# T cell-targeted lentiviral vectors with Cas9/RNP for the in vivo gene therapy of HIV-AIDS

> **NIH NIH R01** · YALE UNIVERSITY · 2023 · $762,160

## Abstract

ABSTRACT/SUMMARY
 Despite suppression by antiretroviral therapy (ART), virus is not eliminated in HIV patients and can
rebound causing full-blown infection upon ART interruption. Thus, a strategy to eliminate the virus reservoir is
urgently needed. The recently discovered gene editing technique called CRISPR has tremendous potential for
eradicating HIV-1. CRISPR is comprised of a Cas9 nuclease and chimeric guide RNA (gRNA). When Cas9
and gRNA designed to target HIV sequences are present in latently-infected cells, it can result in disruption of
the integrated proviral genome, permanently inactivating the virus. The biggest challenge to using the CRISPR
approach for HIV elimination is the absence of an in vivo delivery system for human T cells, the major cellular
of HIV-1.
 This is a R01 application in response to RFA-AI-18-016, “Targeted In Vivo Delivery of Gene
Therapeutics for HIV Cure”. To address the challenge of Cas9/gRNA delivery to human T cells in vivo, we
propose the use of a T cell-targeting lentivirus whose tropism is guided by antibodies to human CD7, a
molecule expressed at high levels on all human T cells, including resting T cells which are a major reservoir for
latent HIV. To address concerns of vector integration and constitutive Cas9 expression, we have generated
lentiviruses that are pre-packaged with Cas9 ribonucleoproteins with no integrating DNA components. Proof-
of-concept studies in virologically-suppressed HIV-infected humanized mice demonstrate that disrupting
CCR5, the coreceptor for HIV-1, with this systemic approach results in ART-free virologic remission.
Importantly, as the approach does not require activation or elimination of the infected cell, it addresses the
limitations of conventional `Shock and Kill' approaches that have yielded promising results in clinical settings.
The proposal has three specific aims-
In Specific Aim 1, we will design and test broad-spectrum gRNAs targeting HIV DNA in two independent
approaches expected to mutate a segment or excise the entire length of the integrated HIV provirus. The
approach will be tested in ART-suppressed humanized mice for impact on virus reservoir and rebound. A
comprehensive investigation of toxicity, off-target effects and virus escape will also be undertaken. In Specific
Aim 2, we will perform functional studies in patient-cell derived humanized mice to determine the effects of
broad-spectrum gRNAs on latent virus quasispecies from HIV+ patients. The studies will employ HIV-1 RNA
Sort-Seq, a novel methodology to quantitate the inducible replication-competent HIV reservoir. In Specific Aim
3, we incorporate strategic changes in the lentiviral vector to reduce vector-associated immunogenicity and
permit a single or multiple but rapid-dosing regimen with enhanced potency.
 The outcome of these proof-of-principle studies is expected to establish a solid platform for future
studies on an approach that could significantly contribute towards a cure for HIV-AIDS.

## Key facts

- **NIH application ID:** 10322128
- **Project number:** 5R01AI145164-04
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Priti Kumar
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $762,160
- **Award type:** 5
- **Project period:** 2019-01-23 → 2024-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10322128

## Citation

> US National Institutes of Health, RePORTER application 10322128, T cell-targeted lentiviral vectors with Cas9/RNP for the in vivo gene therapy of HIV-AIDS (5R01AI145164-04). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10322128. Licensed CC0.

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