| Sly syndrome,beta-D-glucuronic acid product storage,is a typical single-gene genetic disease with metabolic abnormalities caused by beta-glucuronide enzyme(GUSB)deficiency.Existing Beta-glucuronide enzyme substitution therapy,drug treatment and surgical correction are mainly to improve the patient’s condition,relieve the pain,which are so-called "phenotype treatment".The emergence of gene therapy opens a new route for treating this kind of single-gene genetic disease.The gene therapy of single-gene genetic disease is to introduce the therapeutic genes with normal function carried by the corresponding vehicle into the target cells with single-gene defects,which could cure the disease.Although the emergence of gene therapy brought the dawn of permanent cure for the single-gene genetic diseases,there are still some shortcomings in the traditional means of gene therapy.(1)As to taking the strategy of gene therapy for the integration of functional gene in the genome,it exists the drawbacks of lasting expression of foreign functional gene in the genome and the effect of the integration of foreign gene on the expression of genes located the downstream and upstream of the integration site.(2)As to taking the strategy of gene therapy for defected gene in situ repair,the low efficiency of homologous recombination(HR)restricts its application.Researchers established a novel strategy for gene therapy by promoterless site-specific integration in the genome in 2014.The strategy is achieved by site-specific integration of therapeutic gene carrying a self-cleavage peptide T2A at its 5-terminal into the region right before endogenous gene with high level of expression.Therefore,the fusion protein with therapeutic gene and endogenous gene separated by T2A peptide was produced under the strict control of endogenous promoter.Therapeutic protein generated from the fusion protein cleaved by self-cleavage peptide T2A would produce the therapeutic effect.Although the strategy partly resolved the problems present in two protocols of gene therapy above-mentioned,its application of the strategy would be limited to some extent.In addition,exogenous gene HR efficiency is still very low in cells.Therefore,on the basis of the new gene therapy,this topic was achieved by site-specific integration of therapeutic gene in the genome right before the stop codon of albumin gene with the high level expression in the mouse tumor cell line Hepal-6 by inducing the high efficiency of DNA homologous recombination caused by CRISPR/Cas9 system induced DNA double strand breaks(DSB).The therapeutic effect of the novel strategy will be evaluated in mouse cancer cell line Hepal-6 and MEF cell with the deficiency of GUS gene for establishing a safer and more efficient protocol of gene therapy of inherited disease caused by the mutation of single gene.This topic mainly includes three parts.1.Construction of the hCas9 expression vector and design and screening of the specific sgRNAs targeting the region of genome located on the downstream of stop codon of albumin.The codon optimized Cas9 fragment and eukaryotic expression relevant elements were assembled to obtain the hCas9 eukaryotic expression vector.According to the gene information on the NCBI database and related literatures,four sgRNAs targeting the region before the termination codon of Albumin gene were designed,then the sgRNA expressing vectors were constructed.The sgRNA expressing vectors were co-transfected into Hepal-6 cell line with hCas9 expression vector,respectively.The genomic DNA was extracted post 72 hours transfection followed by the specific amplification of sgRNA targeted region using PCR method.The biological activity of sgRNAs was detected by T7E1 assay.2.Construction of the donor vector carrying reporter gene used for specific-integration in the region of mAlb genome.First,the upstream and downstream homologous arms for homologous recombination were designed according to the mAlb genome sequence information and sgRNA sites targeting mAlb.Then the upstream and downstream homologous arms fragments for site-specific integration in the region of mAlb genome and luciferase reporter gene fragment were amplified respectively using PCR technique followed by sequentially ligating them into the basic donor vector.Two donor vectors with and without sgRNA expression cassette were constructed according to the presence or absence of sgRNA expression cassette in the donor vector.The donor vector linearized by intracellular Cas9-sgRNA was constructed by introducing the sgRNA binding site into the donor vector with sgRNA Cassette.Thus,three different donor vectors,the donor vector with sgRNA cassette,the donor vector without sgRNA cassette and the linearizable donor vector with sgRNA cassette,were obtained.Finally,by the homologous recombination efficiency of different donor vectors in Hepa1-6 cell line was detected by using the luciferase report gene detection system while the G422(mouse glioma cell line)which has no Alb promoter activity was used as negative control.3.Construction of the donor vector carrying therapeutic GUSB gene used for specific-integration in the region of mAlb genome and their experimental study in vitro.First,the therapeutic GUSB gene with T2A peptide sequence was obtained by using molecular cloning,then was ligated into the basic donor vector containing the mAlb upstream and downstream homologous arms fragments.Using the similar strategy,three different donor vectors,the donor vector with sgRNA cassette,the donor vector without sgRNA cassette and the linearizable donor vector with sgRNA cassette,were constructed on the basis of the vector generated above.The donor vectors were co-transfected with hCas9 expression vector into Hepa1-6 cell line or mouse MEF cell line with the deficiency of GUS,respectively.Through the Real-Time quantitative PCR,Western Blot and GUS staining techniques,the GUSB mRNA and protein expression level in vitro were detected,which could be used for the evaluation of the potential value of the system.The results were obtained as follows based on the study above.1.The sgRNA targeting the region in Albumin gene before termination codon with high biological activity was screened and obtained.2.Three different donor vectors containing the luciferase report gene for site-specific integration and the homologous recombination were constructed by PCR method,then the efficiency of homologous recombination was detected in vitro.The results indicated that the foreign report gene cannot be expressed after homologous recombination in the G422 cell line without Alb promoter activity.In Hepa1-6 cell line with high activity of the Alb promoter,the homologous recombination efficiency was strongly improved by 10 times using the CRISPR/Cas9 system and resulted in the specific and high expression of report gene.When using the linearizable donor vector with sgRNA cassette,the homologous recombination efficiency was further improved by20 times in the Hepa1-6 cell line.3.The therapeutic GUSB gene with T2A peptide sequence was obtained,then three different donor vectors containing the therapeutic GUSB gene were constructed for site-specific integration.By detecting the GUSB mRNA and protein expression in vitro,this strategy of gene therapy was proven to be able to make the expression of therapeutic gene in the target cells.To sum up,in this topic,the therapeutic GUSB gene carrying self-cleavage peptide T2A at the 5 terminal of the gene was site-specifically integrated before the termination codon of Alb gene which was specifically and highly expressed in mouse liver tissue cells by using the CRISPR/Cas9 genome editing system,resulting in the expression of human beta-glucuronidase with biological activities in the liver cells without affecting the natural expression and secretion of Albumin protein.This topic established a promoterless genome site-specific integration system based on the CRISPR/Cas9 genome editing technology,which laid a foundation for the application of the new strategy in the treatment of single-gene genetic disease. |
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