# Graft Engineering of Allogeneic Hematopoietic Stem Cell Products with Molecular Cascades

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2020 · $665,709

## Abstract

Proposal Summary
Subpopulations of hematopoietic cells with different functions are identified based on the presence or absence
of cell surface markers. Certain subpopulations are hypothesized to mediate forms of graft-vs-host disease
(GVHD) in allogeneic hematopoietic stem cell transplantations (aHSCT). The most advanced efforts to
minimize these side effects, and to have reached clinics, rely on an extended three step immunomagnetic
separation protocol with sequential positive selection (for protection of stem cells from elimination based on
CD34 expression), negative selection (elimination of naïve TN-cells based on CD45RA expression), and
subsequent return of positively selected stem cells to the graft that is then introduced into patients. The
engineered graft, however, is also depleted of B-, NK-, and Treg-cells, which are essential for control of
infectious complications, modulation of GVHD, and relapse control, thus impacting long-term survival after
transplantation. Rapid and efficient single-step positive or negative selections that would minimize handling of
cells out of their natural niches would also improve cell health likely leading to better clinical outcomes. Highl
precision single-step procedures would also provide us with a powerful tool for rigorous and reliable studies of
the impact of high quality preparations of specific subpopulations of cells in animal models of disease.
We have recently developed molecular computing cascades based on oligonucleotides conjugated to targeting
moieties (such as antibodies, their fragments, and aptamers). These are mixtures of targeting moieties that
carry molecular computing elements and produce one outcome, a specific label on a single cell subpopulation,
based on taking into account all targeting moieties. Thus, what would be, for example, a three-step separation
protocol or a three-colored-fluorescence characterization in flow cytometry is condensed into a single step
isolation procedure or a single color in flow cytometry. These computing mixtures of oligonucleotide
conjugates with targeting moieties are thus uniquely suited for single-step `mix-and-separate' cell labelling
protocols for magnetic separations. Our cascades can block isolation based on the presence of a cell surface
marker or they can amplify signal coming from surface markers with low expression levels.
In this project we will ask two principal questions: (1) What are the practical limitations, with metrics being yield,
purity, and health of cells, of molecular computing cascades when applied to isolation of cell-subpopulations for
clinical applications? And, (2) What are the minimal subpopulation of cells that we have to either preserve or
eliminate to minimize induction of various forms of GVHD. In three Aims we will address translational and
mechanistic questions regarding graft engineering and the induction of GVHD using increasingly complex
protocols. We will characterize the impact of cell populations with differen...

## Key facts

- **NIH application ID:** 9872167
- **Project number:** 5R01EB025221-03
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** Markus Y Mapara
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $665,709
- **Award type:** 5
- **Project period:** 2018-06-01 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9872167

## Citation

> US National Institutes of Health, RePORTER application 9872167, Graft Engineering of Allogeneic Hematopoietic Stem Cell Products with Molecular Cascades (5R01EB025221-03). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/9872167. Licensed CC0.

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