Investigator: Afrooz Rashnonejad PhD
Category: Research - Basic
Advancements in CRISPR technology led to a revolution in molecular biology that provided mechanisms to precisely correct desired DNA sequences, thereby paving the way for developing precision gene therapies. The traditional CRISPR-Cas9-mediated gene editing approaches for genetic diseases rely upon cutting mutated DNA sequences at specific chromosomal positions and repairing or replacing edited regions with a new DNA sequence. Ideally, a CRISPR-Cas9 system would be engineered to cut the genome only in the intended location, thereby correcting only the disease-causing lesion while avoiding cutting the genome elsewhere.
Although this method can be highly specific, off-target effects remain a concern, as the human genome contains 3 billion pieces of code, and even a minuscule error rate could cause numerous permanent and unintended genetic changes. This issue of precision is more difficult to achieve with FSHD, which is caused by expression of the toxic DUX4 gene in muscle. The most straightforward method for treating FSHD is to turn the DUX4 gene ‘off’ or mutate it so it is non-functional. However, hundreds of identical or nearly identical DUX4 copies are embedded throughout the human genome within DNA repeats (called D4Z4), and this similarity in DNA sequence makes it challenging to make a precise CRISPR-Cas9 system that would disrupt only the DUX4 copy associated with FSHD. In other words, a DUX4-targeted CRISPR-Cas9 system could potentially cut in hundreds of places genome-wide, or produce shorter arrays of D4Z4 repeats that could actually lead to more – not less - DUX4 being produced in muscles. Thus, extreme care needs to be employed to develop a CRISPR-Cas9 method to treat FSHD.
Instead of using the more traditional CRISPR-Cas9 system to silence the DUX4 gene, here we propose to develop an alternative CRISPR system – based on the Cas13 enzyme – that can be targeted to the DUX4 mRNA instead of DNA. We hypothesize that Cas13-mediated DUX4 mRNA silencing holds advantages because it would still accomplish DUX4 gene silencing using CRISPR but would not nick the genome in potentially hundreds of places. Thus, the goal of this project is to develop a pre-clinical gene therapy approach to silence DUX4 mRNA using CRISPR/Cas13 in human cells and mouse muscles. We have strong preliminary in vitro data showing that loaded Cas13 targets DUX4 transcripts, significantly reduces toxic DUX4 protein and enhances cell viability. Here we propose to further develop this approach in vivo by creating a gene therapy vector that will knockdown DUX4 in an animal model. Upon completion of the aims of this study, we expect to produce pre-clinical data supporting the translation of new AAV-based CRISPR/Cas13 therapy for FSHD.