# Mechanisms of PNA-mediated gene therapy for hematologic disease

> **NIH NIH F30** · YALE UNIVERSITY · 2020 · $30,300

## Abstract

PROJECT SUMMARY
DESCRIPTION: Hematologic disorders such as sickle cell disease, β-thalassemia, and hereditary
spherocytosis are caused by hundreds of characterized monogenetic mutations and affect hundreds of
thousands of newborn infants each year. The consequences of such diseases result in billions in yearly
healthcare costs and an estimated yearly loss of over 15 million disability adjusted life years (DALYs) attributed
to hemoglobinopathies alone. Gene editing technologies hold tremendous potential in the curative treatment of
hematologic, monogenetic disease via site-directed mutation correction of hematopoietic stem and progenitor
cells (HSPCs). While initially promising, CRISPR/Cas9-mediated approaches to treatment have proven difficult
due to low engraftment frequencies after ex vivo treatment and safety concerns related to double strand break
(DSB) induction by the Cas9 nuclease. As an alternative to nuclease-based editing approaches, triplex-forming
peptide nucleic acids (PNAs) have been shown to promote site-directed corrective recombination using co-
delivered short ssDNA templates. Further, when co-encapsulated in polymeric nanoparticles, and in contrast to
Cas9 technologies, PNAs and ssDNA donors can be safely delivered directly in vivo or in utero to create
heritable, disease-ameliorating sequence correction in a murine model of β-thalassemia. To further develop
this technology with the goal of eventual translation to human therapies, it is critical to determine the precise
repair mechanism by which PNAs promote corrective recombination. Our hypothesis is that PNA-mediated
editing takes advantage of non-mutagenic DNA repair pathways that target a single strand of genomic DNA,
thus yielding an efficient and safe mechanistic basis for eventual in vivo therapeutic use in humans. SPECIFIC
AIMS: In order to test our hypothesis and characterize the mechanism of PNA-mediated editing we have set
out two specific aims. First, using an RNAi screen of known DNA repair factors in the genome and a
fluorescent reporter of gene editing, we will elucidate the key repair factors responsible for PNA editing. The
results of aim 1 will reveal implicated repair pathways and allow us to develop a model describing this
phenomenon. Next, in aim 2, I will manipulate DNA repair targets in order to bias pathways of recombination
and improve PNA editing efficiency. I will test small molecule inhibitors, biological inhibitors such as antibodies,
and co-delivered siRNAs against repair targets to rationally improve PNA editing frequency. All results from
both aims will be validated in human CD34+ HSCs.

## Key facts

- **NIH application ID:** 10021412
- **Project number:** 5F30HL149185-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Nicholas George Economos
- **Activity code:** F30 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $30,300
- **Award type:** 5
- **Project period:** 2019-09-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10021412

## Citation

> US National Institutes of Health, RePORTER application 10021412, Mechanisms of PNA-mediated gene therapy for hematologic disease (5F30HL149185-02). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10021412. Licensed CC0.

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