There are no methods currently available for the eradication of the latent reservoir in HIV-infected patients. The long-term goal of the proposed research is to develop a new method based on synthetic molecular sensors that is capable of targeting latently infected cells in HIV-infected patients for destruction. The objective of this particular application is to design constructs known as "Synthetic Molecular Sensors for HIV Eradication (SMaSHEd)" and determine their ability to eradicate latently infected cells from HIV-infected patients or from in vitro latency models. The central hypothesis is that the promoter regions from host genes and the binding sites for miRNAs whose expression can discriminate latently infected from uninfected cells can be used as sensors in SMaSHEd constructs to specifically express a suicide gene in latently infected cells. The rationale for the proposed research is that new methods for eradicating the latent reservoir are required that specifically target latently infected cells, and do not directly target the variable HIV provirus, require activation of HIV replication, or rely on the host immune system to eradicate activated cells. Guided by strong preliminary data, our hypothesis will be tested by pursuing the following specific aims: (1 &2) Identify genes and miRNAs whose expression can discriminate latently infected primary CD4 T cells from uninfected cells, and (3) Determine the ability of SMaSHEd constructs to induce cell death in latently infected primary CD4 T cells. The analyses in Aim 1 &2 will utilize RNA-Seq and smallRNA-Seq to identify the genes and miRNAs, respectively that are differentially expressed between latently infected and uninfected primary memory CD4 T cells derived from the in vitro model of our collaborator Dr. Vincente Planelles.
Our final aim wil utilize procedures well established in the realm of synthetic biology to design SMaSHEd constructs containing the promoters and miRNA binding sites of genes (Aim 1) and miRNAs (Aim 2), respectively, that specifically sense the environment in latently infected cells. The abilty of these constructs to induce the expression of a suicide gene specifically in latently infected primary CD4 T cells will be assessed following transfection or transduction of mixtures of latently infected and uninfected cells from the in vitro primary CD4 T cell latency model of Dr. Planelles and of Dr. O'Doherty, as well as from HIV-infected patients. The research proposed in this application is innovative, in our opinion, because it will utilize the latest advances in synthetic biology and information gained from next generation sequencing platforms (i.e., RNA-Seq and smallRNA-Seq) to develop a novel method for eradicating latently infected cells. This will be significant because it is the first step in a continuum of research that is expected to lea to the development of a new strategy for the eradication of the latent reservoir in HIV-infected patients. Ultimately, SMaSHEd constructs will be developed for use with appropriate vectors (i.e., adeno-associated virus) for the treatment of HIV-infected patients, where, in contrast to gene therapy, they will only require transient expression to induce the destruction of latently infected cells.
This research is relevant to public health since it will develop the knowledge necessary for the design and testing of new therapeutic approaches for the eradication of latently infected cells from HIV-infected patients. Such an approach may eventually lead to a cure for HIV. Thus, the proposed research is relevant to the part of the NIAID's mission that pertains to the better understanding and treatment of infectious diseases.
|Reardon, Brian; Beliakova-Bethell, Nadejda; Spina, Celsa A et al. (2015) Dose-responsive gene expression in suberoylanilide hydroxamic acid-treated resting CD4+ T cells. AIDS 29:2235-44|
|Bonczkowski, Pawel; De Spiegelaere, Ward; Bosque, Alberto et al. (2014) Replication competent virus as an important source of bias in HIV latency models utilizing single round viral constructs. Retrovirology 11:70|