# Development of a "Cell Splicing" Technology Platform

> **NIH NIH R21** · JOHNS HOPKINS UNIVERSITY · 2021 · $245,625

## Abstract

Project Summary:
The general scientific community already separates different layers or subcellular fractions (i.e., membrane vs.
cytoplasm vs. nucleus) as well as subcomponent organelles/machinery (such as mitochondria, lysosomes, etc.)
in order to study and better understand cell function. This Trailblazer research proposal seeks to discover how
we might repurpose such components, with major emphasis presently on nuclear transfer or exchange as part
of a new synthetic biology approach in creating cell-based therapies. Such efforts will lead to the development
of a massively expanded toolbox of interventional therapies with a wide array of potential downstream
applications in biomedicine (i.e., treatments for cancer, genetic and infectious disease, autoimmunity, and tissue
injury and repair). This will be achieved through the following:
 1) Generate methods to efficiently isolate nuclei from macrophage and T cells for fusion into
enucleated red blood cells and platelets. Methods for nuclear isolation will first be optimized using drug and
density centrifugation-induced cellular blebbing and fractionation to isolate nuclei- vs. cytoplasmic component-
containing vesicles, called karyoplasts and cytoplasts, respectfully. Karyoplasts will be derived from innate
immune macrophage and adaptive immune T cells, and then fused (with PEG) into naturally enucleated RBCs
and platelets, and derived cell constructs will be monitored for viability and function over time.
 2) Develop storage, freezing, and thawing requirements to maintain viability of cell-derived
cytoplasts and karyoplasts, and fusion constructs. This will be done by exploring different freezing media
types, constituent chemical concentrations, or altered protocol temperature kinetics to both store (short vs. long-
term) as well as thaw cells or their components with preserved structure and function (Figure 1, middle).
 3) Characterize macrophage- & T cell-derived cytoplasts, as well as new variant cells following
nuclear exchange between enucleated macrophage and T cell bodies. Prior enucleation studies show
modified cell behavior, therefore it is not only of interest to investigate nuclear exchange but also what happens
to enucleated cells. In addition, nuclear exchange will be attempted with both fresh as well as frozen karyoplast
and cytoplast components, with all fusion constructs tested for morphology/viability, proliferation, cytokine
expression, and behaviors either derived or distinct from donor cells. This approach will also allow us to
determine how constructs may be tunable as part of a larger plug and play system.
 4) Test new constructs in functional assays in vitro and in a therapeutic cancer model in vivo. This
strategy will provide a platform to create new cell behaviors related to functional activities like macrophage-
related adherence and phagocytosis, as well as T cell-mediated perforin/granzyme cytolysis. Therefore,
constructs will be tested in vitro in adhesion, migratio...

## Key facts

- **NIH application ID:** 10218482
- **Project number:** 1R21EB030036-01A1
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Joshua Charles Doloff
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $245,625
- **Award type:** 1
- **Project period:** 2021-06-10 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10218482

## Citation

> US National Institutes of Health, RePORTER application 10218482, Development of a "Cell Splicing" Technology Platform (1R21EB030036-01A1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10218482. Licensed CC0.

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