Cellular perturbations to enhance precise therapeutic genome editing to treat FTD/ALS - PROJECT SUMMARY
CRISPR holds great promise toward therapeutically editing pathological mutations in the gene fused in
sarcoma (FUS), known to cause 5% of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD)
cases. The FUS gene is ideal for therapeutic editing, as it is a dominant-negative genetic mutation, allowing for
one allele to produce enough of the FUS protein for cells to survive. However, there are several challenges that
must be addressed before this promise can be realized, including the inefficient delivery of Cas9 and gRNA into
cells and the lack of indel formation after Cas9 cleavage. Towards this, my lab has established a robust neuronal
differentiation protocol via doxycycline-inducible transcription factors. With this protocol, I can derive phenotypic
neurons from iPSCs that recapitulate disease phenotypes within 7 days13. We have additionally engineered
virus-like particles (VLP), that harbor no genetic material, to deliver Cas9 protein and gRNA to neurons12. In this
grant, I propose exploring small perturbations within neurons to gain further insights into how CRISPR therapies
can be designed for the precise editing of neurodegenerative diseases.
I have collected preliminary data with neurons and iPSCs, using the same CRISPR editing strategy, and
found that iPSCs and neurons have divergent editing outcomes. Specifically, we used gRNA designed to cleave
sequences in the NEFL and B2M genes. In both genes, we found that iPSCs had multiple editing outcomes not
present in neurons, commonly large deletions. Editing in neurons frequently resulted in no indel being produced.
The few indels that do occur in neurons appear to be almost exclusively small insertions (+1 indel) are produced
via mutagenic NHEJ. These small indels are ideal to cause frameshifts and induce nonsense-mediated decay
and represent an optimal strategy for therapeutic editing in neurons.
I propose studying how iPSC-neurons can be perturbed to increase the prevalence of our desired editing
outcome. Using our directed differentiation protocol as well as our VLP delivery system, I will explore how indel
frequency and identity are affected by changes in the transcription of the edited gene and the expression and
activity of DNA damage proteins. In Aim 1, I will alter the transcription of genes to explore how interactions with
RNA polymerase change editing frequency and indel formation. In Aim 2, I will investigate how changing the
expression or activity of DNA repair enzymes affects editing outcomes.
The insight gained from the work described in this grant can be used to precisely edit neurons to silence
pathological genetic mutations, like FUS mutations. Future studies would also apply this editing strategy to other
mutated genes in neurons and potentially other post-mitotic cells. The superior training environment provided at
the Gladstone Institutes will allow me the opportunity to explore these aims with expert research mentorship,
support from core facilities, and beneficial collaborations.
