CRISPR/Cas9 is comprised of two components: the Cas9 nuclease and a small guide RNA transcript. level, sustained gene expression achieved with gene therapy vectors. We predict that this design concept will be highly transferrable to most genes in multiple model systems representing a facile cellular engineering platform for promoting gene expression. gene on chromosome 3. is likewise a prototypical large gene and spans ~31 kb and contains 118 exons with an open reading frame of ~9 kb [1,2]. RDEB causative mutations occur over the span of the gene and the resultant phenotype is characterized by diminished/absent type VII collagen (C7) protein causing mucocutaneous disease manifestations. Severe, chronic Cyclosporin A skin blistering occurs along with esophageal strictures, mitten deformities, dental anomalies, corneal scarring, and increased incidence for aggressive squamous cell carcinomas [3]. Therapeutic benefit can be achieved by the delivery of functional C7 protein. Sources of C7 include transplant of allogeneic or gene corrected autologous cells and/or recombinant C7 protein injection. Woodley and colleagues delivered recombinant C7 protein by intravenous injection showing that C7 produced locally or from a distance can mediate a functional benefit [4]. However, repetitive injections of recombinant peptide over the course of a patients lifetime are fiscally burdensome, Cyclosporin A making cellular sources an attractive option. Allogeneic cellular injections have resulted in improved skin integrity; however, the low expression levels of from the endogenous promoter results in poor delivery beyond the site of injection [5]. Further, allogeneic cells may not persist long term due to host immune-mediated clearance [6]. Autologous cellular engineering is highly promising due to the lowered risk of immune rejection, and gene expression has been restored in patient derived cells using gene therapy and gene editing [7,8]. To encode, deliver, and express gene expression. However, the large size of the cDNA can result in lowered titers that can make effective delivery a challenge [5,9,10,11,12]. Efforts have been undertaken to use less size-restricted platforms such as the phiC31 integrase, or Sleeping Beauty, transposon; however, the effective delivery of these vectors can similarly be challenging [5,13,14]. Additionally, the semi-random genomic integration profiles of these systems in the premalignant RDEB phenotype represents a significant safety concern due to insertional mutagenesis [15,16,17]. To capitalize on the precise targeting capabilities afforded by gene editing, we have targeted the gene with transcription activator like effector nucleases (TALEN) and the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system derived from [8,18]. Along with zinc finger nucleases and meganucleases, TALENs and CRISPR/Cas9 represent programmable reagents capable of generating single or double stranded DNA breaks at user-defined loci [19,20]. This stimulates homology directed repair (HDR) from an exogenous template allowing for Rabbit Polyclonal to AML1 (phospho-Ser435) precision genome modification. In situ gene correction maximizes safety but gene control is regulated by the comparatively weak promoter. As such, the systemic therapeutic impact may be incomplete due to the limited distribution of C7 protein. We hypothesized that we could synergize the attributes of gene therapy and gene editing: supraphysiological gene expression and a high degree of specificity. Previous efforts to accomplish this have centered on safe harbor site incorporation of a candidate gene driven by exogenous regulatory elements [21]. Delivering a cargo as large as the ~9 kb cDNA can be challenging making this approach sub-optimal. To address this, we devised a strategy whereby we could incorporate a powerful transcriptional activator into the native locus. This resulted in profound upregulation of the endogenous gene. Because our approach relies on a functional gene embedded in the genome, we pursued our strategy in cells with a favorable immunological profile in Cyclosporin A that they either innately, or can be engineered to, have a low frequency and incidence of immune-based side effects. Umbilical cord blood (UCB) derived hematopoietic stem cells (HSC) are effective for allogeneic therapy and display reduced rates of graft versus host disease (GVHD) [22,23]. Here we show robust gene activation in UCB HSCs with maintenance of their multi-lineage differentiation potential in colony forming assays. In parallel, we pursued T-cell engineering and observed expression levels that surpassed those of wild type keratinocytes. By subsequently ablating the T-cell receptor complex we generated a stable population of T-cells with a low risk of triggering.