Targeted addition of mini-dystrophin into rDNA locus of Duchenne muscular dystrophy patient-derived iPSCs

Zeng, Baitao; Zhou, Miaojin; Liu, Bo; Shen, Fei; Xiao, Rou; Su, Jiasun; Hu, Zhiqing; Zhang, Yiti; Gu, Ao; Wu, Lingqian; Liu, Xionghao

doi:10.1016/j.bbrc.2021.01.056

Cited by 7 publications

(8 citation statements)

References 32 publications

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Section: Rdna-specific Integration Of the Novel F8 Cassette Into Ipsc...mentioning

confidence: 81%

“…Using the donor vector minipHrneo with the help of TALENickases plasmids [22] (TALEN L and TALENickase R D450A), we have achieved an efficient targeted integration of various exogenous genes, including BDDF8, F9, miniDystrophin, IL-24, and TRAIL into the rDNA locus. The stable expression of exogenous genes in vivo and in vitro were confirmed [18,21,[23][24][25]. To explore the enhancer effect of C400 and its application for universal targeted gene therapy for HA in different genetic backgrounds, we nucleofected F8 expression vectors into the rDNA loci of HA-iPSCs and normal human iPSCs (B6-iPSCs), respectively.…”

Section: Rdna-specific Integration Of the Novel F8 Cassette Into Ipsc...mentioning

confidence: 94%

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Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

Xiao,

Chen,

et al. 2024

IJMS

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Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies.

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Section: Rdna-specific Integration Of the Novel F8 Cassette Into Ipsc...mentioning

confidence: 81%

Section: Rdna-specific Integration Of the Novel F8 Cassette Into Ipsc...mentioning

confidence: 94%

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

Xiao,

Chen,

et al. 2024

IJMS

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“…We previously generated an iPSC line derived from the urine cells of a DMD patient with exon-50-deleted (DMD-iPSCs) [ 20 ] that was detected via multiplex ligation-dependent probe amplification (MLPA). Here, exons 49, 50 and 51 in DMD-iPSCs were identified using PCR-amplification, with the exon-spanning primers ( Table S1 ) and normal human iPSCs (hiPSCs) as a positive control ( Figure 1 A).…”

Section: Resultsmentioning

confidence: 99%

“…The normal hiPSCs (DYR0100) were purchased from ATCC. DMD-iPSCs were generated previously [ 20 ]. Briefly, DMD-iPSCs were reprogrammed from urine cells of a severe DMD patient with exon 50 deletion.…”

Section: Methodsmentioning

confidence: 99%

Full-Length Dystrophin Restoration via Targeted Exon Addition in DMD-Patient Specific iPSCs and Cardiomyocytes

Xiao

Zhou

Wang

et al. 2022

IJMS

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Duchenne muscular dystrophy (DMD) is the most common fatal muscle disease, with an estimated incidence of 1/3500–1/5000 male births, and it is associated with mutations in the X-linked DMD gene encoding dystrophin, the largest known human gene. There is currently no cure for DMD. The large size of the DMD gene hampers exogenous gene addition and delivery. The genetic correction of DMD patient-derived induced pluripotent stem cells (DMD-iPSCs) and differentiation into suitable cells for transplantation is a promising autologous therapeutic strategy for DMD. In this study, using CRISPR/Cas9, the full-length dystrophin coding sequence was reconstructed in an exon-50-deleted DMD-iPSCs by the targeted addition of exon 50 at the junction of exon 49 and intron 49 via homologous-directed recombination (HDR), with a high targeting efficiency of 5/15, and the genetically corrected iPSCs were differentiated into cardiomyocytes (iCMs). Importantly, the full-length dystrophin expression and membrane localization were restored in genetically corrected iPSCs and iCMs. Thus, this is the first study demonstrating that full-length dystrophin can be restored in iPSCs and iCMs via targeted exon addition, indicating potential clinical prospects for DMD gene therapy.

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“…HEK-293T cells were purchased from ATCC (Manassas, VA, USA) and cultured on 6 cm plates (Corning Incorporated, Corning, NY, USA) using Dulbecco's Modified Eagle's Medium (DMEM, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher). iPSCs [31] and Duchenne muscular dystrophy (DMD) patient-specific iPSCs (DMD-iPSCs) [32] were previously constructed by our laboratory; all subjects gave their written informed consent. iPSCs were cultured on Matrigel (BD Biosciences, Franklin Lakes, NJ, USA) coated plates (Corning) using high glucose mTeSR-plus cell culture medium (STEMCELL Technologies, Vancouver, Canada) and subcultured every 6 days.…”

Section: Cell Culture and Maintenancementioning

confidence: 99%

An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

Duan

Tang

Zeng

et al. 2021

Life

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(1) Background: Gene editing technology, as represented by CRISPR is a powerful tool used in biomedical science. However, the editing efficiency of such technologies, especially in induced pluripotent stem cells (iPSCs) and other types of stem cells, is low which hinders its application in regenerative medicine; (2) Methods: A gene-editing system, COE, was designed and constructed based on CRISPR/Cas12a and Orip/EBNA1, and its editing efficiency was evaluated in human embryonic kidney 293T (HEK-293T) cells with flow cytometry and restriction fragment length polymorphism (RFLP) analysis. The COE was nucleofected into iPSCs, then, the editing efficiency was verified by a polymerase chain reaction and Sanger sequencing; (3) Results: With the extension of time, COE enables the generation of up to 90% insertion or deletion rates in HEK-293T cells. Furthermore, the deletion of a 2.5 kb fragment containing Exon 51 of the dystrophin gene (DMD) in iPSCs was achieved with high efficiency; out of 14 clones analyzed, 3 were positive. Additionally, the Exon 51-deleted iPSCs derived from cardiomyocytes had similar expression profiles to those of Duchenne muscular dystrophy (DMD) patient-specific iPSCs. Moreover, there was no residue of each component of the plasmid in the editing cells; (4) Conclusions: In this study, a novel, efficient, and safe gene-editing system, COE, was developed, providing a powerful tool for gene editing.

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Targeted addition of mini-dystrophin into rDNA locus of Duchenne muscular dystrophy patient-derived iPSCs

Cited by 7 publications

References 32 publications

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A

Full-Length Dystrophin Restoration via Targeted Exon Addition in DMD-Patient Specific iPSCs and Cardiomyocytes

An Episomal CRISPR/Cas12a System for Mediating Efficient Gene Editing

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