Strategy Study Of Histone Deacetylase Inhibitors In Activating HIV Reservoirs

Background:So far,acquired immune deficiency syndrome(AIDS)is still a serious infectious disease worldwide,endangering people's health.AIDS is caused by the human immunodeficiency virus(HIV)that traps human CD4~+T immune cells.Over the past 20years,with the advent of antiretroviral therapy(ART),AIDS has been transformed from a lethal disease into a controlled chronic disease.In HIV-infected patients treated with ART,the number of CD4~+T cells in the body returns to normal,and the virus in the blood falls below the clinical detection line,and can basically have the same life span as normal people.However,due to the existence of HIV reservoirs,ART cannot completely eliminate the viral genes in HIV-infected individuals,which requires the infected person to take ART for life.The HIV reservoirs are mainly composed of resting CD4~+T cells,and some other cells,such as naive CD4~+T and macrophage cells.In these cells,HIV integrates its own RNA into the genome of the cell by reverse transcription.It does not replicate,but is latent in the genome of the cell.These resting CD4~+T cells,which are latently infected by HIV,can persist in the human body for a long time and are not cleared by ART drugs or the autoimmune system.If an HIV-infected person stops ART,latent-infected HIV will replicate under certain stimuli,causing the virus to recur.Therefore,the biggest obstacle to the cure of AIDS is the HIV reservoirs.In order to completely eliminate the HIV reservoirs,a"shock and kill"strategy was proposed.The strategy is to activate the latent HIV and then kill the virus or host cells by ART or immune cells,so as to completely eliminate the virus.Activation of latent HIV requires latency-reversing agents(LRA)to complete.There are many types of LRA,most of which are in the preclinical research stage.At present,no LRA can completely reverse HIV latency in vivo.HIV gene silencing in resting CD4~+T cells is closely related to histone deacetylation in the long terminal repeat(LTR)region of the HIV promoter.Histone deacetylase(HDAC)recruits to nucleosomes in the LTR region,resulting in transcriptional repression.This transcriptional repression can be reversed by a histone deacetylase inhibitor(HDACI).HDACIs belong to a large class of LRAs,which promote the acetylation of histones in the nucleosomes of the LTR region by inhibiting the activity of HDAC,thereby allowing DNA and nucleosomes to relax and facilitate transcription factors.This process triggers transcriptional activation of latent HIV.Based on the above mechanism,a series of HDACIs have been extensively studied as activators of"shock and kill"therapies.Some of these inhibitors,such as vorinostat(SAHA)and paobinostat,have shown some potential for latent reactivation in vitro and in vivo,and have completed clinical trials.However,their activation of HIV reservoirs is far from being able to achieve a complete cure for HIV.Purpose:In order to solve the problem of low efficiency of HDACI activation,this paper proposes two solutions.One is to find a new and efficient HDACI to meet the needs of the"shock and kill"strategy;the second is to improve the latent HIV activation efficiency of HDACI through nanotechnology.In this study,we conducted separate basic research on the above two solutions in two parts.Method and result:First,chidamide,a novel histone deacetylase inhibitor,has the potential to activate latent HIV.This part provides a preliminary assessment of the latent HIV activation of chidamide.Chidamide is the first self-developed HDACI,approved by the CFDA for the treatment of peripheral T-cell lymphoma.In this study,we demonstrated the reactivation of latent HIV by chidamide at the cell level.(1)We used the histone deacetylase activity assay kit to determine the inhibitory effect of chidamide on HDAC,which laid the foundation for subsequent experiments.(2)We detected the cytotoxicity of chidamide by MTS method,and the results showed that chidamide had less cytotoxicity.(3)Next,we used three different cell models to verify the latent HIV reactivation ability of chidamide at three levels.Viral reactivities of chidamide were accessed in three latent HIV pseudotype virus cell reporter systems(J-Lat Tat-GFP clone A7,J-Lat Tat-GFP clone A72 and TZM-bl),a latently infected full-length HIV virus cell system(U1/HIV),and resting CD4~+T cells from nine HIV-infected patients under ART with undetectable viral load.Chidamide was able to increase HIV expression in each cell line,as evidenced by GFP,luciferase activity and p24,as well as to reactivate latent HIV in primary CD4~+T cells of HIV-infected patients.