ABSTRACT
Gene transfer-based therapeutic approaches have greatly benefited from the ability of some viral vectors to efficiently integrate within the cell genome and ensure persistent transmission of newly acquired transgenes to the target cell progeny. However, integration of provirus has been associated with epigenetic repercussions that may influence the expression of both the transgene and cellular genes close to vector integration loci. The exploitation of genetic insulator elements may overcome both issues through their ability to act as barriers that limit transgene silencing and/or as enhancer-blockers preventing the activation of endogenous genes by the vector enhancer. We established quantitative plasmid-based assay systems to screen enhancer-blocker and barrier genetic elements. Short synthetic insulators that bind to nuclear factor-I protein family transcription factors were identified to exert both enhancer-blocker and barrier functions, and were compared to binding sites for the insulator protein CTCF (CCCTC-binding factor). Gamma-retroviral vectors enclosing these insulator elements were produced at titers similar to their non-insulated counterparts and proved to be less genotoxic in an in vitro immortalization assay, yielding lower activation of Evi1 oncogene expression and reduced clonal expansion of bone marrow cells.
Subject(s)
Gene Transfer Techniques , Genetic Vectors/metabolism , Insulator Elements , NFI Transcription Factors/metabolism , Animals , Binding Sites , Bone Marrow Cells/cytology , Bone Marrow Cells/metabolism , CCCTC-Binding Factor , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Friend murine leukemia virus/genetics , Friend murine leukemia virus/metabolism , Gene Silencing , Genetic Vectors/genetics , HeLa Cells , Humans , MDS1 and EVI1 Complex Locus Protein , Mice , Mice, Inbred C57BL , NFI Transcription Factors/genetics , Plasmids/genetics , Plasmids/metabolism , Proto-Oncogenes/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptional Activation , Transfection , Transgenes , Virus IntegrationABSTRACT
Proteomic analyses encompass a suite of high-throughput technologies for large-scale separation and identification of proteins responsible for execution of physiological processes. As such, proteomics is ideally suited to dissecting developmental complexity and dynamics, an understanding of which is vital to the realization of regenerative therapeutic medicine. Pharmacoproteomics is increasingly targeting characterization of regenerative therapeutic strategies. A perspective on the application of proteomics to further our understanding of cardiac regenerative medicine, in concert with guided cardiogenic programming, is delineated herein.
