# Non-viral delivery of CRISPR/Cas9 for targeted gene replacement

> **NIH NIH R00** · UNIVERSITY OF ARKANSAS AT FAYETTEVILLE · 2020 · $245,989

## Abstract

PROJECT SUMMARY
Hemophilia A and B are monogenic disorders characterized by the loss of factor VIII (F8) or factor IX (F9) leading
to delayed blood clotting and life-long treatment with recombinant clotting factors or blood products. Gene
therapy has held promise for treating monogenetic diseases like hemophilia, however, has yet to deliver a
curative treatment. Gene delivery barriers and the potential requirement of costly repetitive treatments
characterize the drawbacks of technologies currently in the therapeutic pipeline. Gene editing has emerged as
a method to make permanent modifications to the host genome. Specifically, RNA-guided nucleases (RGNs)
have the potential to correct the genetic basis of a broad range of diseases including hemophilia. However,
significant delivery barriers of RGNs or gene-encoded RGNs hinder in vivo gene editing. To date, the most
reported in vivo gene editing approaches have used viral delivery vehicles due to robust expression and available
tissue tropism. An exciting alternative is the generation of effective non-viral delivery vehicles for RGNs that
address the safety concerns of viral delivery vehicles. Therefore, the overall goal of this work is to create a
powerful biomacromolecule delivery vector for genome-editing technologies. The central hypothesis of this work
is that non-viral delivery of gene-editing nucleases can precisely target replacement genes to safe-harbor loci in
the human genome and restore protein expression in hemophilia A or B. The hypothesis will be evaluated with
the following specific aims: 1) Characterize viral and non-viral gene replacement safety and efficacy in murine
liver. 2) Apply viral and non-viral gene replacement in mouse models of hemophilia, and 3) Develop a screening
platform for non-viral gene delivery vehicles. In Aim 1, intravenous delivery of CRISPR/Cas9 will be evaluated
for efficient gene replacement. Promising preliminary data for gene delivery to liver has been demonstrated by
the PI for viral and non-viral delivery. Both viral and non-viral delivery strategies will be pursued in parallel.
Quantitative readouts for single base pair changes, small insertions, and large insertions will be used to assess
the efficacy of the delivery routes and determine immune response and off-target activity. In Aim 2, mouse
models of hemophilia will be evaluated for genomic modification and recovery of the absent clotting factor. Factor
VIII is a model of a large insertion and FIX is a model of a small insertion. In Aim 3, we propose to combine deep
sequencing with gene editing to screen libraries of non-viral compounds in small numbers of animals. This
method overcomes the limitations of high-throughput in vitro screens that cannot recapitulate complex in vivo
delivery barriers. The end result of this work will be an efficient non-viral gene editing toolbox for targeted gene
replacement. In addition, this work will generate new strategies for gene editing and gene therapy to impr...

## Key facts

- **NIH application ID:** 9990774
- **Project number:** 5R00EB023979-03
- **Recipient organization:** UNIVERSITY OF ARKANSAS AT FAYETTEVILLE
- **Principal Investigator:** Christopher Nelson
- **Activity code:** R00 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $245,989
- **Award type:** 5
- **Project period:** 2019-08-15 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9990774

## Citation

> US National Institutes of Health, RePORTER application 9990774, Non-viral delivery of CRISPR/Cas9 for targeted gene replacement (5R00EB023979-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9990774. Licensed CC0.

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