# Enhancing efficacy of virus-specific T cell therapy following hematopoietic stem cell transplantation by CD52 knockout

> **NIH NIH R33** · CHILDREN'S RESEARCH INSTITUTE · 2024 · $561,030

## Abstract

Viral infections are common and can be severe in immunocompromised patients, including those undergoing
hematopoietic stem cell transplantation (HSCT) or solid organ transplantation (SOT). Virus-specific T cells
(VSTs) have been effective in prevention and treatment of viral infections in patients post HSCT or SOT, but are
inactivated or eliminated by immunosuppressive therapies including alemtuzumab, which limits their application
and efficacy. We have demonstrated that CD52 knockout via CRISPR/Cas9 can be performed in CMV-specific
T cells with maintenance of antiviral specificity. The overarching goal of this proposal is to study the impact of
CD52-knockout on the function of multivirus-specific T cells, including antiviral specificity, cytokine profile, and
cytotoxicity, as well as the persistence and safety of CD52-KO VSTs in vivo. In this study, we will address the
following specific aims: 1) To establish the biological activity of multivirus-specific CD52-KO T cells in comparison
with non-edited virus-specific T cells, and 2) To determine if CD52-KO enables T cell persistence and efficacy
in the presence of alemtuzumab in vivo. To evaluate CD52-KO as a preventative cellular therapy, we propose
to optimize CRISPR/Cas9 editing of rapidly-expanded multiviral T cells (targeting cytomegalovirus, Epstein-Barr
virus (EBV), and adenovirus) from healthy donors, followed by extensive characterization of the phenotype and
function of CD52-KO VSTs versus mock-edited VSTs, including surface markers, cytokine profile, single cell
RNA-sequencing, and T cell receptor sequencing. Using a mouse model, we will evaluate the persistence and
safety of CD52-KO VSTs in the presence of alemtuzumab. To assess function in vivo, we will test CD52-KO or
mock-edited VSTs against EBV-lymphoblastoid tumors in mice, in the presence or absence of alemtuzumab.
Finally, we will perform off-targeting analyses of the lead single guide RNA candidates by in silico and orthogonal
testing of CD52-KO and mock-edited VSTs. We hypothesize that CD52-KO will have negligible impact on VST
phenotype and function, and will enable persistence and activity in the presence of alemtuzumab in vivo.
Completion of this study would provide a novel antiviral therapy which could reduce virus-associated morbidity
in HSCT, and will develop a pipeline for production of a next generation of gene-modified VSTs to enhance
efficacy in immunocompromised patients.

## Key facts

- **NIH application ID:** 10991125
- **Project number:** 1R33HL175692-01
- **Recipient organization:** CHILDREN'S RESEARCH INSTITUTE
- **Principal Investigator:** Michael Daniel Keller
- **Activity code:** R33 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $561,030
- **Award type:** 1
- **Project period:** 2024-09-05 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10991125

## Citation

> US National Institutes of Health, RePORTER application 10991125, Enhancing efficacy of virus-specific T cell therapy following hematopoietic stem cell transplantation by CD52 knockout (1R33HL175692-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10991125. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*