Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure. Gene-based therapies represent a promising treatment for HIV-1 infection, as they offer the potential for sustained viral inhibition and reduced treatment interventions. One approach developed here involves using conditionally replicating vectors (CR-vectors). CR-vectors utilize HIV-expressed proteins to replicate and disseminate along with HIV into the budding viral particles, thereby co-infecting target cellular reservoirs. We generated and characterized several CR-vectors carrying various therapeutic payloads of non-coding RNAs targeted to HIV-1, both transcriptionally and post-transcriptionally. Both virus and vector expression was followed in cell culture systems and T cells in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-vectors functionally suppress HIV expression in a long-term stable manner and that transcriptional targeting of and epigenetic silencing of HIV can be passaged to newly infected cells by the action of the CR-vector, ultimately establishing a sustained parasitism of HIV. Our findings suggest that CR-vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term sustained suppression of HIV-1, leading ultimately to a functional cure.
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Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV, as they have the potential for sustained viral inhibition via reduced treatment interventions. One innovative approach developed here involves using conditionally replicating vectors (CR-Vectors), as these vectors utilize HIV-expressed proteins to replicate. These vector payloads can spread along with HIV into the budding viral particles, and co-infect target cells, essentially disseminating to HIV infected cells. We have generated and characterized CR-Vectors carrying therapeutic payloads consisting of various non-coding RNA regulatory expression cassettes, which modulate HIV both transcriptionally and post-transcriptionally, as well as CRISPR directed excision machinery. Notably, we have followed both virus and vector expression in T-cells and in vivo in the presence and absence of mycophenolic acid (MPA) selection. We find here that CR-Vectors functionally suppress HIV expression in a long-term stable and potent manner in both the presence and absence of MPA; and that transcriptional targeting is more potent at modulating stable suppression of HIV than post-transcriptional targeting or CRISPR directed excision of HIV. This suppression may be physiologically relevant, as it appears to drive HIV to a sustained non-pathogenic set point. Our findings suggest that CR-Vectors with modulatory non-coding RNAs may be a viable approach to achieving long-term stable suppression of HIV leading ultimately to a functional cure.
Drug transporters affect antiretroviral therapy (ART) tissue disposition, but quantitative measures of drug transporter protein expression across preclinical species are not available. Our objective was to use proteomics to obtain absolute transporter concentrations and assess agreement with corresponding gene and immunometric protein data.In order to make interspecies comparisons, two humanized mouse [hu-HSC-Rag (n = 41); bone marrow-liver-thymus (n = 13)] and one primate [rhesus macaque (nonhuman primate, n = 12)] models were dosed to steady state with combination ART. Ileum and rectum were collected at necropsy and snap frozen for analysis.Tissues were analyzed for gene (quantitative PCR) and protein [liquid chromatography-mass spectrometry (LC-MS) proteomics and western blot] expression and localization (immunohistochemistry) of ART efflux and uptake transporters. Drug concentrations were measured by LC-MS/MS. Multivariable regression was used to determine the ability of transporter data to predict tissue ART penetration.Analytical methods did not agree, with different trends observed for gene and protein expression. For example, quantitative PCR analysis showed a two-fold increase in permeability glycoprotein expression in nonhuman primates versus mice; however, proteomics showed a 200-fold difference in the opposite direction. Proteomics results were supported by immunohistochemistry staining showing extensive efflux transporter localization on the luminal surface of these tissues. ART tissue concentration was variable between species, and multivariable regression showed poor predictive power of transporter data.Lack of agreement between analytical techniques suggests that resources should be focused on generating downstream measures of protein expression to predict drug exposure. Taken together, these data inform the use of preclinical models for studying ART distribution and the design of targeted therapies for HIV eradication.
Abstract HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a disabling chronic inflammatory disease of the central nervous system (CNS) with similarities to multiple sclerosis (MS). To date, the lack of a suitable small animal model has hindered our quest to understand the immuno- and neuropathogenesis of HTLV-1 in an in vivo system. Previous work from others have established that host immune response plays a critical role in the outcome of HTLV-1 infection, which could be better tested in the context of humanized (hu) mice. Thus, we employ here the Rag1 as well as Bone marrow-Liver-Thymic (BLT) mouse models for engraftment of human CD34+ hematopoietic stem cells and followed HTLV-1 infection. Flow cytometry and histological analyses revealed reconstitution of Rag1 and BLT mice with human immune cells, including macrophages, dendritic cells, T cells and B cells. Proviral load (PVL) was determined in the peripheral blood, spleen, and other organs of neonatal and adult Rag1 and BLT hu-mice by droplet digital PCR. Tax showed peak expression at 14 wpi in Rag1 mice with continued expression until 16 weeks. Within blood, PVL and viral protein Tax was detected as early as 2 wks post-infection (wpi) in Rag-1 and BLT hu-mice. Successful infection was followed by immune activation and Tax expression within lymphoid organs. Moreover, lymphocytic infiltration with concomitant Tax expression and resulting myelin disruption were observed in the spinal cord of the infected mice. These data represents the first attempt to establish HTLV-1 neuropathogenesis in the context of RAG1 and BLT mice suggesting possibility of developing a small animal model of HAM/TSP in humanized mice.
Gene-based therapies represent a promising therapeutic paradigm for the treatment of HIV-1, as they have the potential to maintain sustained viral inhibition with reduced treatment interventions.Such an option may represent a long-term treatment alternative to highly active antiretroviral therapy.Methods: We previously described a therapeutic approach, referred to as transcriptional gene silencing (TGS), whereby small noncoding RNAs directly inhibit the transcriptional activity of HIV-1 by targeting sites within the viral promoter, specifically the 5' long terminal repeat (LTR).TGS differs from traditional RNA interference (RNAi) in that it is characterized by concomitant silent-state epigenetic marks on histones and DNA.To deliver TGS-inducing RNAs, we developed functional RNA conjugates based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV-1 siRNA (dsiRNA), LTR-362. Results:We demonstrate here that high levels of processed guide RNAs localize to the nucleus in infected T lymphoblastoid CEM cell line and primary human CD4+ T-cells.Treatment of the aptamer-siRNA conjugates induced TGS with an ~10-fold suppression of viral p24 levels as measured at day 12 post infection.To explore the silencing efficacy of aptamer-siRNA conjugates in vivo, HIV-1-infected humanized NOD/SCID/IL2 rγ null mice (hu-NSG) were treated with the aptamer-siRNA conjugates.Systemic delivery of the A-1-stick-LTR-362 27-mer siRNA conjugates suppressed HIV-1 infection and protected CD4+ T cell levels in viremia hu-NSG mice.Principle conclusions: Collectively these data suggest that the gp120 aptamer-dsiRNA conjugate design is suitable for systemic delivery of small RNAs that can be used to suppress HIV-1.
