# Human genetic supplementation without donor DNA or a DNA break

> **NIH NIH DP1** · UNIVERSITY OF CALIFORNIA BERKELEY · 2022 · $79,652

## Abstract

ABSTRACT
Human genome engineering has widely anticipated promise as a healthcare strategy, but current technologies
are unlikely to provide the safe, efficient, and broadly useful implementation of transgene introduction essential
to complete the next big leap forward for gene therapy. CRISPR-based approaches for transgene integration
have major impediments, including the need for donor DNA delivery, the propensity of that DNA to undergo
non-specific integration, and the low efficiency of repair by homologous recombination relative to sloppy
rejoining of the broken DNA ends. Also severely limiting is the fact that slowly proliferating cells are rarely in a
cell cycle phase favorable for homologous recombination, and just the presence of a DNA break can be toxic.
The alternative approach of adeno-associated virus introduction of a transgene also has limitations, among
others including the small transgene size permitted by the virus capsid and the challenges of engineering virus
uptake into different cell types. It remains an unmet need to have a non-mutagenic, non-toxic approach for
gene introduction to the human genome. Therapy for many loss-of-function pathologies hinges on this missing
technology. Also, only transgene introduction offers the opportunity for non-native control of protein expression,
isoform selectivity, and myriad other clinically useful outcomes.
 Starkly missing from current efforts to develop transgene introduction techniques is an approach exploiting
the gene insertion strategy widespread endogenously across eukaryotes: cDNA synthesis. The ancestral,
evolutionarily persistent type of eukaryotic LINE/non-LTR retroelement integrates by nick-primed reverse
transcription that is rigorous both it its sequence specificity of target site selection and in its specificity for use
of an RNA transcript with the retroelement 3’ UTR as template. The biochemical activities required for target
site selection, introduction of precisely positioned nick, and cDNA synthesis are carried out by a single protein.
Any RNA sequence flanked by 5’ and 3’ regions of the retroelement genome should assemble with a favorably
modified retroelement protein, and this RNP would then seek its native insertion site. Because several
LINE/non-LTR retroelement families target highly conserved, repetitive sequences invariant across
multicellular eukaryotes, there is no need to re-engineer DNA site-specificity of these retroelement proteins,
although that may become of interest to undertake. The simple architecture of the non-LTR retroelements begs
to be exploited for developing an approach to human genome supplementation with genes of therapeutic
impact. The novelty of this approach demands continuous innovation and obliges high risk of failure to reach
the goal of delivering an engineered RNP capable of transgene introduction into human cells. Success of this
strategy would usher in a new modality of therapeutic treatment for loss-of-function diseases.

## Key facts

- **NIH application ID:** 10683044
- **Project number:** 3DP1HL156819-03S1
- **Recipient organization:** UNIVERSITY OF CALIFORNIA BERKELEY
- **Principal Investigator:** Kathleen Collins
- **Activity code:** DP1 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $79,652
- **Award type:** 3
- **Project period:** 2020-09-30 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10683044

## Citation

> US National Institutes of Health, RePORTER application 10683044, Human genetic supplementation without donor DNA or a DNA break (3DP1HL156819-03S1). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10683044. Licensed CC0.

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