Highly active antiretroviral therapy (HAART) has dramatically changed the prognosis for individuals infected with HIV-1. Yet, even when HIV-1 viremia has been well controlled by these drugs for years, termination of HAART results in viral rebound, most likely coming from latent provirus in long-lived memory CD4+ T cells. Most efforts to eradicate latent HIV-1 proviruses have focused on reactivation of proviral transcription to potentiate the elimination of reservoir cells harboring HIV-1 provirus, but these efforts have largely been unsuccessful. Alternative approaches for proviral elimination are therefore needed. Technological advances in gene editing tools provide a potential method for direct inactivation of latent HIV-1 provirus within reservoir cells. Specifically, the Cas9/CRISPR programmable nuclease system, a versatile platform for the generation of targeted double-strand breaks within the genome, has been shown to excise HIV-1 provirus in cell lines. However, the activity and precision of the Cas9/CRISPR system is suboptimal for clinical application. We have developed a novel nuclease architecture that combines the favorable cleavage activity of Cas9 with the targeting specificity of Transcription Activator-Like Effector (TALE) domains. This Cas9-TALE system dramatically improves the activity and precision of DNA cleavage. We propose to optimize this nuclease platform for the inactivation of HIV-1 provirus in memory CD4+ T cells, one of the primary cellular reservoirs of latent provirus. Development of a robust nuclease system for the efficient neutralization of provirus will also require a detailed map of the local chromatin landscape of the provirus in quiescent reservoir cells to identify areas at which it is particularly vulnerable to attack. However, obtaining the quantities of latently-infected primary cells needed for these analyses is not feasible. Consequently, in Aim 1 we will employ genome-editing technologies to generate populations of CD34+ cells containing a proviral insertion at a specific site and orientation within the genome. Once engrafted in NSG-BLT mice, these CD34+ cells will generate populations of resting CD4+ cells with uniform proviral integration sites for i-depth characterization, which may reveal important and unexpected drivers of latency in reservoir cells.
In Aim 2, we will optimize the Cas9-TALE system to efficiently and precisely inactivate HIV-1 provirus with a high degree of precision. Initial optimization will be performed i Jurkat-based cell lines and then will move to an in vitro model of latently-infected central memory CD4+ cells.
In Aim 3, these optimized nucleases will be evaluated on latent HIV provirus in a HAART-suppressed humanized mouse model of HIV-1 infection, and on resting CD4+ cells from patients undergoing HAART. Validated nucleases from our studies will be able to inactivate provirus efficiently from quiescent cells without compromising the host genome.

Public Health Relevance

Relevance of this Project to Public Health Our goal is to develop precise nucleases that will permit the targeted inactivation of latent HIV-1 provirus in patients. We have assembled a world-class group of investigators with expertise in a broad range of specialties who will leverage novel programmable nuclease technology, detailed analysis of the chromatin landscape of the provirus, and a humanized mouse model to achieve this end.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI117839-02
Application #
9010933
Study Section
Special Emphasis Panel (ZAI1)
Program Officer
Sanders, Brigitte E
Project Start
2015-02-15
Project End
2020-01-31
Budget Start
2016-02-01
Budget End
2017-01-31
Support Year
2
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Other Basic Sciences
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
Bolukbasi, Mehmet Fatih; Liu, Pengpeng; Luk, Kevin et al. (2018) Orthogonal Cas9-Cas9 chimeras provide a versatile platform for genome editing. Nat Commun 9:4856
Donnard, Elisa; Vangala, Pranitha; Afik, Shaked et al. (2018) Comparative Analysis of Immune Cells Reveals a Conserved Regulatory Lexicon. Cell Syst 6:381-394.e7
McCauley, Sean Matthew; Kim, Kyusik; Nowosielska, Anetta et al. (2018) Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines. Nat Commun 9:5305
Yurkovetskiy, Leonid; Guney, Mehmet Hakan; Kim, Kyusik et al. (2018) Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex. Nat Microbiol 3:1354-1361
Belaghzal, Houda; Dekker, Job; Gibcus, Johan H (2017) Hi-C 2.0: An optimized Hi-C procedure for high-resolution genome-wide mapping of chromosome conformation. Methods 123:56-65
Oomen, Marlies E; Dekker, Job (2017) Epigenetic characteristics of the mitotic chromosome in 1D and 3D. Crit Rev Biochem Mol Biol 52:185-204
Nora, Elphège P; Goloborodko, Anton; Valton, Anne-Laure et al. (2017) Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization. Cell 169:930-944.e22
Zhu, Lihua Julie; Lawrence, Michael; Gupta, Ankit et al. (2017) GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases. BMC Genomics 18:379
Bolukbasi, Mehmet Fatih; Gupta, Ankit; Wolfe, Scot A (2016) Creating and evaluating accurate CRISPR-Cas9 scalpels for genomic surgery. Nat Methods 13:41-50
Valton, Anne-Laure; Dekker, Job (2016) TAD disruption as oncogenic driver. Curr Opin Genet Dev 36:34-40

Showing the most recent 10 out of 14 publications