Linking Order of RAX and OTX2 Following CRX in Polycistronic Vectors Can Partially Determine Efficacy of Direct Conversion to Photoreceptor-like Cells In Vitro.

Redirecting differentiation of somatic cells by overexpression of transcription factors can be a promising, simple, low-cost approach to generate target cells from somatic cells. Our research group previously demonstrated that a combination of CRX, NeuroD1 and RAX is necessary to reprogram photoreceptor-like cells (PRLs) from human dermal fibroblasts, where OTX2 plays a role as an amplifier. However, relatively low induction-efficiency and heterogeneity in maturity of induced PRLs and cellular damage by the retroviral-based delivery of those transcription factors are problematic. Polycistronic vectors consisting of four transcription factors, CRX (C), RAX (R), NeuroD1 (N) and OTX2 (O) in place of the previously used mixture of monocistronic vectors, may overcome such limitations. Three kinds of polycistronic retroviral vectors including genes of four transcription factors in different orders, CNOR, CNRO or CRNO, or a mixture of monocistronic retroviral vectors for the four transcription factors, were transduced into a commercially available human dermal fibroblast cell line, HDF-a. Expression levels of transcription factors were assessed by immunocytochemistry (ICC). Induction efficiency in PRLs was assessed using qRT-PCR and calcium imaging. Expression levels of the four transcription factors were not significantly different among cells transduced with pMXs-CNOR-IP, pMXs-CNRO-IP and pMXs-CRNO-IP by ICC. Expression of blue opsin, S-antigen, GNAT2, PDE6C, CNGA3 and recoverin was examined at post-transduction day (PTD) 10, 14 and 28 using qRT-PCR. All genes were clearly expressed in cells transduced with pMXs-CNRO-IP and pMXs-CRNO-IP vector-transduced cells, while S-antigen, PDE6C and GNAT2 were expressed at low levels in pMXs-CNOR-IP vector-transduced cells throughout the period we examined. Ca2+ influx was significantly higher in PRLs induced by any of the three polycistronic vectors than in non-transduced cells. The order of transcription factors on polycistronic vectors can affect the expression levels of photoreceptor-related genes. CNRO or CRNO, may be preferable to CNOR for photoreceptor induction, suggesting that the order of RAX and OTX2 on polycistronic vectors might be critical for the control of induction efficacy of PRLs in vitro. Further study is warranted to conclude whether these transcriptional factors interact with each other when they are expressed in polycistronic vectors.