B5 - Neurologic Diseases (excluding Ophthalmic and Auditory Diseases)
1598: Disruption of Aberrant Splicing of STMN2 by Gene Editing with a Type V CRISPR-Cas Enzyme as a Potential Treatment for ALS
Type: Poster Session
Poster Board Number: 1598
Presentation Details
Session Title: Friday Posters: Neurologic Diseases
TDP-43 is a nuclear RNA-binding protein that regulates RNA processing. In many neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTD), TDP-43 is mis-localized and accumulated in the cytoplasm, a pathological hallmark of these conditions. The lack of nuclear TDP-43 in disease contexts results in aberrant splicing and processing of numerous genes. One of the most highly down-regulated is STMN2, which encodes for a microtubule-binding protein that controls axonal outgrowth and is highly expressed in motor neurons. Prior studies suggest that nuclear depletion of TDP-43 results in the aberrant splicing of STMN2 and loss of full-length functional STMN2, which may contribute to the pathology in ALS. To explore a potential treatment for ALS, we used gene editing with a Type V CRISPR-Cas enzyme to disrupt aberrant splicing of STMN2 by creating larger deletions of ~10 base pairs. We first screened in HEK cells multiple type V CRISPR nucleases and guides spanning the 310 base pair aberrantly spliced novel exon of STMN2 (exon 2A) that includes cis-acting elements such as a splice acceptor site, a TDP-43 binding region and a polyadenylation site. Nuclease/guide pairs with efficient indel formations from the HEK screening were tested in SH-SY5Y cells under TDP-43 knockdown to identify the pairs that were capable of reducing aberrant splicing and promoting an increase of full-length STMN2 mRNA. Several nuclease/guide pairs disrupting the splice acceptor site resulted in the effective reversal of aberrant splicing and increase of full-length STMN2 mRNA, but not the other cis-acting elements, thus defining the splice acceptor as the relevant target. These findings were replicated in human induced pluripotent stem cell (iPSC)-derived motor neurons. Moreover, the disruption of aberrant splicing using the nuclease/guide pairs in human motor neurons reversed the axonal loss caused by TDP-43 knockdown. Finally, the most potent nuclease/guide pairs, ABR-001 variant and gX, and ABR-004 variant and gY, were packaged into a rh10 serotype AAV vector and injected into neonatal mice transgenic for the human STMN2 exon 2A locus. In these mice, efficient editing as well as the reversal of STMN2 aberrant splicing were observed. In summary, we have identified multiple type V nuclease/guide pairs that reverse the aberrant splicing of STMN2 and the disease-relevant phenotypic consequences due to TDP-43 nuclear depletion in a cell line, in motor neurons and in a mouse model. Together, these findings have the potential to pave a path towards a CRISPR gene editing therapeutic for ALS.
Plain Language Summary
In many neurodegenerative diseases, TDP-43, an RNA/DNA-binding protein normally found in the cell nucleus, accumulates in the cytoplasm. This mis-localization impairs the processing of genes, including STMN2. The protein produced by STMN2 controls nerve fibers (axons) growth and is highly expressed in cells controlling motor functions. Prior studies suggest that errors in STMN2 may contribute to the onset and progression of amyotrophic lateral sclerosis (ALS). This work outlines a gene editing approach designed to disrupt the formation of errors in the STMN2 gene by deleting targeted regions of the genome. Gene editors contain several components to enable edits to specific genomic targets. After confirming the most effective target region with our gene editors, we identified combinations of components to reverse the occurrence of errors in the STMN2 gene in cellular and animal models. These findings pave the path towards a proof of concept for therapeutic CRISPR gene editing in ALS.
Priscilla D. Negraes1, Peter D. Buckett1, Jace Jones-Tabah1, Anthony J. Garrity1, Mishti Bose1, Sevda Lule1, Andrew Park1, Nolan Sheppard1, Linh B. Truong1, Qiyuan Yang1, Ivan A. Kristanto1, Venkata Sai Sinduri Bachu1, Maximiliano F. Presa2, Jennifer Ryan2, Erin Merkel1, Christina Noe1, Jenny V. Tobin1, Lijun Liu1, Florie Borel1, Cathleen Lutz2, John E. Murphy1, Chee Yeun Chung1
1Arbor Biotechnologies, Cambridge, MA,2The Jackson Laboratory, Bar Harbor, ME"
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