Abstract: Although associations between DNA demethylation and gene expression were established four decades ago, the causal role of DNA demethylation in activation of gene expression remains unresolved. Different strategies to address this question were developed; however, confounds involved in these approaches may complicate the disentangling of cause and effect. We developed here a highly effective new method using only nuclease-dead Cas9 (dCas9) and gRNA site-specific targeting to physically block DNA methylation at specific targets in the absence of a confounding flexibly-tethered enzymatic activity, which is common with flexibly-tethered TET.dCas9 constructs, enabling examination of the role of DNA demethylation per se in living cells. We find no evidence of off-target DNA demethylation as a consequence of gRNA binding. We show that the extensive induction of gene expression achieved by previously described TET/dCas9-based targeting vectors is confounded by DNA methylation-independent activities, inflating the role of DNA methylation in the promoter region. Using this new method, we probe a small number of inducible promoters and find that in these instances the main effect of DNA methylation is silencing basal promoter activity. Thus, the effect of demethylation of the promoter region in the tested genes is small, while induction of gene expression by several pharmacological inducers is large and independent of DNA methylation. In contrast, targeting demethylation to the pathologically silenced FMR1 gene elicits more robust induction of gene expression. Once applied to a comprehensive set of genomic regions, this new method could be used to reveal the true extent, nature, and diverse contribution of DNA methylation to gene regulation. Moreover, this method could potentially be applied to site specific demethylation of silenced genes for therapeutic purposes in future (epi)gene therapy.

Abstract: In addition to discussing some of the ways people conceptualize the applications of gene editing and the subsequent impact on permissibility. We will discuss the impact of human germline editing. We will examine the decisional challenges of genetic editing, the potential ethical dilemmas, the economic and societal impacts and the realistic approaches to public discourse

Abstract: Translational control of gene expression is an important regulator of adult stem cell quiescence, activation and self-renewal. In skeletal muscle, quiescent muscle stem cells maintain low levels of protein synthesis, mediated in part through the phosphorylation of eIF2a (P-eIF2a). Muscle stem cells unable to phosphorylate eIF2α exit quiescence, activate the myogenic program, and differentiate, but do not self-renew. P-eIF2α ensures in part the robust translational silencing of accumulating mRNAs that is needed to prevent the activation of muscle stem cells. Additionally, P-eIF2α-dependent translation of mRNAs regulated by upstream open reading frames (uORFs) contributes to the molecular signature of stemness. Pharmacological inhibition of eIF2α dephosphorylation enhances skeletal muscle stem cell self-renewal and regenerative capacity as revealed by engraftment into a preclinical mouse model of Duchenne muscular dystrophy.

Abstract: Approximately 40% of people will be diagnosed with cancer at some point during their lifetimes. Cancer is not a single disease, rather it represents the disease conditions sharing similar properties created by the intimate interaction between malignant cells and their environment.  Although our understanding of cancer has been advanced for the last few decades and new therapies have developed, we are still far from conquering cancer.

Animal cancer modeling has been an important part of cancer research. Various types of cancer models have been developed such as germline genetically modified cancer models, cancer cell lines transplant models and Patient-derived Xenograft (PDX) models. Each has advantages and limitations. Development of CRISPR-mediated genome editing created new opportunities to generate novel cancer mouse models, somatic genetically modified cancer models. My group has recently developed a unique strategy to generate mouse ovarian cancer models using a combination of in vivo fallopian tube electroporation, Cre-mediated lineage tracing and CRISPR mediated genome editing. Our strategy has high flexibility in gene mutation combinations, targeting areas and the number of cells electroporated. Cre-mediated lineage tracing and CRISPR-mediated retrospective lineage tracing allow us to track micrometastasis and clonal evolution, respectively, in immune competent animals. Thus, somatic cancer modeling is a novel powerful strategy to dissect out the intimate interaction between malignant cells and their environment.

Abstract: Cellular therapies have made immense progress over the last decade, hoped to provide unique opportunities to target a host of diseases. However, the development of stem cell derived authentic cellular therapy poses unique challenges to fulfill these promises and bring such options to the clinic. During this presentation, we will take a look at how Bluerock is tackling these challenges, with the example of the clinical development of stem-cell derive dopaminergic neurons for the treatment of Parkinson’s Disease.