# Development of a novel model to study the effects of LINE-1 retrotransposons in disease and normal physiology using nanobodies

> **NIH NIH R21** · MERCER UNIVERSITY MACON · 2024 · $176,238

## Abstract

LINE-1 retrotransposons encode a multicistronic enzymatic complex with three open reading frames. Thousands of copies
of LINE-1 are embedded throughout the genome, and the enzyme activity of LINE-1 has generated approximately one third
of the human genome via the insertion of LINE-1 and SINES, another type of retrotransposon that does not encode its own
proteins. Although LINE-1 is largely silenced in most healthy somatic cells, it is reactivated in a large number of diseases
where it is hypothesized to play a role in pathogenesis and disease progression, and some researchers have suggested the
LINE-1 may also have a necessary biological role. Although LINE-1 reactivation can affect cells through multiple
mechanisms, including mutation of genomic DNA, the effects of LINE-1-encoded proteins on LINE-1-associated diseases
have been particularly hard to dissect owing to a lack of reliable knock down models. The lack of reliable knockdown
models arises from i) the large number of LINE-1 copies in the genome, which makes conventional gene editing unfeasible,
including Prime, and ii) the complex and poorly understood interactions and crosstalk between LINE-1, RNAi, and
interferon pathways, which makes the use of shRNA or siRNA difficult to interpret. We propose herein to establish a novel
model to knock down LINE-1 proteins using intracellular functionalized nanobodies, also known as intrabodies. We will
use phage display to isolate nanobodies with high-affinity to LINE-1 proteins from a synthetic nanobody library. These
nanobody sequences will then be fused to GFP or Fboxes to enable live-cell tracking and kinetic experiments (GFP-
nanobodies) or knock down of LINE-1 proteins (Fboxes). Notably, Fbox-nanobody fusions have achieved 100% knockout
of target proteins via rapid ubiquitination through the recruitment of E3 ubiquitin ligase, resulting in proteasomal
degradation. We will then test the ability of these functionalized, LINE-1-specific nanobodies to facilitate live-cell
localization of LINE-1 proteins and to eliminate LINE-1 proteins. We will also perform an initial phenotypic
characterization of cells -/+ knockdown of the LINE-1 protein ORF1p, the most highly expressed LINE-1 protein.
Successful completion of these aims will advance the LINE-1 field and enable more robust hypothesis-testing to determine
the roles of LINE-1 proteins in disease as well as rigorously testing their proposed role in mammalian development.

## Key facts

- **NIH application ID:** 10953070
- **Project number:** 1R21GM155785-01
- **Recipient organization:** MERCER UNIVERSITY MACON
- **Principal Investigator:** Pamela Cook
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $176,238
- **Award type:** 1
- **Project period:** 2024-08-01 → 2026-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10953070

## Citation

> US National Institutes of Health, RePORTER application 10953070, Development of a novel model to study the effects of LINE-1 retrotransposons in disease and normal physiology using nanobodies (1R21GM155785-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10953070. Licensed CC0.

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