New Strategy Of CRISPR-mediated CCR5 Ablation In Hematopoietic Stem Cells For An AIDS Control

AIDS is a global public health and social problem.Currently,the main treatment is antiretroviral therapy,which can effectively control AIDS but can not completely remove the latent HIV(Human Immunodeficiency Virus)and accompanied by serious drug resistance problems.Therefore,scientistsintend to find a new anti-HIV strategy.In recent years,research on AIDS has made a breakthrough.In 2007,a German medical team transplanted CCR5(C-C chemokine receptor type 5)gene-deficient hematopoietic stem cells into an AIDS patient who developed leukemia as well.After years of observation,HIV cannot be detected in the patient.The success of this case suggests that CCR5 may be an ideal target for AIDS treatment.In addition,hematopoietic stem cells(HSC)transplantation has been successfully applied in the treatment of a variety of hematopoietic cancer and immune disorders,providing technical basis for genome editing based cell therapy.Therefore,this study intends to combine elimination of CCR5 gene onhematopoietic stem cell with hematopoietic stem cell transplantation to establish a new idea of a functional cure AIDS.In order to achieve the goal of a functional cure for AIDS,we conducted in-depth research on a wide range of content.In this study,a highly specific CRISPR(Clustered regularly interspaced short palindromic repeats)technology platform was established to specifically knock out the HIV-assisted receptor gene CCR5 on HSC.HIV infection on humanized mouse was applied for safety and effectiveness evaluation.The CRISPR system consists of two parts: the sgRNA(single guide RNA)and the Cas9 protein responsible for cleavage.CRIPSR has the characteristics of high cutting efficiency and easy operation.We only need to design for each site gRNA as Cas9 protein is universal.CRISPR with high cleavage ability and high specificity of CCR5 locus was screened in vitro.The safety and efficacy of CRISPR were further verified by mouse model transplantation.It was verified that CCR5-modified cells had the ability to resist HIV infection.We will have a safety-proven CRISPR gene modification system targeting the CCR5 gene that is resistant to HIV infection and has laid a solid foundation for clinical research and application.In this study,we will use CCR5 as the target,combined with CRISPR / Cas9 technology and hematopoietic stem cell transplantation treatment program,to establish a new strategy for functional cure of AIDS.With a view to establishing a safer and more effective new strategy for HIV treatment.First,we designed and constructed a number of gRNA expression vectors targeting the CCR5 gene to establish highly purified,endotoxin-free Cas9 and gRNA vectors,to establish techniques for introducing cells,and to target gene mutation rate detection systems.Second,the protocol of isolation and purification of Hematopoietic Stem / Progenitor Cells(HSPCs)was optimized.In addition,the establishment of hematopoietic stem cell gene knockout and efficiency detection platform.Third,the humanized mouse model was optimized to establish a highly humanized immune system to reconstitute the mouse model of HIV infection detection technology.Next,CRISPR with high CCR5 targeting efficiency,high specificity,and low cytotoxicity to cells were screened.Finally,the resistance to HIV-1 infection was assessed on the CCR5 mutant hematopoietic stem cell transplantation mouse model.Theviral load and CD4 + T cell count were compared with those of the control group.The selection of CCR5 mutant cells in mice was observed.We have established a new CRISPR / Cas9 gene editing system.We designed 45 recognition sites for CCR5 gene,detected CRISPR cutting efficiency and specificity on 293 T and K562 cells,and screened CRISPR combinations with high specificity and high cutting efficiency.The plasmid required for CRISPR was transfected into 293 T cells by calcium phosphate transfection.After three days of culturing,the genome was extracted and the relevant modified fragment was purified by PCR.The cleavage efficiency was detected by T7 endonuclease.After testing,CRISPR cutting efficiency can reach about 50%.The optimal CRISPR was selected for subsequent hematopoietic stem cell transfection experiments.By optimizing the existing CRISPER system,two gRNAs were directed to Cas9 to cut the target sequence,thereby improving the knockout efficiency of the target sequence.In the same batch of CD34 + cells,the optimized system could cut up to 32% of the CCR5 sequence.We conducted a safety evaluation on human HSPCs modified with CCR5 gene.