Construction and Development of a Cardiac Tissue-Specific and Hypoxia-Inducible Expression Vector

Abstract

Purpose: Cardiovascular gene therapy is a sophisticated approach, thanks to the safety of vectors, stable transgene expression, delivery method, and different layers of the heart. To date, numerous expression vectors have been introduced in biotechnology and biopharmacy industries in relation to genetic manipulation. Despite the rapid growth of these modalities, they must be intelligently designed, addressing the cardiac-specific transgene expression and less side effects. Herein, we conducted a pilot project aiming to design a cardiac-specific hypoxia-inducible expression cassette.Methods: We explored a new approach to design an expression cassette containing cardiac specific enhancer, hypoxia response elements (HRE), cardiac specific promoter, internal ribosome entry site (IRES), and beta globin poly A sequence to elicit specific and inducible expression of the gene of interest. Enhanced green fluorescent protein (eGFP) was sub-cloned by BglII and NotI into the cassette. The specificity and inducible expression of the cassette was determined in both mouse myoblast C2C12 and mammary glandular tumor 4T1 as ‘twin’ cells. eGFP expression was evaluated by immunofluorescence microscope and flow cytometry at 520 nm emission peak.Results: Our data revealed that the designed expression cassette provided tissue specific and hypoxia inducible (O2<1%) transgene expression.Conclusion: It is suggested that cardiac-specific enhancer combined with cardiac-specific promoter are efficient for myoblast specific gene expression. As well, this is for the first time that HRE are derived from three well known hypoxia-regulated promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter.

Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

“…26 (3) Several reports that used CRM did not compare CRM-containing vectors to vectors that differed only in lacking the CRM. [37][38][39]41 In one case, this comparison revealed that addition of a cardiomyocyte-speci c CRM to the cardiac troponin T promoter either had no effect or decreased transgene expression in mouse hearts. 40 (4) When compared to enhancer-promoter combinations that lack identi ed CRMs, CRM-containing vectors have not always yielded the highest levels of expression.…”

Transcriptional Targeting Approaches in Cardiac Gene Transfer Using AAV Vectors

“…The regulatory element mainly boosts the performance of the αMHC promoter. Combined with other (cardiac)-muscle-specific promoters like SPc5-12, cTnT, MLC2v, and SLN, the CS-CRM4 element predominantly increased the specificity of cardiac gene expression [9,36,37,57]. In a large-scale approach, a library comprising 400 bp sequences of potentially cardio-specific enhancer elements was screened for identification of novel enhancer sequences in mouse hearts in vivo using an AAV9-based assay [58].…”

Novel expression cassettes for increasing apolipoprotein AI transgene expression in vascular endothelial cells

“…The activity of the CRM in these initial reports was impressive, increasing in vivo transgene expression from plasmids and AAV vectors by up to 200-400-fold and preserving tissue specificity [16][17][18] . However, this early work on CRM-containing vectors from the Chuah/VandenDriessche group and others [28][29][30][31][32][33][34] also revealed limitations: (1) Some of the initially described CRM had no effect or decreased expression when placed upstream of the transthyretin promoter 16 . (2) CRM activity depends both on the identity of the adjacent promoter and on the transgene that is expressed, with up to 10-fold differences in CRM activity depending on the promoter [16][17][18]29 , and similar transgene-dependent variability 18 .…”

“…(2) CRM activity depends both on the identity of the adjacent promoter and on the transgene that is expressed, with up to 10-fold differences in CRM activity depending on the promoter [16][17][18]29 , and similar transgene-dependent variability 18 . (3) Several reports that used CRM did not compare CRM-containing vectors to vectors that differed only in lacking the CRM [29][30][31]33 . In one case, this comparison revealed that addition of a cardiomyocyte-specific CRM to the cardiac troponin T promoter either had no effect or decreased transgene expression in mouse hearts 32 .…”