humanized-nodscidil2rnull-hu-nsg-mouse-model-for-hiv-replication

Humanized NOD/SCID/IL2r&#947;<sup>null</sup> (hu-NSG) Mouse Model for HIV Replication and Latency Studies

Ethical regulations and technical challenges for research in human pathology, immunology, and therapeutic development have placed small animal models in ...

Beckman Research Institute of City of Hope

The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here, we combine the live-cell-based SELEX with high-throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as small interfering RNA [siRNA] delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5-expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4(+) T cells with a nanomolar inhibitory concentration 50%. G-3 was also capable of transferring functional siRNAs to CCR5-expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties.

Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

Although current combinatorial antiretroviral therapy (cART) is therapeutically effective in the majority of HIV patients, interruption of therapy can cause a rapid rebound in viremia, demonstrating the existence of a stable reservoir of latently infected cells. HIV latency is therefore considered a primary barrier to HIV eradication. Identifying, quantifying, and purging the HIV reservoir is crucial to effectively curing patients and relieving them from the lifelong requirement for therapy. Latently infected transformed cell models have been used to investigate HIV latency; however, these models cannot accurately represent the quiescent cellular environment of primary latently infected cells  For this reason,  humanized murine models have been developed for screening antiviral agents, identifying latently infected T cells, and establishing treatment approaches for HIV research. Such models include humanized bone marrow/liver/thymus mice and SCID-hu-thy/liv mice, which are repopulated with human immune cells and implanted human tissues through laborious surgical manipulation. However, no one has utilized the human hematopoietic stem cell-engrafted NOD/SCID/IL2rγ (NSG) model (hu-NSG) for this purpose. Therefore, in the present study, we used the HIV-infected hu-NSG mouse to recapitulate the key aspects of HIV infection and pathogenesis  Moreover, we evaluated the ability of HIV-infected human cells isolated from HIV-infected hu-NSG mice on suppressive cART to act as a latent HIV reservoir. Our results demonstrate that the hu-NSG model is an effective surgery-free  system in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions. HIV can establish a stably integrated, nonproductive state of infection at the level of individual cells, known as HIV latency, which is considered a primary barrier to curing HIV. A complete understanding of the establishment and role of HIV latency  would greatly enhance attempts to develop novel HIV purging strategies. An ideal animal model for this purpose should be easy to work with, should have a shortened disease course so that efficacy testing can be completed in a reasonable time, and should have immune correlates that are easily translatable to humans. We therefore describe a novel application of the hematopoietic stem cell-transplanted humanized NSG model for dynamically testing antiretroviral treatment, supporting HIV infection, establishing HIV latency  The hu-NSG model could be a facile alternative to humanized bone marrow/liver/thymus or SCID-hu-thy/liv mice in which laborious surgical manipulation and time-consuming human cell reconstitution is required.

HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy.

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.  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.  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 , HIV-1-infected humanized NOD/SCID/IL2 rγ 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.  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.

Article	Year
Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity. Chemistry & biology\| PubMed ID: 25754473	2015
HIV Replication and Latency in a Humanized NSG Mouse Model during Suppressive Oral Combinational Antiretroviral Therapy. Journal of virology\| PubMed ID: 29343582	2018
Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1. Theranostics\| PubMed ID: 29556342	2018

Sangeetha Satheesan

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