Genome-wide Computational Analysis Reveals Cardiomyocyte-specific Transcriptional Cis-regulatory Motifs That Enable Efficient Cardiac Gene Therapy

Abstract

Gene therapy is a promising emerging therapeutic modality for the treatment of cardiovascular diseases and hereditary diseases that afflict the heart. Hence, there is a need to develop robust cardiac-specific expression modules that allow for stable expression of the gene of interest in cardiomyocytes. We therefore explored a new approach based on a genome-wide bioinformatics strategy that revealed novel cardiac-specific cis-acting regulatory modules (CS-CRMs). These transcriptional modules contained evolutionary-conserved clusters of putative transcription factor binding sites that correspond to a “molecular signature” associated with robust gene expression in the heart. We then validated these CS-CRMs in vivo using an adeno-associated viral vector serotype 9 that drives a reporter gene from a quintessential cardiac-specific α-myosin heavy chain promoter. Most de novo designed CS-CRMs resulted in a >10-fold increase in cardiac gene expression. The most robust CRMs enhanced cardiac-specific transcription 70- to 100-fold. Expression was sustained and restricted to cardiomyocytes. We then combined the most potent CS-CRM4 with a synthetic heart and muscle-specific promoter (SPc5-12) and obtained a significant 20-fold increase in cardiac gene expression compared to the cytomegalovirus promoter. This study underscores the potential of rational vector design to improve the robustness of cardiac gene therapy.

Enhancing atrial‐specific gene expression using a calsequestrin cis‐regulatory module 4 with a sarcolipin promoter

“…Various enhancer sequences have been reviewed, although CRM4 was the most promising candidate as a result of its superior cardiac specificity. Thus, we have manipulated the promoter by adding CRM4 , a cardiomyocyte‐specific enhancer, to improve transcriptional regulation of SLN …”

“…10 With the current research on transcriptional regulation, we designed a combined a novel combination with synergistic effects to obtain enhanced atrial specificity. 21 Various enhancer sequences have been reviewed, [26][27][28][29][30] although CRM4 was the most promising candidate as a result of its superior cardiac specificity. Thus, we have manipulated the promoter by adding CRM4, a cardiomyocyte-specific enhancer, to improve transcriptional regulation of SLN.…”

“…Thus, we have manipulated the promoter by adding CRM4, a cardiomyocyte-specific enhancer, to improve transcriptional regulation of SLN. 21 In the present study, we evaluated an atrial-specific promoter sequence enhanced by a cis-regulatory module in vitro and in vivo. Initially, to evaluate promoter efficiency, we observed expression in HL1 cells and confirmed that the CRM4 within the gene cassette significantly improved luciferase activity driven by the SLN promoter in atrial cells (p < 0.005) ( Figure 3A).…”

“…Alone, the SLN promoter activity is unsubstantial compared to that of ubiquitous promoters. Thus, the addition of calsequestrin 2 cis‐ regulatory module 4 ( CRM4 ), a cardiomyocyte‐specific enhancer, which showed superior activity in the heart, was used to heighten transgene expression of SLN within the heart …”

“…Thus, the addition of calsequestrin 2 cis-regulatory module 4 (CRM4), a cardiomyocyte-specific enhancer, which showed superior activity in the heart, was used to heighten transgene expression of SLN within the heart. 21 In addition to cardiac tissue, high SLN levels are found in the skeletal muscle and diaphragm. 22,23 The addition of CRM4 was thus hypothesized to improve cardiac specificity.…”