Inhibiting The Infection Of Pseudotyped HIV-1 Virus By Gene Targeted Editing Of CCR5 Based On TALEN

Acquired Immunodeficiency Syndrome(AIDS)is a kind of chronic infectious disease which resulted from the Human Immunodeficiency Virus(HIV).With the development of antiretroviral therapy,HIV-1 infection is now manageable as a chronic disease.However,HIV-1 still remains a global epidemic responsible for considerable morbidity and mortality.Although Highly Active Antiretroviral Therapy(HAART)is effective in suppressing viral replication and reducing viral loads in HIV patients,it has limitations including high cost,the lifetime treatment and side effects of long-term therapy,as well as emergence of cumulative toxicities and viral resistance.Moreover,although HARRT extends the lives of HIV-1 infected patients,it does not offer a permanent cure,since interruption of therapy leads to rapid rebound of viremia from latent reservoirs.Therefore,with the increasing knowledge of mechanisms that allow control of HIV infection,there is a need to develop more effective countermeasures for HIV infection either as a stand-alone approach or as an adjuvant to HAART.Objects To explore the feasibility of inhibiting the infection of pseudotyped HIV-1 virus by targeted editing of CC-Chemokine Receptor 5(CCR5)in human Jurkat T cells,based on engineered Transcription Activator-like Effector Nuclease(TALEN).Methods We constructed a pair of TALEN and a homologous recombination recombinated vectors targeting close to the sequence of the 32 bp deletions(TALEN CCR5?32)polymorphism in the human CCR5 gene.Assessment on the specificity and activity of TALEN CCR5?32 was evaluated by sequence in human Jurkat T cells.Real-time PCR assay was used to test the infection efficiency of pseudotyped HIV-1 virus.Results 1.The total efficiency of CCR5 gene knockout was 78.3%(47/60).16 clones had a knockout of TALEN CCR5?32,and the efficiency was 26.7%(16/60).2.The average expression of p24 of the treated group was 47.9% after TALEN CCR5?32 vectors transfected T cells.3.The average CCR5 expression of protein in Jurkat T cells by TALEN CCR5?32 was 60.6%.The inhibitions of CCR5 expression of protein was only 39.4% while the decrease expression of p24 was 52.1%.TALEN CCR5?32 effectively recognized the CCR5?32 region,inducing a frame shift of CCR5 coding region that resulted in the partial absence of CCR5 expression of protein at the cell surface and inhibited the infection of Jurkat T cells by pseudotyped HIV-1 virus.Conclusion TALEN is an effective tool for gene editing and disruption that has the potential for revolutionizing the field of HIV gene therapy.It provides powerful means to disrupt CCR5 and other host factors necessary for HIV infection as well as to delete the integrated HIV genome as a means to eradicate HIV latency.Towards this end,TALEN CCR5?32 vectors with HIV-1 inhibiting activity were successfully constructed which could be used for better serving the infection mechanism study of HIV-1.