The Study Of Two Novel Series Of Drugs On Targeting Gene Therapy Against HIV/SIV

HIV is the unique pathogeny of AIDS. There are two existing forms of HIV in AIDS patients: both existing in the infected CD4+ cells and existing in the blood circulation as free HIV. Cleaning away such two forms of HV, AIDS should be cured from the cause of disease. Historically, there was a genetic engineered design regarding CD4-PE40( Domain II-III of Pseudomonas aeruginosa, exotoxin) was tried to reach this aim, but due to the very strong immunogenicity of CD4 protein getting from engineered E.coli and PE40 as a foreign protein for human, this attempt was concluded to the failure. In view of above negative experiences, this project tried to lead the expression DNA recombinant regarding PEIIImut (Domain III mutant of Pseudomonas aeruginosa exotoxin) which possesses vary strong killing function to cells into cells infected by HIV to express PEIII mutant protein so as to kill the cells infected by HIV as a targeting way. Such a design could avoid the immunogenicity as well as avoid s toxicity to kill any other kind of cells including healthy cells.To realize the above-mentioned purpose, we have designed three kinds of fusion protein in two series respectively which can be acquired using genetic engineering. One part is the segment of the receptors of HIV including CD4, CXCR4 and CCR5 which lie on the surface of CD4+ cells and bind the Env gp120 which only appears on cells infected by the HIV; The other part was expressed from the DNA fragment, which is rich in positive amino acids. The first part of this fusion protein will combine PEIIImut expressing DNA recombinant. The second part of this fusion protein (for example, CD4, CXCR4,CCR5) will carry the whole DNA-protein comlex and combine with the gp120 of the surface of the cells infected by HIV, so that it will make the whole complex enter such kind of cells and express PEIII protein, and kill the cells infected by HIV. In the meantime, the second part of the fusion protein will also combine the free HIV virus existing in the blood stream, making it lose infectivity because of neutralization.This program has designed and implemented in two series of plans of this kind protocol. Cell activity experiment has been done with SIV as its subject in terms of the parallel relationship of the HIV and SIV(simian immunodeficiency virus )in biological behavior.There are two parts in this dissertation.Part ONEThe Targeting Gene Therapy of AIDS as the H TypeIt is known that there is gp120/gp41 on the surfaces of the infected cells by HIV/SIV, and the gp120/gp41 can be used as the carrier for the targeting gene therapy of AIDS. With DNA recombinant technique, a series of expression recombinants were constructed in vitro based on methylotrophic yeast vector pPIC9K, which express a group of fusion proteins consisting of one targeting domain and one DNA-binding domain in yeast, Pichia pastoris(KM71). The targeting domain is one of the following proteins: CXCR4,CCR5 and CD4V1V2. On the other hand, the DNA-binding domain is a fragment of histone 1, which is rich in positive amino acids. The fusion proteins were isolated and purified from SDS-PAGE gels, and the SDS in which was removed by acid acetone. DNA-protein complexes were made according to the charge ratios of CD4V1V2- H1,CCR5-H1 or CXCR4-H1 with a recombinant pA-PEâ…¢mut(1:0.9), which contains the mutated format of domainâ…¢of Pseudomonas aeruginosa exotoxin A, PEâ…¢_mut(pA- PEâ…¢_mut or pYL-PEâ…¢_mut), in which PEâ…¢_mut were regulated by CMV promoter or by CMV promoter and HIV LTR, respectively.A series of DNA-protein complexes were made as the drugs. The killing effects of these complexes on the cells infected by SIV and the neutralizing effects of the complexes on the free SIV during the infection of SIV on CEMX-174 cells, one kind of human T cell lines, were observed.The results showed that the killing rates of the complexes alone or in combined manner in different dosages(2,4,6μg, calculated in DNA) are as follows: the killing rates of CD4V1V2-H1₈₆/pA-PEâ…¢_mut were 46.2%, 52.2%, 62.2%, respectively; the killing rates of CCR5-H1₂₄/pA-PEâ…¢_mut were 33.1%, 41.2%, 48.3%, respectively; the killing rates of CXCR4-H1₈₆/PA-PEâ…¢_mut were 42.8%, 51.2%, 63.5%, respectively; the killing rates of CD4V1V2-H1₈₆/CCR5-H1₂₄/pA-PEâ…¢_mut were 47.2%, 50.7% and 61.9%, respectively, and the killing rates of D4V1V2-H1₈₆/CXCR4-H1/pA- PEâ…¢_mut were 52.2%, 60.4% and 71.9%, respectively. Moreover, the