Date Published: March 18, 2018
Publisher: Tabriz University of Medical Sciences
Author(s): Shahrooz Ghaderi, Neda Alidadiani, Jafar Soleimani Rad, Hamid Reza Heidari, Nafi Dilaver, Behzad Mansoori, Reza Rhabarghazi, Rezayat Parvizi, Vahid Khaze Shahgoli, Behzad Baradaran.
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.
Since identification and description of hypoxia-inducible factor 1 (HIF-1) by Wang et al.,1 more than 200,000 articles have been published in scientific literatures. HIF-1 is an inducible transcription factor, which binds to hypoxia response elements (HREs) or enhancer elements during hypoxia. HREs are located in the upstream of promoter region. HIF-1 regulates several genes, such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO).2 hypoxia inducible factor-1 alpha (HlF-1) is comprised of a heterodimer basic helix-loop-helix (bHLH) transcriptional complex and is divided into distinct subsets, including HIF-1α and aryl hydrocarbon receptor nuclear translocator (ARNT), encoding a protein that is referred to beta (β) subunit. HIF-1 belongs to a conserved subfamily of PER-ARNT-SIM (PAS), which functions as oxygen sensors. The PAS domain is a subfamily of bHLH, which is a transcription factor. The HIF family consists of HIF-1α, -1β, -2α, -2β, -3α, -3β.3,4 In normoxia, HIF-1α is degraded by ubiquitin-mediated proteolysis activity, while being transcribed into mature RNA under hypoxic conditions, leading to an increase in oxygen delivery to the tissues. Therefore, HIF-1α has an essential role both in physiologic and pathologic conditions, including, but not limited to, myocardial ischemia, coronary artery disease, organ rejection and some cancers.5,6
The results obtained through the experiments support some of our hypotheses. The current study explored the application of bioinformatics to improve the efficiency of cardiac gene therapy. Based on in silico analysis, we chose a cardiac specific cis-regulatory conserved motif that belonged to calsequestrin 2 (CASQ2) gene. The corresponding gene is specifically expressed in the heart. Tissue-specific cis-regulatory elements and correlated transcription factors, including myogenic regulatory factors (MRFs) like myogenic differentiation 1 (MYOD1), myogenic factor 5 (MYF5), myogenin and myogenin regulator factor 4 (MRF4), myogenic factor 6 (MYF6) interact with other transcription factors, particularly T box protein 2 (TBX2) and NK2 homeobox 5 (Nkx2.5).,21-24 which play a pivotal role in the correct differentiation and progressive formation of cardiac muscle (Table 3). This summary helps us to understand the role of specific cis-regulatory elements in designing tissue-specific expression cassettes. Both CASQ2 and MLC2v have a specific transcription factor binding site (TFBSs), which is efficient for cardiac-specific transgene expression.
In conclusion, combination of the cardiac/muscle specific cis-regulatory elements, CASQ2, and myosin light chain-2 (MLC2v) have a significant specificity for myocytes. Both CASQ2 and MLC2v have a specific TFBS, which is efficient for cardiac-specific transgene expression. Sp1 binding sites are located near the HRE regions on the promoter. Therefore, Sp1 and HIF-1α have binding sites to a far distance of HREs derived from three promoters. Therefore, there is no longer need to overlap PCR process for one repeated sequence just in one promoter. This expression cassette well-designed for cardiac specific hypoxia inducible gene expression.
This work was a part of Ph.D. thesis and supported by the Research Council, Tabriz University of Medical Sciences. The authors thank the personnel of Immunology Research Center, Department of Pharmaceutical biotechnology for their kind guidance.
The authors declare “no” conflict of interest.
HREs: hypoxia response elements, HIF-1α: hypoxia inducible factor-1 alpha, VEGF-A: vascular endothelial growth factor-A, PGK-1: phosphoglycerate kinase 1, MLC2v: myosin light chain, EPO: erythropoietin, ARNT: aryl hydrocarbon receptor nuclear translocator, eGFP: enhanced green fluorescent protein, CASQ2: calsequestrin 2, bHLH: basic helix-loop-helix , PAS: PER-ARNT-SIM, EMCV: encephalomyocarditis virus, UTR: untranslated region, IRES: internal ribosome entry site, MRFs: myogenic regulatory factors, MYOD1: myogenic differentiation 1, MYF5: myogenic factor 5, MYF6: myogenic factor 6, MHC-α: myosin heavy chain- alpha, TBX2: T box protein 2, NKX2-5: NK2 homeobox 5.