Cincinnati Children's Hospital Medical Center
333 Burnet Avenue
Cincinnati, OH 45229
Gene Therapy using Gene Editing
Current genetic therapies use viral vectors to insert a therapeutic gene randomly in the genome. However, with the advent of CRISPR/Cas9, we can edit the genome at specific sites, using a ‘fix and run’ approach. Gene editing has the potential to correct a wide range of genetic diseases. The gene editing nuclease Cas9 can be targeted to create chromosomal double strand breaks, that the cell repairs either using an error-prone non-homologous/microhomology-mediated end-joining (NHEJ/MMEJ) DNA repair pathways, or by homologous recombination (HR), that fixes the disease mutation seamlessly. Currently, NHEJ repair far exceeds HR-mediated repair. We have developed a novel Cas9 that suppresses NHEJ where Cas9 cuts DNA, so that HR occurs. We seek funding to refine this technology to a level that can fix a variety of disease mutations that lead to blood disorders at a clinically relevant level. These studies will have wide application for all human genetic diseases.
Program Goals:
We hypothesized that if error-prone repair was specifically inhibited only at the DSB generated by Cas9, HR efficiency and fidelity would be enhanced without compromising genome integrity, thereby promoting cellular homeostasis, cellular viability and clinical safety. Specifically, we will utilize a dominant negative (DN) version of 53BP1 fused to Cas9, to block NHEJ events only at the Cas9 cut site. 53BP1, a DNA repair adaptor protein, binds altered chromatin at DSB, and recruits all downstream NHEJ pathway proteins, and additionally inhibits DNA end-resection that is required for initiation of HR. We will specifically,
Aim 1: Determine the ability of the DN53BP1-Cas9 fusion to specifically block NHEJ at Cas9-induced DSBs. We will compare the localization and function of DN-53BP1 alone, DN-53BP1-Cas9 fusion, or DN1-53BP1 fused to a catalytically dead Cas9 (DN53BP1-dCas9) and WT 53BP1-Cas9 fusion.
Aim 2: Determine the ability of DN53BP1-Cas9 to improve HR in human HSC. Here, we will determine if DN-53BP1-Cas9 skews the DNA-repair pathway choice towards HR in human HSC in vitro and in xenografts, and assess its toxicity and safety.
Aim 3: Determine if inhibition of MMEJ, along with site-specific NHEJ inhibition with DN-Cas9 further increases HR in human HSC. We propose PolQ inhibition to augment HR by DN-Cas9.
Note: This grant is Part 1 of a five-year, $250,000 commitment. The Dater Foundation has made similar commitment for two decades.
Website:
http://www.cincinnatichildrens.org Amount: $50,000
Date: June 2022
