All-in-one CRISPR inhibition for FSHD1 and FSHD2
Budget
- $100,000 USD
- FSHD Canada Foundation Contribution: US$75,000
Timeline
- 3 months from December, 2019
Lay Abstract
Investigator: Charis L. Himeda, PhD, Research Assistant Professor
Research Institution: Peter Jones Lab, University of Nevada, Reno School of Medicine, Reno NV USA
The most direct path to an FSHD therapy is suppressing expression of DUX4 mRNA. CRISPR technology provides an avenue for effectively targeting and manipulating specific regions of the human genome, and the potential to cure the root cause of a disease, rather than merely treating symptoms. Therefore, our lab is developing CRISPR-based approaches to therapeutically reduce DUX4 expression in FSHD. CRISPR technology consists of two components working together: 1) Cas9, the protein that performs the desired activity (editing or repression), and 2) a single guide RNA (sgRNA), which directs Cas9 to a specific site in the genome. We have shown proof-of-principle that a nuclease-deficient “dead” Cas9 protein (dCas9) fused to a small transcriptional repressor can be targeted to the FSHD locus to repress DUX4, a technique referred to as CRISPR inhibition (CRISPRi). A major impediment to taking this technology to the clinic is efficient systemic delivery of CRISPRi components. Fortunately, a recently described ortholog, SaCas9, is significantly smaller than the original and can be packaged and expressed by adeno-associated virus (AAV), presenting a viable route for introducing the reagents into FSHD muscle.
In order to achieve stable, long-term repression of DUX4, we have engineered new CRISPRi vectors. We first designed a smaller, muscle-specific regulatory cassette to allow larger therapeutic components to be delivered in vivo. This allowed us to build constructs containing dSaCas9 fused to four different epigenetic repressors that were previously too large to fit into therapeutic vectors. In primary FSHD myocytes, we have shown that our new system for CRISPRi reduces expression of DUX4 and DUX4 targets without affecting normal muscle differentiation, and that it returns the chromatin at the disease locus to a more normal state of repression.
Recently, we have re-engineered our vectors to accommodate all CRISPRi components (dSaCas9 fused to an epigenetic regulator and sgRNA) in a single vector. This is critical for bringing CRISPRi to the clinic, as it eliminates the need for two viruses, thus: 1) increasing the efficiency of delivery, 2) reducing the high cost of therapy, and 3) reducing the immunotoxicity caused by delivering high viral doses. The primary goal of this bridge-funding period is to test our new all-in-one AAV CRISPRi technology in primary human muscle cells and in FSHD-like mouse models. Our new CRISPRi constructs will be tested in primary FSHD myocytes for reduction of DUX4 and DUX4 target gene expression, as well as global effects on gene expression. They will also be tested in our lab’s FSHD transgenic mouse model, in which DUX4 expression can be induced specifically in skeletal muscles to varying degrees of severity. This model will be ideal for testing reversal of pathology and functional improvements in muscle strength.
Upon successful completion of this 3 month bridge phase, we will test our all-in-one CRISPRi system in FSHD xenograft mice developed by the Bloch lab, pending funding, which contain FSHD patient-derived muscle in their hindlimbs. Because these human grafts contain an intact FSHD locus, this xenograft model will be important for testing the long-term stability of DUX4 repression mediated by CRISPRi.
Deliverables
- Successful DUX4 silencing in human myogenic cells
- Production of high titer AAV
- AAV treatment and silencing of DUX4 in the FSHD-like mouse model