numbers of survival cells in the different groups after different treatment were significantly lower than those of the control one(p＜0.05), respectively.On the other hand, the neutralizing effects of the complexes on the free SIV are as follows: the rates of CD4V1V2-H1₈₆/pA-PEâ…¢mut are 52%, 55.5%, 62%, respectively; the rates of CXCR4-H1₈₆/pA-PEâ…¢mut were 48.3%, 55.1%, 59.2%, respectively; the rates of CCR5-H1₂₄/pA-PEâ…¢mut were 44.2%, 51.1%, 53.6%, respectively; the rates of CD4V1V2-H1₈₆/CXCR4-H1₈₆/pA-PEâ…¢mut were 57.9%, 62.6%, 71.7% respectively,; and the rates of CD4V1V2-H1₈₆/CCR5-H1₂₄/pA-PEâ…¢mut were 52.3%, 57.0%, 62.3%, respectively. Moreover, the numbers of fluorescent cells and the titers of SIV infectivity in different groups were markedly lower than those of the control one(SIV+Normal cells) (p＜0.05), respectively.PART TWOThe targeting gene therapy of AIDS as the S TypeIn order to enhance the expression level of the fusion protein, we have modified the composition of the above-mentioned fusion genes. Based on the knowledge, the key binding regions of CD4, CXCR4 and CCR5 with gp120/gp41 are V1 domain containing 100 amino acids of CD4. the second extracellular loop containing 27 amino acids of CXCR4 and the N terminal region containing 30 amino acids of CCR5, respectively. Moreover, the DNA-binding region H1 in the above mentioned fusion proteins was replaced by another fragment of DNA-binding protein, SON spanning 72 amino acids which is rich with the positive amino acids, for example, Glu and Arg. According to the rule of codon bias of DNA sequence, those non-bias codons were changed into the bias ones in E.coli system with PCR and DNA re-synthesis. These fusion genes were inserted into one expression vector, pCW111, whose promoter is controlled by temperature and the recombinant were constructed successfully. After the induction and harvest of the engineered bacteria, the new kinds of fusion proteins were isolated and purified from the SDS-PAGE. The expression rates of the fusion proteins, CD4V1SON, XESON and RNSON expressed in the host cells were at about 25%, 21% and 14%, respectively. Mixing one of these proteins with pA-PEâ…¢_mut recombinant, respectively. A series of DNA- protein complexes were made as drugs. The killing effects of these complexes on the cells infected by SIV and the neutralizing effects of the complexes on the free SIV during the infection of SIV on the normal CEMX-174 cells were observed, respectively.The results showed that the killing rates of the complexes in single and combined manner in different dosages(2,4,6μg, calculated in DNA) were as follows: the killing rates of CD4V1-SON/pA-PEâ…¢_mut were 43%, 47.5%, 59.1%, respectively; RN-SON/pA-PEâ…¢_mut were35.7%, 42.8%, 47.2%, respectively: XE-SON/ pA-PEâ…¢_mut were 39.4%, 43.8%, 53.3%, respectively: CD4V1-SON/RN-SON/pA-PEâ…¢_mut were 38.3%, 43.8%, 57.2%, respectively; CD4V1-SON/XE-SON/pA-PEâ…¢_mut were 43.8%, 50.9% and 60.9%,respectively. Moreover, the numbers of survival cells in the different groups aider different treatment were significantly lower than those of the control one(p＜0.05), respectively.On the other hand, the neutralizing effects of the complexes on the free SIV are as follows: the rates of CD4V1-SON/pA-PEâ…¢_mut were 47.4%, 55.5%, 59.8%; XE-SON/pA-PEâ…¢_mut were 45.8%, 48.3%,57.0%, respectively; RN-SON/pA-PEâ…¢_mut were 45.8%,50.8%,57.3%, respectively: CD4V1-SON/XE-SON/pA-PEâ…¢_mut were 2.0%, 57.6%, 61.7%, respectively, and CD4V1-SON/ RN-SON/pA-PEâ…¢_mut were 44.5%, 51.7%, 57.3%, respectively. Moreover, the numbers of fluorescent cells and the titers of SIV infectivity in different groups were markedly lower than those of the control one(SIV+Normal cells) (p＜0.05), respectively.According to the above results, it is suggested that the fusion proteins can effectively mediate the recombinant pA-PEâ…¢mut entry into the target cells, and then express the target toxin protein, PEâ…¢mut effectively. The toxin protein could kill the cells infected by SIV efficiently. Moreover, the data showed that the fusion proteins could neutralize the free SIV with effect resulting in the decrease of the infectivity of SIV markedly in different treatment groups.Such a kind of protocols used in this study is a novel strategy for HIV therapy, and it is very likely to be extended for the efficient therapy of other diseases, if the targeting protein which binds to receptor could be changed to other suitable kind of ones, for example, cancer, autoimmune diseases.