Biology subjects

Tremblay, J. P.

Publications and source records attributed to Tremblay, J. P..

7 recordsLinked to original sources

bioRxiv2025

Prime Editing Enables High-Efficiency Correction of the Ryr1 T4706M Mutation: A Promising Therapeutic Approach for RyR1-Related Myopathies

Prime editing has emerged as a powerful genome-editing tool for precise correction of pathogenic mutations, offering a promising therapeutic approach for genetic myopathies. Here, we evaluate the correction efficiency of the T4706M mutation in the Ryr1 gene, which is implicated in severe skeletal muscle dysfunction. Using an optimized epegRNA design and RNA electroporation, we achieved a remarkable 80% editing efficiency in immortalized C2C12 myoblasts and 37% correction in primary myoblasts derived from the RYR1TM/TM mouse model. Our results demonstrate that the PE6 prime editing strategy, combined with rationally designed epegRNAs, significantly enhances editing efficiency in unselected cell populations. These findings establish a critical ex vivo foundation for the development of in vivo Ryr1 gene therapy in preclinical mouse models. They also provide a validated editing design that can support delivery-focused applications in both academic and industry settings.

molecular biology↗

bioRxiv2025

Split Cas12a protospacer engineering enables ultra-specific, PAM-free detection

ABSTRACTCRISPR-Cas12a is a programmable, RNA-guided endonuclease that has revolutionized biotechnology, with applications in genome engineering and diagnostics. To induce nuclease activity, Cas12a must first interact with the target dsDNA duplex by associating with a short protospacer adjacent motif (PAM) in the sequence. In this study we have split this target duplex to create PAM-proximal and PAM-distal duplex regions, which has allowed us to regulate trans-cleavage activity when these regions are included in combination or separately. These observations on Cas12a activity led to hypotheses into the related functional mechanisms, which we have tested and that have highlighted DNA/protein interactions during Cas12a complex assembly that were not otherwise apparent. Selective destabilization of the nucleic acid complexes appears to drive greater reliance on the Cas12a protein for complex stability. We have exploited this to provide significant improvements in both structural selectivity and nucleotide specificity in PAM-proximal and PAM-distal duplex regions, respectively. The result is an architecture that shows promise as a PAM-free ultra-specific platform to resolve single nucleotide polymorphisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/667643v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b1771dorg.highwire.dtl.DTLVardef@103197aorg.highwire.dtl.DTLVardef@7dd393org.highwire.dtl.DTLVardef@cd812d_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Base editing strategy allows insertion of the A673T mutation in APP gene to prevent the development of Alzheimer's disease

Amyloid precursor protein (APP), a membrane protein mostly found in neurons, is preferentially cut by the -secretase enzyme, however, abnormal cleavage by {beta}-secretase leads to the formation of {beta}-amyloid peptide plaque in the brains of Alzheimers patients. Genome analysis of an Icelandic population that did not appear to show symptoms of Alzheimers at advanced age led to the discovery of the A673T mutation, reducing {beta}-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in a patients genome could be an effective and sustainable method to slow down or prevent the progression of familial and sporadic forms of Alzheimers disease. We have thus modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9n-deaminase enzyme, which changes a cytosine into a thymine, thus converting the alanine codon to a threonine. Several Cas9n-deaminase variants were tested to compare their efficiency of conversion. The results were characterized and quantified by deep sequencing. We successfully modified the APP gene in up to 56.7% of the HEK293T cells. Our approach aimed to attest to the efficiency of base editing in the development of treatments against genetic diseases as well as provide a new strategy for the treatment of Alzheimers.

bioengineering↗

bioRxiv2020

Specific mutations in genes responsible for Alzheimer and for Duchenne Muscular Dystrophy introduced by Base editing and PRIME editing.

Base editing technique and PRIME editing techniques derived from the CRISPR/Cas9 discovery permit to modify selected nucleotides. We initially used the base editing technique to introduce in the APP gene the A673T mutation, which prevents the development of Alzheimers disease. Although the desired cytidine to thymidine mutation was inserted in up to 17% of the APP gene in HEK393T, there were also modifications of up to 20% of other nearby cytidines. More specific mutations of the APP gene were obtained with the PRIME editing technique. However, the best percentage of mutations was only 5.8%. The efficiency of the PRIME editing treatment was initially tested on the EMX1 gene. A single treatment produced the desired modification in 36% of the EMX1 gene. Three consecutive treatments increased the percentage of mutations to 50%. The PRIME editing technique was also used to insert specific point mutations in exons 9 and 35 of the DMD gene coding for the dystrophin gene and which is mutated in Duchenne Muscular Dystrophy (DMD). Up to 10% desired mutations of the DMD gene were obtained. Three repeated treatments increased the percentage of specific mutations to 16%. Given that there are thousands of nuclei inside a human muscle fiber and that the dystrophin nuclear domain is about 500 m, this level of modifications would be sufficient to produce a phenotype improvement in DMD patients.

