Humanized NOD/SCID/IL2rγnull (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 high demand. With a close genetic and behavioral resemblance to humans, small animals such as the mouse are good candidates for human disease models, through which human-like symptoms and responses can be recapitulated. Further, the mouse genetic background can be altered to accommodate diverse demands. The NOD/SCID/IL2rγnull (NSG) mouse is one of the most widely used immunocompromised mouse strains; it allows engraftment with human hematopoietic stem cells and/or human tissues and the subsequent development of a functional human immune system. This is a critical milestone in understanding the prognosis and pathophysiology of human-specific diseases such as HIV/AIDS and aiding the search for a cure. Herein, we report a detailed protocol for generating a humanized NSG mouse model (hu-NSG) by hematopoietic stem cell transplantation into a radiation-conditioned neonatal NSG mouse. The hu-NSG mouse model shows multi-lineage development of transplanted human stem cells and susceptibility to HIV-1 viral infection. It also recapitulates key biological characteristics in response to combinatorial antiretroviral therapy (cART).

Receptor-targeted aptamer-siRNA conjugate-directed transcriptional regulation of HIV-1.

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 rγnull 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.

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

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 in vivo For this reason, in vivo 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/IL2rγnull (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 in vivo 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 in vivo system in which to efficiently evaluate HIV replication, antiretroviral therapy, latency and persistence, and eradication interventions.IMPORTANCE 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 in vivo 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 in vivo 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.

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

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.