Current Grant
02/25 – 01/26

Dr. Tremblay’s team plans to continue characterising the R1925X mouse model by performing functional evaluation of the R1925X mice, comparing them to WT and BlaJ mice.

The project’s long-term aim is to develop a treatment for DYSF related myopathies by correcting mutations directly in the muscles in vivo. The present project aims to improve the efficiency of two factors important to in vivo editing:

  • the percentage of nucleotide modification by PE (improving nCas9, RT activity and optimizing the pegRNA and nsgRNA position).
  • the in vivo delivery of the PE components (comparing dual AAV, VLP, LNP and EVs) to correct the mutation in R1925X mice.

Also, in collaboration with clinicians in Quebec Province, the project will conduct a census of Quebec LGMD mutations.

Grant Duration
08/23 – 01/25

Dr. Tremblay’s team built plasmids coding for epegRNAs (which target the site to be edited) with various RTT and PBS lengths to correct the dysferlin mutations R1905X, E1833X, Q1010X and W1968X. They found that each target mutation has its own optimal RTT and PBS length and that shorter RTT and PBS lengths (10 nucleotides or less) do not achieve very efficient editing. They noticed that PE3 doubles the editing efficiency compared to PE2. They reached 31% editing efficiency in HEK293T cells, and 11% in patient-derived myoblasts, but no significant editing in patient-derived fibroblasts.  The same trend was also noticed when trying to edit fibroblasts from patients affected by other myopathies. The hypothesis is that the target genes are mostly expressed in muscles but not in skin.  Therefore, the DNA does not seem to be accessible for modification in fibroblasts but is available in myoblasts. Cas-OFFinder was used to predict the possibility of targeting other locations in the genome, which could cause off-target modifications. No off-target sequences were found similar to the protospacer correcting the R1905X, E1833X, Q1010X or W1968X mutations in the human genome.

Tremblay’s team verified that the recently created R1925X mouse model (equivalent to the human R1905X mutation) carries the desired mutation. They did so by amplifying a 322 bp region around the mutation and did Sanger sequencing. The amplified section was indeed of approximately 300 bp and the sequencing showed the presence of the expected CGT to TGA mutation in all mice. They also confirmed the absence of dysferlin expression in muscle by both immunohistochemistry and Western blot. During the mouse dissection, they also noticed a fat accumulation in the lower abdomen and upper leg region in female R1925X mice.  The R1925X mice are planned to be used later in the project for in vivo editing.