genetics↗

bioRxiv2020

Protective mutation A673T as a potential gene therapy for most forms of APP Familial Alzheimer's Disease

The accumulation of plaque in the brain leads to the onset and development of Alzheimers disease. The Amyloid precursor protein (APP) is usually cut by -secretase, however an abnormal cleavage profile by {beta}-secretase (BACE1) leads to the accumulation of A{beta} peptides, which forms these plaques. Numerous APP gene mutations favor plaque accumulation, causing Familial Alzheimer Disease (FAD). However, a variant of the APP gene (A673T) in Icelanders reduces BACE1 cleavage by 40 %. A library of plasmids containing APP genes with 29 FAD mutations with or without the additional A673T mutation was generated and transfected in neuroblastomas to assess the effect of this mutation on A{beta} peptide production. In most cases the production of A{beta} peptides was decreased by the co-dominant A673T mutation. The reduction of A{beta} peptide concentrations for the London mutation (V717I) even reached the same level as A673T carriers. These results suggest that the insertion of A673T in the APP gene of genetically susceptible FAD patients may prevent the onset of, slow down, or stop the progression of the disease.

neuroscience↗

bioRxiv2019

Immune Responses to Human Induced Pluripotent Stem Cells and their Derived Myogenic Progenitors Are Mediated by Different Mechanisms in Humanized Mice

It is still unclear if immune responses will compromise the large scale utilization of cell therapies derived from human induced pluripotent stem cells (hiPSCs). To answer this question, we used humanized mouse models and evaluated the engraftment in skeletal muscle of myoblasts derived either directly from a muscle biopsy or differentiated from hiPSCs or fibroblasts. Our results showed that while allogeneic grafts were rejected, engraftment of autologous cells was tolerated, indicating reprogramming and differentiation procedures are not immunogenic. We also demonstrated that hiPSC-derived myogenic progenitors, in opposition to hiPSCs, are not targeted by natural killer (NK) cells both in vitro and in vivo. Yet, adoptive transfer of NK cells can prevent the formation of hiPSC-derived teratoma. Overall, our findings suggest that hiPSC-derived muscular therapies will be tolerated in presence of a competent human immune system and highlight the risk of forming a teratoma if using partially differentiated autologous human cells. HighlightsO_LIhiPSC-derived myofibers are tolerated in autologous humanized mouse models C_LIO_LIInfiltration of autologous T cells is not predictive of successful skeletal muscle engraftment C_LIO_LIAdoptive transfer of NK cells prevents the formation of hiPSCs derived teratomas C_LIO_LINK cells are unable to reject established teratomas C_LI

cell biology↗

bioRxiv2018

CRISPR-induced deletion with SaCas9 restores dystrophin expression in dystrophic models in vitro and in vivo.

Duchenne Muscular Dystrophy (DMD), a severe hereditary disease, affecting 1 boy out of 3500, mainly results from the deletion of one or more exons leading to a reading frame shift of the DMD gene that abrogates dystrophin protein synthesis. We used the Cas9 of Staphylococcus aureus (SaCas9) to edit the human DMD gene. Pairs of sgRNAs were meticulously chosen to induce a genomic deletion to not only restore the reading frame but also produced a dystrophin protein with normally phased spectrin-like repeats. The formation of a dystrophin protein with spectrin-like repeats normally phased is not usually obtained by skipping or by deletion of complete exons. This can however be obtained in rare instances where the exon/intron borders of the beginning and the end of the complete deletion (patient deletion plus CRISPR-induced deletion are at similar positions in the spectrin-like repeat. We used pairs of sgRNAs, targeting exons 47 and 58 and a normal reading frame was restored in 67 to 86% of the resulting hybrid exons in myoblasts derived from muscle biopsies of 4 DMD patients with different exon deletions. The restoration of the DMD reading frame and restoration of the dystrophin expression was also obtained in vivo in the heart of the del52hDMD/mx. Our results provide a proof-of-principle that SaCas9 could be used to edit the human DMD gene and could be considered for the further development of a therapy for DMD.

molecular biology↗