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Budd A. Tucker, Kristin R. Anfinson, Jeanean L. Andorf, Luan M. Streb, Todd Scheetz, Robert F. Mullins, Edwin M. Stone; Targeting Human Mak Mutant iPSCs For In Vitro Gene Replacement. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2693.
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© ARVO (1962-2015); The Authors (2016-present)
The gene male germ cell associated kinase (MAK) encodes a protein involved in regulation of photoreceptor cilia length. By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we recently found that a mutation in exon 9 of the MAK gene resulted in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa. The purpose of this study was to determine if a lentiviral vector designed to drive expression of a retinal MAK transcript could be used to treat mutant human iPSCs and restore proper MAK expression and photoreceptor structure post-differentiation.
iPSCs were generated via viral transduction of human keratinocytes obtained from patients with MAK-associated retinitis pigmentosa using the transcription factors Oct4, Sox2, C-Myc and KLF4. iPSC potency was analyzed via ICC, WB, teratoma formation and embryoid body generation. Immunocytochemistry and rt-PCR analysis were used to detect transgene expression post-viral transduction.
Three separate patient specific lines of MAK mutant RP iPSCs each harboring the previously identified ALU insertion (Tucker et. al. 2011) were generated. Each of these lines were expanded in feeder free conditions and determined to be pluripotent based on immunocytochemical and rt-PCR pluripotency marker expression (Nanog, SSEA3, SSEA4, Tra-1-60, and Tra-1-81) and ability to generate tissues specific to all three germ layers (determined via teratoma and embryoid body formation). Full-length retinal MAK cDNA, generated via rt-PCR amplification from total human retinal RNA, was TA cloned and subsequently ligated into a lentiviral vector containing a GFP reporter driven under control of the EF1α promoter. MAK virus driven gfp expression could be detected at 1-week post-iPSC viral transduction. Restoration of the normal retinal MAK transcript could be detected via rt-PCR at this time.
We have successfully developed a MAK lentiviral based gene replacement strategy that has been validated in human iPSCs. This study demonstrates that in addition to their utility for cell replacement therapy, human iPSCs are also a valuable tool for analysis of the pathogenic mechanism of specific mutations and in vitro evaluation of gene replacement therapy.
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