12/24/2023 0 Comments Sequential artHuman hematopoietic stem cells (HSC) reconstituted NOD.Cg-Prkdc scid Il2rgt m1Wjl/SzJ (NSG) mice produce human T cells, that are broadly susceptible to HIV-1 infection 23, 24, 25, 26, 27, 28, 29, 30. This is based both on known species restrictions for HIV-1 infection and long-term establishment of tissue reservoirs of infection. With the knowledge that few small animal models of HIV-1 reflect actual viral reservoirs and long-term infections, another system for study is required. We conclude that viral elimination by a combination of LASER ART and gene editing strategy is possible.Ĭreation and characterization of HIV-1 infected humanized mice This is confirmed in those mice using ultrasensitive HIV-1 nucleic acid detection methods by the absence of post-treatment viral rebound and by the inability to transfer virus from those infected and dual-treated mice to replicate uninfected untreated mice. Viral clearance is achieved from HIV-1 infected spleen and lymphoid tissues as well as a broad range of solid organs from documented prior infected humanized mice treated with LASER ART and AAV 9-CRISPR-Cas9. Here we demonstrate elimination of replication competent HIV-1 in an experimental model of human infectious disease. Thus, the two approaches are combined to examine whether LASER ART and CRISPR-Cas9 treatments could provide combinatorial benefit for viral elimination. We realize that CRISPR-Cas9-based technologies could be most effective in the setting of maximal viral restriction and substantive reductions in the absolute proviral DNA load. Thus, in parallel, we develop CRISPR-Cas9 based gene editing technology using AAV 9 delivery that specifically and efficiently excises fragments of integrated HIV-1 proviral DNA from the host genome 19, 20, 21, 22, 23, 24. However, LASER ART alone cannot rid the infected host of latent HIV-1 no matter how successful the drugs may prove to be at restricting viral infection. Macrophages enable uptake of significant amounts of intracellular antiretroviral drug crystals and tightly control ongoing viral replication by the cells’ slow drug release and transfer to adjacent CD4 + T cells during cell-to-cell contact or through direct drug uptake 13, 14, 16, 17, 18. These reduce disease co-morbidity in small animals and maintains effective antiretroviral drug concentrations in blood and tissue viral reservoirs from days to weeks 12, 13, 14, 15, 16. LASER ART properties are defined by slow drug dissolution, enhanced lipophilicity, improved bioavailability and limited off-target toxicities, which directly affect the frequency of ART administration from daily to weeks. To address this need and design a suitable therapeutic strategy, our laboratories produce highly hydrophobic lipophilic viral reservoir penetrating antiretroviral prodrugs coined as long-acting slow-effective release ART (LASER ART). Therefore, multimodal robust pharmaceutic strategies are needed for complete elimination of HIV-1 if no viral resurgence after cessation of ART is to be achieved. This underscores that early intervention with potent antiretroviral medicines may help to further reduce the size of the reservoir and ultimately facilitate viral elimination 11. Yet, another obstacle towards elimination of infection is that viral latency is established after infection onset and precedes peak viremia. All single or combination therapeutic approaches preclude HIV-1 cure as viral rebound universally follows ART cessation 6, 7, 8, 9, 10. This includes inadequate therapeutic access to viral reservoirs, rapid spread of infection by continuous sources of virus and susceptible cells and a failure to eliminate residual latent integrated proviral DNA. There are several reasons why success has not yet been realized. However, elimination of HIV-1 infection in its infected human host is documented only in two individuals 4, 5. Thus, a major issue for any HIV-1 curative strategy is the means to eliminate either integrated proviral DNA or the cells that harbor virus without collateral cytotoxic reactions. As such, virus persists in a latent state within infectious reservoirs and ART cessation readily leads to viral reactivation and disease progression to acquired immunodeficiency syndrome (AIDS) 3. However, ART fails to eliminate integrated copies of HIV-1 proviral DNA from the host genome 1, 2. In the clinic, antiretroviral therapy (ART) restricts viral infection by stalling various steps of the viral life cycle. According to UNAIDS, it is estimated that more than 36.7 million people worldwide are infected with the human immunodeficiency virus type one (HIV-1) and >5000 individuals worldwide are newly infected each day.
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