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Validation of gene transfer vectors containing tissue-specific promoters in cell-based functional assays poses a formidable challenge for gene therapy product development. Here, we describe a novel approach based on CRISPR/dCas9 transcriptional activation to achieve robust transgene expression from transgene cassettes containing tissue or cell type-specific promoters after infection with AAV vectors in cell-based systems. Guide RNA sequences targeting two promoters that are highly active within mammalian photoreceptors were screened in a novel promoter activation assay. Using this screen, we generated and characterized stable cell lines that co-express dCas9.VPR and top-performing guide RNA candidates. These cells exhibit potent activation of proviral plasmids after transfection or after infection with AAV vectors delivering transgene cassettes carrying photoreceptor-specific promoters. In addition, we interrogated mechanisms to optimize this platform through the addition of multiple guide RNA sequences and co-expression of the universal adeno-associated virus receptor (AAVR). Collectively, this investigation identifies a rapid and broadly applicable strategy to enhance in vitro expression and to evaluate potency of AAV vectors that rely upon cell or tissue-specific regulatory elements. Keywords: AAV, CRISPR, Cas9, dCas9, retina, photoreceptor

BackgroundVariants inPRPF31, which encodes pre-mRNA processing factor 31 homolog, are known to cause autosomal-dominant retinitis pigmentosa (adRP) with incomplete penetrance. However, the majority of mutations cause null alleles, with only two proven pathogenic missense mutations. We identified a novel missense mutation inPRPF31in a family with adRP.MethodsWe performed whole exome sequencing to identify possible pathogenic mutations in the proband of a family with adRP. Available affected family members had a full ophthalmological evaluation including kinetic and two-colour dark adapted static perimetry, electroretinography and multimodal imaging of the retina. Two patients had evaluations covering nearly 20 years. We carried out segregation analysis of the probable mutation,PRPF31c.590T>C. We evaluated the cellular localisation of the PRPF31 variant (p.Leu197Pro) compared with the wildtype PRPF31 protein.ResultsPRPF31c.590T>C segregated with the disease in this four-generation autosomal dominant pedigree. There was intrafamilial variability in disease severity. Nyctalopia and mid-peripheral scotomas presented from the second to the fourth decade of life. There was severe rod >cone dysfunction. Visual acuity (VA) was relatively intact and was maintained until later in life, although with marked interocular asymmetries. Laboratory studies showed that the mutant PRPF31 protein (p.Leu197Pro) does not localise to the nucleus, unlike the wildtype PRPF31 protein. Instead, mutant protein resulted in punctate localisation to the cytoplasm.Conclusionsc.590T>C is a novel pathogenic variant inPRPF31causing adRP with incomplete penetrance. Disease may be due to protein misfolding and associated abnormal protein trafficking to the nucleus.

Triple negative breast cancer (TNBC) remains one of the most lethal breast cancers while only accounting for 10-20% of all breast cancers. Mortality rates are a staggering 50%, with high likelihood of metastasis to other tissues if left untreated. This is due to this cancers heterogeneous nature and differentiation from other breast cancers, negatively staining for common mutations in estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor 2 (HER2). The patient population is shifted more towards African Americans (AA) with increased incidence and mortality rates. To date the nature of this statistic remains multifaceted with no clear therapeutic regiment. Through the identification of methylation as viable cause for TNBC, the exploration of environmental, genetic, and socioeconomic risk factors serve as an important aspect of overall mortality rate. This review seeks to investigate the relationship between AA with TNBC and potentially important DNA methylation markers that change in response to multiple risk factors. [Med-Science 2017; 6(4.000): 787-792]