elements

The development of efficient and safe muscle-directed gene therapy is an unmet medical need. One of the bottlenecks in muscle-directed gene therapy is the high levels of muscle-targeted transcription required in these afflicted target tissues. To circumvent this problem, novel transcriptional cis-regulatory elements (CREs) were identified by transcriptome-wide data-mining that led to a significant increase of transgene expression in skeletal muscle, heart, and diaphragm after adeno-associated viral vector 9 (AAV9) gene transfer. The expression achieved with this CRE arrays outperformed that obtained with several quintessential muscle-targeted promoters, such as the synthetic SPc5-12 and MHCK7 promoters, used in various muscle-targeted gene therapy clinical trials. Incorporation of these CRE arrays led up to a robust 20- to 30-fold increase in luciferase reporter gene expression when compared with the SPc5-12 and MHCK7 promoters. To validate their therapeutic efficacy, AAV9 vectors containing CREs and encoding α-glucosidase (<i>GAA</i>) were administered to <i>GAA</i>-/- knockout mice that mimic Pompe disease (glycogen storage disease type II) in human subjects. The CRE arrays resulted in a significant 25-fold increase in GAA protein and <i>GAA</i> mRNA expression in different skeletal muscles, leading to GAA activity levels comparable with those of wild-type mice. Subsequently, this led to a significant decrease in glycogen accumulation and a restoration of centronuclear localization similar to those of wild-type levels. Most importantly, long-term correction of skeletal muscle, diaphragm, and cardiac function was achieved in <i>GAA</i>-/- knockout mice treated with the CRE-containing AAV9 vectors yielding normal phenotypes indistinguishable from wild type. This robust phenotypic correction was demonstrated based on grip and hanging tests, cardiac conductance assays as reflected by PR interval prolongation, and diaphragm contractility function tests. The current study has broad implications for improving outcomes of future clinical trials in Pompe patients and other genetic disorders that affect skeletal muscle, heart, and diaphragm.