Development of a novel model to study the effects of line-1 retrotransposons in disease and normal physiology using nanobodies - line-1 retrotransposons encode a multicistronic enzymatic complex…

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