Researchers at the Beijing Institute of Life Sciences for the first time use in vitro mature Cas9 / sgRNA ribosomal complexes in patient-specific recessive dystrophic bullous epidermolysis bullosa (RDEB) mouse epidermal stem cells The adult gene repair, so as to RDEB disease and other hereditary skin disease cure provides a new program. The results of this research are published in the PNAS magazine.
Recessive malnutrition bullous epidermolysis is caused by the skin of type VII collagen (Collagen VII) protein dysfunction caused by recessive skin genetic disease. In normal skin, Collagen VII secreted by Keratinocyte and fibroblast in the dermis forms a homotrimer and is then assembled into anchoring fibrils, making the epidermis and dermis Firmly combined. In RDEB patients, some mutations in the Col7a1 gene make the type VII collagen dysfunction, leading to detachment of the epidermis and dermis, resulting in blood blister.
Gene therapy is the only treatment that can cure such diseases. The CRISPR / Cas9 system is an efficient gene editing system that has been used in recent years to treat some of the genetic diseases that were previously untreated. However, efficient in vivo introduction of CRISPR / Cas9 transport system has been constrained by this technology to clinical applications. <BR> <BR> In order to explore the use of CRISPR / Cas9 system to achieve adult gene editing to cure RDEB disease, Beijing Life Sciences Institute Chen Ting laboratory researchers first established patient-specific RDEB mouse model. Mutant homozygous (Col7a1 c.6485G & gt; A mut / mut) mice appeared to be similar to the patient's bloodstained phenotype after birth. While the heterozygous (Col7a1 c.6485G & gt; A wt / mut) mice were intact with no symptoms. Tissue biopsy results show that the skin and epidermis are separated from the dermis in the presence of a blister. Even in the absence of blister's back skin and the skin on the tail, the epidermis and leather connection loose, but also showing the epidermis and dermis separation of phenotype. Collagen VII immunofluorescence staining showed that in the wild-type mouse skin Collagen VII was linearly distributed in the basal layer between the epidermis and the dermis. In the mutant homozygous, the Collagen VII protein is dispersed in the basal cells of the epidermis and in the dermal cells adjacent to the basal layer.
Collagen VII protein according to its structural characteristics, can be divided into N-terminal NC1, located in the middle of the central helical region (central helical region) and C-side NC2 domain. Wherein the central helical domain is composed of a plurality of "Gly-X-Y" repeats in the one-dimensional structure of the protein. Theoretically, the removal of the mutant 80th exon will only lead to a slight reduction in the length of the Collagen VII protein without affecting its protein function. To verify the safety of exonectomy at position 80, the investigators once again produced the 80th exon-deficient mouse (Col7a1 ΔExon80) by the NHEJ cell repair pathway mediated by the CRISPR / Cas9 system. Hybrid and pure and mutant mice were consistent with wild-type mice, and neonatal and adult mice did not have a hematopoetic phenotype of RDEB disease. Collagen VII proteins are linearly distributed in the basal layer, both in mutant hybrids and in homozygotes. Tissue sections also showed that the mutant hybrids were homogeneous with the homozygous mouse skin tissue, and the epidermis and dermis were intact. The researchers then screened out sgRNAs that specifically excipiently excised at the 80th exon, and demonstrated that sgRNA was able to successfully excite the 80th exon resection in an in vitro epidermal stem cell line.
Subsequently, the researchers established the intradermal introduction system of Cas9 / sgRNA ribosomal protein complex mediated by electric shock. In order to accurately and rigorously demonstrate that the CRISPR / Cas9 system is transported into the skin cells and is able to function properly, the researchers use the plasmids expressing the Cas9 protein and two sgRNAs that specifically recognize the Flox site to activate the skin cells by electroporation Rosa26-stop-tdTomato mouse tail skin cells and found that almost all RFP + cells were distributed in differentiated keratinocytes. When the plasmid is replaced with the Cas9 / sgRNA Ai14-L / R ribosomal protein complex, the Cas9 / sgRNA ribosomal protein complex can be transported to the differentiated keratinocytes and can be efficiently transported into basal cells. Through a long track observation. The researchers confirmed that RFP + basal cells not only can be amplified over time, but also can be divided into epidermal cells.
In order to demonstrate that the Cas9 / sgRNA ribosomal protein complex was excised in the mouse from the Col7a1 gene and the 80-deleted exon-deleted mRNA was synthesized. The investigators were examined in Col7a1 c.6485G & gt; A mut / mut, Rosa26- (SgRNA col7a1-L / R, sgRNA-Ai14-L / R) ribosomal protein complex in the stop-tdTomato fl / wt mice, the exonectomy at the 80th day of Col7a1 was performed to activate Ai14 cells simultaneously. In enrichment of RFP + cells, the researchers successfully detected genomic DNA and mRNA-sequenced products in mice, and were validated by sequencing. More importantly, the researchers analyzed the immunofluorescence staining of Collagen VII in the repaired mouse skin. The Collagen VII protein was significantly increased at the basal layer once a intradermal injection of the Cas9 / sgRNA-col7a1-L / R ribosomal protein complex was performed and subjected to electrical shock. In addition, the percentage of epidermal and dermal adhesion increased from ~ 30% to ~ 60% in the colaa c c. 6485G & gt; A-mut / mut mice after gene repair.
In summary, the Cas9 / sgRNA ribonucleoprotein complex was able to mediate the expression of the Col7a1 gene by electroporation, thereby recovering the Collagen VII protein function and repairing the RDEB phenotype.
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