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Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users

Award Number: R01DA059551
ORGANIZATION: NATIONAL INSTITUTE ON DRUG ABUSE
OPDIV: NIH
AWARD CLASS: DISCRETIONARY
AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)
PERIOD OF PERFORMANCE START DATE: 09/15/2023
PERIOD OF PERFORMANCE END DATE: 07/31/2028

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Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users - ABSTRACT Opioid users are prime beneficiaries of cure strategies for HIV due to their medical and social vulnerabilities and low adherence to antiretroviral therapies, but the nature of the latent reservoir in people using morphine or heroin is unknown. The central hypothesis of this multi-PI proposal is that HIV DNA+ cells from opioid users show unique transcriptional and proteomic signatures that provide fundamental insight into initiation, establishment and maintenance of the latent reservoir in drug users. This hypothesis is based on our recent data in Nature characterizing HIV DNA+ CD4+ T cells without prior activation using a new sorting and sequencing strategy called FIND-Seq. We further show that specific silencing and cell survival pathways are altered in latently infected cells and identify 55 differentially expressed genes (DEGs) implicated in the underlying biology of HIV latency. Here we propose to extend these studies to tissue reservoirs from opioid users, test the functional relevance of DEGs on latency biology, and develop important genomic, proteomic and biochemical advancements of FIND-Seq. The central hypothesis will be tested in four specific aims: 1) Define the cellular mechanisms of HIV persistence and their modulation by morphine in HIV-infected T cells and microglia from Last Gift participants. We will use FIND-seq to sort and RNA sequence HIV+ cells from the spleen, gut, and brain of Last Gift participants with and without morphine to elucidate the role of opioids on latently infected T cells and microglia across different tissues. 2) Determine how DEGs and opioids regulate HIV latency in T cells and microglia. We will perform ex vivo CRISPR activation/interference experiments with isolated CD4+ T cells and microglia derived from induced pluripotent stem cells to determine the functional relevance of identified DEGs for latency establishment with and without morphine or heroin. 3) Perform a proteogenomic analysis of HIV+CD4+ T cells from the spleen of Last Gift participants. We will use DAb-seq, which allows high-coverage genome sequencing and simultaneous analysis of cell-surface protein expression on CD4+ T cells from spleens from Last Gift participants with or without morphine to determine the intactness of the provirus, its integration site, and changes in protein expression of latently infected cells induced by opioid use. 4) Simultaneously sequence the RNA, protein, and full HIV genome from HIV DNA+ cells. We will develop FIND-Seq into a multimodal assay that sorts HIV DNA+ cells on a flow cytometer and simultaneously performs genomic, transcriptomic, and proteomic analysis on recovered cells. We have assembled a highly complementary team of world experts in the biology and advanced investigation of HIV latency with cutting-edge microfluidics, CRISPR and multi-omics sequencing technologies combined with leading experts in clinical care of people living with HIV and opioid use from the Last Gift Cohort and the Johns Hopkins HIV Clinical Cohort. We expect to gain paradigm-shifting insight into the “underlying molecular mechanisms by which HIV latency is initiated, established, and maintained in the CNS, lymphoid and myeloid tissues and how substance use might influence these processes” and are fully aligned with this RFA.


Issue Date FY	Funding FY	Legal Entity Name	Legal Entity Address	Legal Entity City	Legal Entity State	Legal Entity Zip Code	Legal Entity COUNTY	Legal Entity COUNTRY	Assistance Listing	Award Code	Budget Year	Action Date	Action Type	Action Amount

Issue Date FY: 2026 ( Subtotal = $1,550,268 )
2026	2026	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Drug Use and Addiction Research Programs	000	4	7/28/2026	NON-COMPETING CONTINUATION	$1,550,268
														Subtotal = $1,550,268

Issue Date FY: 2025 ( Subtotal = $1,557,466 )
2025	2025	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Drug Use and Addiction Research Programs	001	3	8/11/2025	NON-COMPETING CONTINUATION	$1,557,466
2025	2024	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Drug Use and Addiction Research Programs	000	2	8/11/2025	NON-COMPETING CONTINUATION	$0
														Subtotal = $1,557,466

Issue Date FY: 2024 ( Subtotal = $1,560,507 )
2024	2024	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Drug Use and Addiction Research Programs	000	2	7/30/2024	NON-COMPETING CONTINUATION	$1,560,507
														Subtotal = $1,560,507

Issue Date FY: 2023 ( Subtotal = $1,630,854 )
2023	2023	J.DAVID GLADSTONE INSTITUTES	1650 OWENS ST	SAN FRANCISCO	CA	94158	SAN FRANCISCO	USA	Drug Use and Addiction Research Programs	000	1	9/15/2023	NEW	$1,630,854
														Subtotal = $1,630,854

Grand Total All Awards = $6,299,095

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Sorting and Sequencing Latent Reservoirs in HIV+ Opioid Users

Award Number: R01DA059551

ORGANIZATION: NATIONAL INSTITUTE ON DRUG ABUSE

OPDIV: NIH

AWARD CLASS: DISCRETIONARY

AWARD ACTIVITY TYPE: SCIENTIFIC/HEALTH RESEARCH (INCLUDES SURVEYS)

PERIOD OF PERFORMANCE START DATE: 09/15/2023

PERIOD OF PERFORMANCE END DATE: 07/31/2028

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