Fig. 2

A, B rt-PCR analysis performed on RNA isolated from patient-specific photoreceptor precursor cells two-weeks following transduction with AAV5-CMV-MAK^CI, AAV5-CMV-MAK^RI, AAV5-EF1α-MAK^CI, or AAV5-EF1α-MAK^RI (MOI = 10⁴vg/cell). Compared to untransduced cultures (UT), which lack expression of wildtype exon-9-containing MAK transcript, cells transduced with each of the AAV5 constructs expressed exon-9-containing MAK transcripts. Only AAV5-CMV-MAK^RI and AAV5-EF1α-MAK^RI were capable of driving expression of exon-12-containing retinal MAK (B). C Western blot of patient-specific photoreceptor precursor cells transduced with AAV5-CMV-MAK^CI, AAV5-CMV-MAK^RI, AAV5-EF1α-MAK^CI, or AAV5-EF1α-MAK^RI (MOI = 10⁴vg/cell). Only the constructs driving MAK exon 12 restored expression of full-length retinal-specific MAK protein. D-G Immunocytochemical analysis of patient iPSC-derived photoreceptor precursor cells following AAV5 transduction using antibodies targeted against MAK and OTX2 (photoreceptor precursor cell marker). Although MAK was detected in cultures transduced with AAV5 vectors carrying the canonical isoform (AAV5-CMV-MAK^CI (D) and AAV5-EF1α-MAK^CI (F)), pronounced expression throughout the cell body and neurites was only detected in cultures transduced with vectors carrying the retinal isoform (AAV5-CMV-MAK^RI (E) or AAV5-EF1α-MAK^RI (G)). MAK – green, OTX2 – blue. Scale bars = 200 μm. Arrows denote rt-PCR primer positions.