. Latent HIV infection within the CNS remains a major problem. Precisely identifying the latently infected cell types, the mechanisms of viral reactivation, and the approach for elimination of HIV-1 in the CNS have been the topics of much research over the years. CRISPR/Cas9 has shown promise in removing productively infect- ed cells from the periphery, but its penetration to and impact in the brain has yet to be investigated. The salient feature of this method lies in its ability to edit the viral genome and permanently inactivate it in the latently in- fected cells by removing a large fragment of viral DNA with no requirement for latent virus activa- tion/reawakening. The Long-Term Goal is to establish a basis for eradication of HIV from reservoirs, including the CNS. The Objective of this Application is to develop a strategy for, and a means of measuring effective- ness of, removing virus from latently infected cells using CRISPR/Cas9, without killing the infected cell. The Central Hypothesis is that removal of key stretches of integrated DNA from the host cell would render the virus replication incompetent, and that this would result in elimination of viral protein production, which has a neuro- toxic effect, i.e. Tat, gp120 and then secretion in the brain environment. This is based this on previous work from the interdisciplinary team of neuroimmunology (Khalili & MacLean), HIV-1 molecular geneticist (Khalili), glia activation in brains in the absence of viral replication (MacLean), experience with reactivation of latently in- fected T cells (Ling), and gene therapy based approaches to chronic diseases (Bunnell). We will employ CRISPR/Cas9 gene editing strategy to excise various regions of SIV-1 including the sequences between the 5'-LTR and gag, as well as the entire viral coding sequence located between the 5'- and 3'-LTRs. The Ra- tionale for these studies, is that once this project is completed, it will be possible to eradicate HIV from CNS reservoirs without causing deleterious neuroinflammation.
Two Specific Aims are proposed:
Specific Aim 1 : Determine the phenotype of latently-infected cells in the CNS. Microglia and astrocytes are the major res- ervoirs for viral latency in the CNS. We hypothesize that latent infection (integrated DNA) of these cells results in irreversible activation or other phenotypic changes that can distinguish latently infected cells in the CNS. Our central hypothesis for this aim is that CRISPR/Cas9 gene editing will reduce or even reverse this activation with regard to innate immune activation, and expression of proinflammtory cytokines and viral proteins. Specif- ic Aim 2: Deliver CRISPR/Cas9 to the CNS using an adeno-associated viral delivery vector. Our hypothe- sis here is that AAV will facilitate delivery of CRISPR/Cas9 through the blood-brain barrier to latently infected cells following SIV infection, and by inference, HIV infection. Our central hypothesis is that the removal of longer sequences of virus will substantially reduce viral reservoirs, and the inflammatory profile of CNS cells in HIV infected individuals.

Public Health Relevance

HIV infection results in CNS complications, including those on combined antiretroviral therapies. Removing viral-infected cells in the brain, without inducing further inflammation has proven problematic, although CRISPR/Cas9 has shown promise in removing productively infected cells from the periphery. This application seeks to utilize our expertise in primate gene therapy to improve CRISPR/Cas9 penetration into the brain of SIV-infected monkeys.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS104016-02
Application #
9473820
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Wong, May
Project Start
2017-05-01
Project End
2022-01-31
Budget Start
2018-02-01
Budget End
2019-01-31
Support Year
2
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Tulane University
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
053785812
City
New Orleans
State
LA
Country
United States
Zip Code
70118
Peterson, Tiffany A; MacLean, Andrew G (2018) Current and Future Therapeutic Strategies for Lentiviral Eradication from Macrophage Reservoirs. J Neuroimmune Pharmacol :
Delery, Elizabeth C; MacLean, Andrew G (2018) Chronic Viral Neuroinflammation: Speculation on Underlying Mechanisms. Viral Immunol :
Xu, Wanli; Luo, Zhenwu; Alekseyenko, Alexander V et al. (2018) Distinct systemic microbiome and microbial translocation are associated with plasma level of anti-CD4 autoantibody in HIV infection. Sci Rep 8:12863
Kumar, Vinay; Mansfield, Joshua; Fan, Rong et al. (2018) miR-130a and miR-212 Disrupt the Intestinal Epithelial Barrier through Modulation of PPAR? and Occludin Expression in Chronic Simian Immunodeficiency Virus-Infected Rhesus Macaques. J Immunol 200:2677-2689
Ramsey, J; Martin, E C; Purcell, O M et al. (2018) Self-injurious behaviours in rhesus macaques: Potential glial mechanisms. J Intellect Disabil Res 62:1008-1017
Jiang, Wei; Luo, Zhenwu; Martin, Lisa et al. (2018) Drug Use is Associated with Anti-CD4 IgG-mediated CD4+ T Cell Death and Poor CD4+ T Cell Recovery in Viral-suppressive HIV-infected Individuals Under Antiretroviral Therapy. Curr HIV Res 16:143-150
Perez, Stefanie; Johnson, Ann-Marie; Xiang, Shi-Hua et al. (2018) Persistence of SIV in the brain of SIV-infected Chinese rhesus macaques with or without antiretroviral therapy. J Neurovirol 24:62-74
Veazey, Ronald S; Ling, Binhua (2017) Short Communication: Comparative Susceptibility of Rhesus Macaques of Indian and Chinese Origin to Vaginal Simian-Human Immunodeficiency Virus Transmission as Models for HIV Prevention Research. AIDS Res Hum Retroviruses 33:1199-1201