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.
