Development of an in vitro model of dysferlinopathy via crispr/cas-mediated transcriptional activation of the dysf gene

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Scientists need cell models from human tissues to develop methods of gene therapy and genome editing for monogenic diseases. It is preferable to use minimally invasive methods to obtain samples; these tissues can be applied for further screening in order to select the most effective approach to restore the synthesis of the target protein. We used the CRISPR/Cas9-SAM transcriptional activation system, which ensures expression of the DYSF gene in HEK293Т cells, as well as in fibroblasts from patients with dysferlinopathy (c.2779delG (Ala927LeufsX21)). After targeted activation of DYSF, it was possible to detect the main gene products: mRNA and protein (HEK293Т_ТА) and mRNA (fibroblasts). Transcriptionally activated dysferlin-deficient fibroblasts and HEK293 cells can be used to evaluate the in vitro efficacy of gene therapy for dysferlinopathies.

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作者简介

I. Yakovlev

Artgene Biotech; OOO Genotarget, Skolkovo Innovation Center

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Email: mail@genotarget.com
俄罗斯联邦, Moscow; Moscow

Y. Slesarenko

OOO Genotarget, Skolkovo Innovation Center

Email: mail@genotarget.com
俄罗斯联邦, Moscow

I. Starostina

Kazan (Volga Region) Federal University

Email: mail@genotarget.com
俄罗斯联邦, Kazan

A. Shaimardanova

Kazan (Volga Region) Federal University

Email: mail@genotarget.com
俄罗斯联邦, Kazan

V. Solovyova

Kazan (Volga Region) Federal University

Email: mail@genotarget.com
俄罗斯联邦, Kazan

P. Bobrovsky

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

Email: mail@genotarget.com
俄罗斯联邦, Moscow

E. Grafskaia

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

Email: mail@genotarget.com
俄罗斯联邦, Moscow

L. Belikova

Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine

Email: mail@genotarget.com
俄罗斯联邦, Moscow

S. Bardakov

Artgene Biotech

Email: mail@genotarget.com
俄罗斯联邦, Moscow

A. Rizvanov

Kazan (Volga Region) Federal University; Division of Medical and Biological Sciences, Tatarstan Academy of Sciences

Email: mail@genotarget.com
俄罗斯联邦, Kazan; Kazan

A. Isaev

Artgene Biotech

Email: mail@genotarget.com
俄罗斯联邦, Moscow

R. Deev

Artgene Biotech; OOO Genotarget, Skolkovo Innovation Center; Avtsyn Research Institute of Human Morphology, Petrovsky National Research Centre of Surgery

Email: mail@genotarget.com
俄罗斯联邦, Moscow; Moscow; Moscow

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2. Fig. 1. CRISPR/Cas9-SAM transcriptional activation system.

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3. Fig. 2. Distribution of fibroblasts isolated from the gums of a patient with dysferlinopathy by surface markers (immunophenotyping). Vertical axis – cell number; horizontal axis – glow intensity of the probe bound to the corresponding antibody. a – Cells negative for markers CD4 5, CD34, CD 1 1b, CD 19 and HLA-DR; b – CD90+ cells (94.4%), b – CD 10 5+ cells (90.5%), d – CD73+ cells (91.5%); d, e – histogram of cell population distribution by GFP fluorescent signal intensity before and after sorting, respectively. Flow cytometry.

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4. Fig. 3. Detection of p53 knockout in fibroblasts. a – Fibroblasts from a patient with dysferlinopathy transduced with LVUHshp53-gfp.3 lentivirus. b – Western blot: KO – immortalized skin fibroblasts from a patient with dysferlinopathy obtained by knockout of the p53 tumor suppressor gene (53 kDa), WT – skin fibroblasts from a patient with dysferlinopathy. M – molecular marker.

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5. Fig. 4. Detection of DYSF mRNA and protein in fibroblasts and HEK 293T cells before (wo TA) and after (TA) transcriptional activation of DYSF using CRISPR/dCas9 SAM. a – Relative expression of dysferlin mRNA (ΔΔCT, RT-PCR) in human myoblasts without a mutation in the DYSF gene used as control cells (HMb), patient fibroblasts after transcriptional activation (TA) of DYSF (HF-mut26-p53_TA) and patient fibroblasts lacking a component of the activation system (HF-mut26-p53 wo TA). b – Relative (ΔΔCT) expression of dysferlin mRNA in HEK 293T cells before and after TA DYSF and C 2C 1 2 cells, which served as a positive control. c, d – Western blots with antibodies to the dysferlin protein and their densitograms (D – optical density), respectively. a, b: Data are presented as mean ± standard error of the mean.

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