(1)Soft agar culture method is often used to detect the growth of tumor cells and transformed cell lines,normal cell culture can not growth in the soft agar suspension state.Compared with the control group,cancer cells proliferated significantly,while CCR5 gene modified CD34 + cells did not appear signs of proliferation,proved that it does not have in vitro tumorigenicity.(2)Through the grouping of 12 nude mice,Raji cells were injected into 6 nude mice in the control group,and lymphoma was observed.Six mice in the experimental group were injected with CCR5 gene modified CD34 + cells.No lymphoma and its symptoms were detected.(3)By detecting the expression of proto-oncogene Myc and HRAS,it was found that the expression of CCR5 gene in hematopoietic stem cells was not improved,and the expression of proto-oncogene was not increased.In the CCR5 knockout hematopoietic stem cells,Myc and HRAS,the expression of BMI1 was significantly increased,and the expression of GAPDH did not change significantly.(4)CCR5 gene-modified CD34 + cells in the experimental group did not show karyotype abnormalities by chromosome karyotype detection.(5)Through the guinea pig allergy test,the experimental group CCR5 gene modified CD34 + cells in the animal infusion did not appear allergic symptoms,the control group infusion of BSA animals were obvious allergic symptoms.CCR5-modified CD34 + cells are capable of reconstituting human hematopoietic cells in immunodeficient mice.The modified CD34 + HSPCs were transplanted into immunodeficient mice.After 8 weeks,the percentage of CD45 + cells in lymphocytes was detected in the peripheral blood of transplanted mice.The rats were examined for 8 weeks,10 weeks,12 weeks and 16 weeks respectively.The long-term average reconstitution efficiency of human CD45 cells was 40%,and the highest reconsitution up to 90%.At the same time,the CCR5 knockout ratio was 32.2% in the peripheral blood of mice 12 weeks after transplantation.To further assess the proportion of CCR5 deletions in HSC with long-term reconstitution capacity,we detected the gene modification of 31.2% and 24.7% in peripheral blood samples from mice aged 30-47 weeks andsecondary transplanted mice.In addition,we successfully achieved long-term,effective infection of CCR5 tropism of Ba L-1 strain in humanized mice.Virus-infected mice had a viral load that reached the desired level and maintained long-term stability.To detect the antiviral effect of this strategy in vivo,we tested Bal-1 virus infection in transplanted mice at 12 weeks,and the viral load was measured at 2,4,6 and 8 weeks after infection.Compared with the control group,the viral load of the experimental group decreased significantly in the 8th week.At the same time,CD4 + T cells were significantly enriched before and after challenge,and the proportion of CCR5 was significantly increased.In conclusion,we achieved efficient CCR5 knockout in vitro and in vivo.CCR5 knockout was achieved with CRISPR / Cas9 in CD34 + cells with high knockout efficiency while achieving high stability.Second,we transplanted CD34 + cells to mice,and can efficiently reconstitute human hematopoietic system,demonstrating that our in vitro modification strategy does not affect the ability of hematopoietic stem cell remodeling.In addition,blood cells with CCR5 knockout were detected in the hematopoietic system of the reconstituted human,and the target of knocking out the CCR5 gene in vivo was achieved.In addition,we in the body,in vitro to complete the routine safety experiments,proved the safety of this strategy.Before the clinical trial,we conducted a comprehensive assessment of the safety of the treatment strategy to prove that the use of CRISPR technology in CD34 + cells to achieve CCR5 gene knockout is a safe gene modification program.The results showed that the viral load of mice infected with R5 virulent HIV-1 virus at the 8th week was higher than that in the control group.The viral load in peripheral blood of the mice transplanted with treated HSPCs was significantly declined.The results of this study show that humanized mice reconstituted with CRISPR modified hematopoietic stem cell CCR5 gene have the ability to resist HIV-1 virus infection.Therefore,we have established a CRSPR-based CCR5 gene knockout hematopoietic stem cell transplantation for the treatment of AIDS technology system,the system has the ability to resist HIV-1 infection,providing a new strategy for a functional cure of AIDS.