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D - Synthetic/Molecular Conjugates and Physical Methods for Delivery of Gene Therapeutics -> Synthetic/Molecular Conjugates and Physical Methods for Delivery of Gene Therapeutics (including exosomes)

164: Development of a Monoparticle-Based CRISPR Gene Editing and Intraparenchymal Delivery System for Neurological Applications

Type: Oral Abstract Session

Presentation Details
Session Title: Synthetic/Molecular Conjugates and Physical Methods for Delivery of Gene Therapeutics II
Location: Petree Hall C
Start Time: 5/18/2023 17:15
End Time: 5/18/2023 17:30

Introduction. CRISPR-based gene editing within the central nervous system (CNS) has the potential to revolutionize the treatment of neurological disorders, including currently-incurable monogenic neurodegenerative diseases such as Huntington’s, Alzheimer's, and Parkinson's diseases. However, developing CRISPR-based therapeutics for the CNS has been challenging, due in large part to drug delivery. Two key obstacles need to be overcome before gene editing in the human brain is feasible: (1) efficient and safe delivery of Cas9 and gRNA into neurons, and (2) establishment of strategies that enable targeted brain regions to be efficiently edited. The goal of this study is to develop formulations of Cas9 ribonucleoprotein (RNP) enzymes that can edit large volumes of targeted brain structures after intracranial injection. Our team - a collaboration of the Wilson, Murthy, and Bankiewicz laboratories - has developed a non-viral delivery system involving peptide-mediated intracellular delivery of a RNP complex administered via intrastriatal injection using convection-enhanced delivery (CED). This approach offers several advantages, including ease of production, transient CRISPR activity, and robust tissue distribution enabled by exceptionally small particle size. Material and Methods. We have developed a nuclear localization signal (NLS)-rich Cas9 nuclease protein combined with sgRNAs resulting in small particles (≤20nm), or “monoparticles.” A series of different monoparticle formulations were developed that contained NLS-rich Cas9-RNPs and amphipathic peptides promoting intracellular delivery. A pair of gRNAs were used to target the cassette repressing tdTomato fluorescent reporter expression in transgenic Ai9 reporter mice. The various monoparticle (RNP:peptide) formulations were screened using a cell-based in vitro system. Lead Cas9 formulations were then assessed in vivo by administration into the striatum of Ai9 mice via CED. Three weeks following infusion, animals were euthanized and their brains were processed for immunohistological analysis of distribution and efficiency of neuronal genomic editing within the target structure. Results and Conclusion. Our best performing RNP:peptide monoparticle formulation exhibited an editing efficiency of approximately 82% of neurons within a coverage area involving over 33% of the striatum in Ai9 mice. This optimal formulation was then combined with sgRNAs targeting the gene expressing green fluorescent protein in transgenic GFP mice, where we observed knockdown of GFP expression in 76% of the neurons in the target area. We conclude that this monoparticle-based CRISPR reagent delivery system is efficacious in genomic editing of CNS neurons following intracranial delivery to target brain regions. We are currently advancing this technology to large animals utilizing MRI-guided CED for intraparenchymal delivery. This approach holds the promise to accelerate successful translation of CRISPR-Cas9 to the clinic for the treatment of brain diseases.

Victor S. Van Laar1, Brigette Manohar2, Vikas Munjal1, Allison Clark1, Meika Travis1, Rohit Sharma2, Sheng Zhao2, Piotr Hadaczek3, Lluis Samaranch3, Niren Murthy2, Ross Wilson4, Krystof Bankiewicz3

1Department of Neurological Surgery, The Ohio State University, Columbus, OH,2Innovative Genomics Institute, University of California Berkeley, Berkeley, CA,3The Ohio State University, Columbus, OH,4University of California Berkeley, Berkeley, CA
 V.S. Van Laar: None.

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