(4)The mechanism of action of chidamide was investigated by chromatin immunoprecipitation(ChIP).The experiment results demonstrated that chidamide activated the transcription of latent HIV by inducing histone acetylation in the HIV promoter region.Through the study in this chapter,we have completed the evaluation of chidamide at the cellular level,demonstrating the potential of chidamide to activate HIV reservoirs,which lays the foundation for cytology for subsequent clinical studies.Second,the use of nanotechnology to improve the ability of HDACI to activate latent HIV.Panobinostat is one of the most advanced HDACIs in the field of latent HIV reactivation,and has completed relevant clinical trials.However,there are still many shortcomings,such as insufficient activation efficiency to meet the treatment requirements,poor solubility,and high toxic side effects.Therefore,there is an urgent need to solve these problems and help improve the activation efficiency.A promising approach is to develop drug delivery vehicles for existing molecules that enhance activation.The advantages of nanocarrier delivery systems over many traditional drug delivery methods have been well documented in cancer therapy.Several nanomedicines have been approved by the FDA and other countries for cancer.In this chapter,we applied nanotransport technology to the field of latent HIV reactivation.To this end,we synthesized an amphiphilic polypeptide(cholesterol-KG2R9)with cell-penetrating effect,which can self-assemble in aqueous solution to form stable nanoparticles,and can encapsulate small molecule hydrophobic drugs,such as panobinostat.The panobinostat-loaded nanoparticles(PNP-P)enter the cell by endocytosis,releasing panobinostat,which enhances the cellular uptake of HIV-infected cells.The released panobinostat will enter the nucleus and act on HDAC to promote transcription of the latent HIV.Newly produced HIV may be killed by ART to achieve the goal of completely eliminating HIV.In order to verify the above hypothesis,the following experiments were performed.(1)We first characterized PNP-P,observed the morphology of the nanoparticles by transmission electron microscopy(TEM),and measured the particle size and potential of PNP-P by dynamic light scattering(DLS).High performance liquid chromatography(HPLC)was used to detect the drug loading rate and drug release in vitro,and the stability of PNP-P was determined by DiO staining.Through the above tests,we successfully constructed a spherical nanomedicine PNP-P with a positive charge,good stability and sustained release with a diameter of about 80 nm.(2)We examined the cellular uptake of PNP-P by flow cytometry and laser confocal microscopy.It was found that the nanomedicine was more likely to be taken up by HIV-infected cells than free panobinostat.The cellular uptake is improved,which is beneficial to increase the activation efficiency of the drug.(3)After evaluating the cellular uptake of PNP-P,we examined the cytotoxicity of nanomedicine by flow cytometry.The cells treated with PNP-P still had a survival rate of about 90%,indicating that PNP-P is less cytotoxic.(4)Promoting histone acetylation is the key to activating latent HIV.We examined the histone acetylation after PNP-P treatment by flow cytometry.The experiment results showed that PNP-P could increase the histone acetylation and provide a theoretical basis for latent HIV activation.(5)The ability of PNP-P to activate latent HIV was verified by a three-level cell model in vitro.Viral reactivities of PNP-P were accessed in two latent HIV pseudotype virus cell reporter systems(J-Lat Tat-GFP clone A7 and J-Lat Tat-GFP clone A72),two latently infected full-length HIV virus cell systems(ACH2 and U1/HIV),and PBMCs from two HIV-infected patients under ART with undetectable viral load.PNP-P was able to increase HIV expression in each cell line,as evidenced by GFP,CD69 and p24,as well as to reactivate latent HIV in PBMCs of HIV-infected patients.These results confirmed that our nano-delivery system can help drugs increase the ability to activate latent HIV in vitro.(6)To test the latent HIV activation of PNP-P in vivo,we conducted related animal experiments.J-Lat Tat-GFP clone A72 cells were injected into a combined severely immunodeficient mouse(M-NSG)and administered with various drug formulations.Twenty-four hours later,J-Lat Tat-GFP clone A72 cells were isolated from mice,and GFP was detected by flow cytometry.The experiment results demonstrated that PNP-P was able to activate J-Lat Tat-GFP clone A72 cells in vivo and the activation efficiency was much higher than that of free panobinostat.At the same time,we also evaluated the biosafety of PNP-P.We collected mouse serum and various organs for biochemical indicators and H&E staining.The results of biochemical indicators showed that PNP-P treatment did not affect the function of heart,liver and kidney;H&E staining did not find any abnormalities in heart,liver,spleen,lung and kidney,indicating that the nano system had good biocompatibility and no toxic side effects.Through the study in this chapter,we demonstrated that PNP-P had a latent HIV activation that is superior to free panobinostat.The study suggests that nanotechnology can help existing LRAs improve the activation efficiency of latent HIV,and this study lays the foundation for further development of LRA delivery systems for HIV reactivation.Conclusions:In summary,this paper provides two solutions to solve the problem of low efficiency of HDACI activation,namely new effective drugs and new drug delivery strategies.This study confirmed the ability of chidamide as a new LRA to activate latent HIV,and clarified that nanotechnology was able to improve the application prospects of existing LRA(panobinostat)to reactivate HIV reservoirs.The study has laid an important theoretical basis and laboratory basis for the complete elimination of HIV reservoirs.