Back to basics: Optimization of DNA and RNA transfer in muscle cells using recent transfection reagents

Cocchiararo, Ilaria; Cornut, Mélanie; Soldati, Hadrien; Bonavoglia, Alessandro; Castets, Perrine

doi:10.1016/j.yexcr.2022.113392

Cited by 7 publications

(6 citation statements)

References 14 publications

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“…Our findings demonstrate that these loci can drive the expression of promoterless GFP and enhance RNA expression of promoterless hF9 in C2C12 cells upon plasmid transfection using lipofection. However, due to the limited efficiency of lipofection in myoblasts, compounded by the generally low efficiency of integration, we confirmed that the efficiency of transgene integration at the genomic level is approximately 3% through the UDiTaS sequencing approach (46,47). Despite this modest level of integration, we observed the expression of promoterless GFP and increased RNA expression of hF9 compared to a control, representing the episomal vector without Cas9.…”

Section: Discussionsupporting

confidence: 60%

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Padmaswari,

Bulliard,

Agrawal

et al. 2024

Preprint

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Gene replacement therapies in genetic medicine primarily rely on adeno-associated viral (AAV) vectors for transgene expression. However, episomal expression can decline over time due to epigenetic silencing. CRISPR-based integration methods offer promise for long-term transgene insertion. While the development of transgene integration methods has made substantial progress, identifying optimal insertion loci remains challenging. Skeletal muscle is a promising tissue for gene replacement owing to the ease of access, relative proportion of body mass, the multinucleated nature of muscle, and the potential for reduced adverse effects. Leveraging endogenous promoters in skeletal muscle, we evaluated two high-expressing loci using homology-independent targeted integration (HITI) to integrate reporter or therapeutic genes in mouse myoblasts. We hijacked the muscle creatine kinase (Ckm) and myoglobin (Mb) promoters by co-delivering CRISPR-Cas9 and a donor plasmid with promoterless constructs encoding green fluorescent protein (GFP) or human Factor IX (hFIX). Additionally, we deeply profiled our genome and transcriptome outcomes from targeted integration and evaluated the safety of the proposed sites. This study introduces a proof-of-concept technology for achieving high-level therapeutic gene expression in skeletal muscle, with potential applications in targeted integration-based medicine and synthetic biology.

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Section: Discussionsupporting

confidence: 60%

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Padmaswari,

Bulliard,

Agrawal

et al. 2024

Preprint

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“…Nonetheless, CRO32TMI performed equally tantamount to VNc owing to their similar photothermal profile (Figure 2). Despite this, PDA‐PEI‐CRO32TMI showed a promising prospect for enhancing transfection efficiency in the difficult‐to‐transfect C2C12 cell line as it is a widely used cell line with low transfection efficiency with Lipofectamine reagents [16] …”

Section: Resultsmentioning

confidence: 99%

Harnessing Croconaine Organic Photosensitizers for a Milder Surface‐Mediated Transfection

Ferdinandus,

Tan,

Tan

et al. 2024

Chemistry An Asian Journal

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Polydopamine‐based materials, notably Polydopamine‐polyethyleneimine (PDA‐PEI), have gained considerable interest for surface‐mediated transfection. However, the high laser power density required to achieve high transfection efficiency poses a significant challenge in preserving cell viability. An organic photosensitizer CRO32TMI was developed to improve the photothermal conversion capability of PDA‐PEI. The modified PDA‐PEI‐CRO32TMI exhibited remarkable photothermal and photostability properties upon NIR irradiation, enabling it to achieve better transfection efficiency at lower laser power density as compared to the traditional solution or lipid‐based transfection methods.

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“…Plasmids were amplified in competent DH5α E. coli strain and extracted with the endotoxin-free NucleoBond® Xtra Maxi kit (Macherey Nagel). Transfection of C2C12 cells was done with C2C12 Cell Avalanche (EZ Biosystems) or JetOptimus (Polyplus-transfection), as previously described [ 52 ].…”

Section: Methodsmentioning

confidence: 99%

Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis

Cocchiararo,

Cattaneo,

Rajendran

et al. 2023

Human Molecular Genetics

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Defective lysosomal acidification is responsible for a large range of multi-systemic disorders associated with impaired autophagy. Diseases caused by mutations in the VMA21 gene stand as exceptions, specifically affecting skeletal muscle (X-linked Myopathy with Excessive Autophagy, XMEA) or liver (Congenital Disorder of Glycosylation). VMA21 chaperones vacuolar (v-) ATPase assembly, which is ubiquitously required for proper lysosomal acidification. The reason VMA21 deficiencies affect specific, but divergent tissues remains unknown. Here, we show that VMA21 encodes a yet-unreported long protein isoform, in addition to the previously described short isoform, which we name VMA21-120 and VMA21-101, respectively. In contrast to the ubiquitous pattern of VMA21-101, VMA21-120 was predominantly expressed in skeletal muscle, and rapidly up-regulated upon differentiation of mouse and human muscle precursors. Accordingly, VMA21-120 accumulated during development, regeneration and denervation of mouse skeletal muscle. In contrast, neither induction nor blockade of autophagy, in vitro and in vivo, strongly affected VMA21 isoform expression. Interestingly, VMA21-101 and VMA21-120 both localized to the sarcoplasmic reticulum of muscle cells, and interacted with the v-ATPase. While VMA21 deficiency impairs autophagy, VMA21-101 or VMA21-120 overexpression had limited impact on autophagic flux in muscle cells. Importantly, XMEA-associated mutations lead to both VMA21-101 deficiency and loss of VMA21-120 expression. These results provide important insights into the clinical diversity of VMA21-related diseases and uncover a muscle-specific VMA21 isoform that potently contributes to XMEA pathogenesis.

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Back to basics: Optimization of DNA and RNA transfer in muscle cells using recent transfection reagents

Cited by 7 publications

References 14 publications

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Precision and efficacy of RNA-guided DNA integration in high-expressing muscle loci

Harnessing Croconaine Organic Photosensitizers for a Milder Surface‐Mediated Transfection

Identification of a muscle-specific isoform of VMA21 as a potent actor in X-linked myopathy with excessive autophagy pathogenesis

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