CRISPR technology provides an avenue for targeting and correcting virtually any sequence in the human genome, with long-term or permanent beneficial effects. Thus, it has the potential to cure the root cause of a disease such as FSHD, rather than merely treating symptoms. Although typically used for gene editing (in which DNA is cut and replaced, allowing conversion of a disease-causing mutation to the normal sequence), CRISPR can also be used to change the expression level of a disease-causing gene (e.g., CRISPR inhibition turns a gene off) without cutting the genome. FSHD is caused by mis-expression of the DUX4 gene in skeletal muscle, and could potentially be cured by either CRISPR editing or CRISPR inhibition. Because FSHD is associated with highly repetitive DNA sequences, there are a number of concerns with a CRISPR editing approach that introduces many cuts into the genome. CRISPR inhibition circumvents these problems and is a viable approach for FSHD. The goal of this project is to test the efficacy, specificity, and stability of this approach in two novel mouse models of FSHD, including a xenograft model that will allow us to study the effects of CRISPR inhibition in human muscle derived from FSHD patient cells. This project is being jointly supported by the FSHD Global Research Foundation and FSHD Canada Foundation.

PROGRESS REPORTS

Update September 2019

Grant 47 – Pre-clinical testing for FSHD CRISPR-inhibition therapy.

CRISPR technology provides an avenue for targeting and correcting virtually any sequence in the human genome, with long-term or permanent beneficial effects. Thus, it has the potential to cure the root cause of a disease such as FSHD, rather than merely treating symptoms. In the CRISPR inhibition (CRISPRi) system, a guide RNA (sgRNA) recruits an enzymatically dead version of the Cas9 protein fused to a transcriptional repressor (dCas9-KRAB) to a desired genomic location. We have successfully used this system to repress pathogenic expression of the DUX4 gene in human FSHD muscle cells in culture. This proposal funded by FSHD Global Research Foundation will take FSHD CRISPRi to the next level and closer to the clinic. The goal is to test the efficacy, specificity and stability of this approach, first in primary lines of human FSHD myocytes (Aim 1) and then, using the best combination of CRISPRi, in two novel mouse models of FSHD (Aim 2). We will be using the FSHD-like FLExDUX4 mouse, created by the Jones lab, that will allow us to investigate systemic and long-term inhibition, and a xenograft model, created by the Bloch lab, that will allow us to study the effects of CRISPR inhibition in human muscle derived from FSHD patient cells.

In order to take CRISPRi to the clinic, we need to be able to fit all the DNAs into the small available genome of adeno-associated virus (AAV) for systemic delivery to all patient skeletal muscle cells. Thus, we have redesigned the components to best fit into AAV. We have engineered a new skeletal muscle promoter to express the dCas9-KRAB and we have switched to the smaller version of dCas9, sadCas9,
which has different sequence requirements for sgRNAs. We have shown that our new optimised AAV system works very well, as follows:
1) Our new more compact promoter expresses well in human skeletal muscle.
2) We have designed and tested new sgRNAs that are compatible with the specialized sadCas9-KRAB and which show increased efficacy in repressing DUX4 expression in primary FSHD muscle cells.
3) We are now engaged in experiments leading up to a global analysis of transcriptional, which include CRISPRi infections of primary FSHD muscle cells, harvesting of cells, and preparation and validation of RNAs. This last part of Aim 1, which would be Milestone 1, will be completed by the end of September.