I will speak specifically on the therapeutics platform epigenome therapies that we developed in our lab. The therapeutic platform we have been developing is based on all-in-one AAV vectors that comprise CRISPR-deactivated Cas fused with a synthetic repressor molecule and driven by an engineered microglia-specific promoter or regulatory sequence. The knowledge from our single-cell multiomics discovery informs us how to refine the next generation of the epigenome therapeutics and how to tailor it to repress APOE specifically and in the particular cell type of interest, which is microglia in this case...
I will speak specifically on the therapeutics platform epigenome therapies that we developed in our lab. The therapeutic platform we have been developing is based on all-in-one AAV vectors that comprise CRISPR-deactivated Cas fused with a synthetic repressor molecule and driven by an engineered microglia-specific promoter or regulatory sequence. The knowledge from our single-cell multiomics discovery informs us how to refine the next generation of the epigenome therapeutics and how to tailor it to repress APOE specifically and in the particular cell type of interest, which is microglia in this case. And for example, defining the candidate target regulatory sequences of our therapeutic platform, we perform by compiling a dataset from diverse populations, from African and European ancestry groups, a dataset that we have on single-cell multiomics from our lab, where we replicated the result that increased ApoE expression in the microglia cluster is associated with disease state. Then we look into the proximate regulatory element across both ancestries that regulate APOE expression, and we identified overlapping candidate cis-regulatory sequences. We use these sequences to design the guide RNA that navigates the therapeutic vector precisely to the element that regulates APOE expression in the microglia and in disease state. We also utilize knowledge coming from the multiomics discovery to direct the expression of the therapeutic vector specifically to the cell types that mediate the pathogenic effect, microglia in this case, particularly for the APOE-targeted therapies that we are developing. So when we evaluate the therapeutics vector in vitro using human-induced pluripotent stem cell-derived microglia, we demonstrate a significant microglia-specific reduction in APOE messenger RNA level, amounting to about 30% lower level compared to the control vector. The repression of the APOE E4, this cell is homozygous for E4 expression in this iPSC-derived microglia, resulted in a significant increase in A-beta clearance, such as the increase in A-beta uptake was very similar to that that we see in the isogenic APOE3 homozygous microglia cells. So in addition, when we evaluate inflammatory response and activated microglia marker, we validated that the reduction in APOE4 expression rescues neuroinflammation that characterizes this iPSC-derived microglia cell from a patient that was homozygous for the E4 allele. Then we move on to animal study. We use our humanized ApoE mouse model in which the entire mouse region of the ApoE was targeted and replaced with the human ApoE, including the flanking regulatory regions. The result of our study demonstrates a robust and efficient APOE repression also in vivo. Very interestingly, we saw differences between the effect in male and female mice. But the results are very promising to move forward this therapeutic, APOE-targeted therapeutics invention.
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