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.

Project Narrative DNA is the biomolecule in cells that carries instructions for making the proteins, lipids, and other molecules that make up living cells and tissues. In addition to the instructions carried in our DNA for making normal cellular proteins, we have many stretches of DNA in our genome that encode retrotransposons, which are similar to retroviruses, but they don’t infect other cells. A special ability of retrotransposons is that they can take copies of their DNA and insert those copies into new locations in our genome. This process can create mutations in our DNA, leading to cancer and other diseases. In addition, retrotransposon DNA, just like our DNA, makes proteins. Unfortunately, we know very little about the effects of these retrotransposon proteins in our cells, but we know that many types of diseased cells make much more of these proteins than healthy cells. Retrotransposon proteins also increase in cells as we age. The diseases associated with increased retrotransposon proteins include neurodegeneration, autoimmune disease, and others. The primary reason we know so little about retrotransposon proteins and their effects in our cells is because we lack reliable methods to study them. The lack of methods to study these proteins arises from the fact that there are so many copies of retrotransposon DNA in our genome, which makes conventional DNA editing technologies unfeasible. We propose to develop a novel method to eliminate retrotransposon proteins using molecules called “nanobodies”, which can be modified and introduced into cells to eliminate specific proteins. We can also use nanobodies to track retrotransposon proteins in cells to see where they go and what other proteins they interact with. Developing this new system will advance our ability to study and understand the effects of retrotransposon proteins on our health and how they influence aging and disease.