Abstract
Despite advancements in gene therapy methods for the treatment of genetic diseases, there is still an unmet need for neurological afflictions specifically due to the physical blood-brain barrier and overall complexity of the central nervous system. While antisense oligonucleotides and adeno-associated vectors have been promising forms of central nervous system gene therapy, they are restricted in their versatility and have limited carrying capacity respectively. As an alternative, we proposed engineering mesenchymal stem cells (MSCs) to deliver therapeutics through the blood-brain barrier.
Preliminary data from our lab indicate that MSCs can be successfully engineered to express and secrete DNA-binding-domain (DBD) proteins like zinc finger (ZF) and transcription activator-like effectors (TALEs) that can elicit targeted alterations of gene expression in neurons. Through the use of novel Fink lab secretion machinery constructs fused to DBD proteins, MSCs have been shown to successfully secrete zinc fingers (ZFs) and transcription activator-like effectors (TALEs), acting as a bio-factory for these therapeutic proteins. However, the secretion of CRISPR/dCas9 proteins, which are substantially larger, had not been studied and remains a system of interest due to its targeting versatility.
To test the feasibility of the MSC secretion platform, we planned to use Huntington’s disease models. Huntington’s disease (HD) is an autosomal dominant neurodegenerative disease afflicting approximately 1 in 10,000 people in the United States. HD is caused by an expansion of CAG repeats in Exon 1 of Chromosome 4 which produces mutant huntingtin protein that localizes to neurons and delivers neurotoxic effects resulting in cell death.
In this study, we sought to engineer mesenchymal stem cells to secrete Cas9 protein. By incorporating a murine IgK leader sequence, MSCs were engineered to secrete a novel Cas9 variant, dxiCas9, that has increased targeting versatility and epigenetic modifying capability when compared to ZFs and TALEs. Plasmids containing dxiCas9 were cloned into the established ZF and TALE lentiviral secretion backbone and subsequently packaged using Lenti-X 293 cells. MSCs were transduced and evaluated for dxiCas9 expression via western blot, and results indicated successful expression of dxiCas9 protein inside engineered MSCs. However, for the delivery platform to be successful, MSCs must demonstrate the ability to secrete dxiCas9 into the extracellular space. Conditioned media from the engineered MSCs were evaluated via western blot using an anti-CRISPR antibody for the presence of dxiCas9 in the extracellular space. Western blot analysis did not indicate secretion of dxiCas9 into the conditioned media of MSCs. However, current endeavors in the Fink lab are working towards modifying the secretion constructs to improve the MSC secretion platform for dxiCas9, serving as an exciting potential treatment avenue for genetically linked neurological diseases.