626. Directing Skeletal Myogenic Progenitor Cell Lineage Specification with CRISPR/Cas9 Transcriptional Activators

Abstract

Induced pluripotent stem cells (iPSCs) are a promising source for autologous cell-based therapies, disease modeling, and drug discovery in pathologies of muscular disease and wasting. The field of regenerative medicine has made significant strides in the successful reprogramming of iPSCs into various specialized cell types, including skeletal muscle cells. Genetic reprogramming of iPSCs into skeletal muscle progenitors and myocytes has been previously demonstrated by cDNA expression of exogenous myogenic transcription factors, including Pax7 and MyoD, to achieve varying levels of conversion toward the myogenic lineage.Recent advances in genome engineering technologies have established the CRISPR/Cas9 system as a programmable transcriptional regulator capable of targeted activation or repression of endogenous genes. The nuclease-deactivated dCas9 protein can be fused to a variety of transcriptional activation domains, such as the histone acetyltransferase p300 and the transactivation domain VP64, to potently activate genes in their natural chromosomal context. In contrast to ectopic expression of transgenes, activation of endogenous genes can facilitate chromatin remodeling and can also capture the full complexity of transcript isoforms, mRNA localization and stability, and other effects of non-coding regulatory elements.Here, we use VP64dCas9VP64 and dCas9-p300 constructs for targeted activation of the endogenous myogenic transcription factor Pax7 in human iPSCs to direct differentiation into skeletal muscle progenitors. We hypothesize that Pax7 activation will be sufficient to induce the myogenic program, as it plays a key role in myogenesis through regulation of muscle regeneration and the function of muscle progenitor cells. Lentiviral transduction of the dCas9 transcriptional activators under a constitutive promoter along with gRNAs targeted to the Pax7 promoter resulted in increased Pax7 transcript levels as assessed by qRT-PCR at 4 days after transduction. Additionally, widespread expression of Pax7 protein was detected by immunofluorescence staining by 13 days post transduction, indicating a high efficiency of endogenous gene activation by dCas9 effector constructs. Current efforts aim to achieve transient induction of high levels of Pax7 to examine progressive differentiation into mature myotubes expressing downstream myogenic markers. Future experiments will focus on assessing the in vivo skeletal muscle regenerative potential of Pax7+ human iPSC-derived myogenic progenitor cells by examining engraftment and self-renewal in injured muscle tissues of immunodeficient mice. These studies aim to introduce a novel method for the ex vivo derivation and expansion of myogenic precursor cells from patient-derived iPSCs, which will create new opportunities for disease modeling and cell therapies in disorders of skeletal muscle regeneration including muscular dystrophies, sarcopenia, and cachexia.