# Nonenzymatic Gene Editing in Treatment of Heredity Spherocytosis

> **NIH NIH R01** · YALE UNIVERSITY · 2020 · $598,421

## Abstract

Project Summary/Abstract
Approximately a quarter of patients with hereditary spherocytosis, a common inherited anemia, suffer from alpha-
spectrin linked recessive HS (rHS), the most severe form of the disease. rHS patients present in infancy with
life-threatening hemolytic anemia, many are transfusion-dependent. In these patients, splenectomy is only
palliative; the only cure is hematopoietic stem cell (HSC) transplant. Development of genome engineering has
expanded the repertoire of potential strategies for treatment of inherited erythrocyte disorders. We have
developed a non-nuclease based gene editing technique using biocompatible and biodegradable nanoparticles
(NP) encapsulating chemically modified peptide nucleic acids (PNAs) and donor DNAs. Using this strategy, we
have cured a murine model of beta-thalassemia using IV injections of NP-PNAs, achieving gene correction of
6% after a single treatment with unmodified PNA, establishing proof-of-principle and demonstrating feasibility of
our approach. This technique avoids ex vivo manipulation and its associated challenges, as well as obviates
most of the genotoxicity associated with nuclease-based methods. This project leverages a multi-PI collaborative
effort to develop the NP-PNA approach for gene editing in sph mice, a spontaneous murine model of rHS due to
a point mutation in the alpha-spectrin gene. It addresses the hypothesis that NP-PNAs and donor DNAs can be
used to correct the alpha-spectrin mutation in an in vivo model of HS at clinically relevant frequencies sufficient
to ameliorate the HS disease phenotype with minimal toxicity and extremely low genomic off-target effects. We
will establish robust protocols for in vivo DNA modification in hematopoietic cells after simple IV administration
of NP-PNAs, providing a facile, non-toxic strategy for treatment of HS without the need for complex
transplantation procedures or ex vivo manipulation. The goal of aim one is to optimize triplex-forming PNAs for
site-specific gene editing of the alpha-spectrin gene. Relevant studies include assays of gene editing, off target
effects, and genotoxicity. The goal of aim two is to identify and develop nanoparticle formulations for systemic in
vivo editing of the alpha-spectrin gene in hematopoietic stem and progenitor cells (HSPCs). Relevant studies
include development and characterization of NPs with novel size and polymer composition to improve target
delivery of PNAs after systemic administration to HSPCs as well as improving penetration of the bone marrow
compartment. The goal of aim three is the establishment of robust gene editing protocols for in vivo modification
of the alpha-spectrin gene in HSPCs in alpha-spectrin deficient sph/sph mice to ameliorate or cure the HS
phenotype. Results will be monitored by detailed laboratory analyses of the HS phenotype, including functional
analyses of the erythrocyte membrane. Gene editing efficiency in hematopoietic stem cells and genotoxicity will
also be ...

## Key facts

- **NIH application ID:** 9851927
- **Project number:** 5R01HL139756-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** PATRICK G GALLAGHER
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $598,421
- **Award type:** 5
- **Project period:** 2019-02-01 → 2022-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9851927

## Citation

> US National Institutes of Health, RePORTER application 9851927, Nonenzymatic Gene Editing in Treatment of Heredity Spherocytosis (5R01HL139756-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9851927. Licensed CC0.

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