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B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

1507: Enhancing CRISPR Genome Editing by Extensive Chemical Modification and Self-Delivery of Guide RNAs

Type: Poster Session

Poster Board Number: 1507
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Therapeutic genome editing using CRISPR-based reagents are rapidly moving to the clinic. Ex vivo genome editing can be potent with standard genome editing reagents (e.g. Cas9 protein or mRNA co-delivered with synthetic end-protected guide RNAs) and is currently being used successfully in clinical trials. However, therapeutic treatment of many genetic diseases will require the editing of cells and tissues directly within the patient’s body. The in vivo activity of CRISPR reagents can be improved through partial chemical modifications of the backbone that stabilize the guide RNA from hydrolysis by endogenous nucleases (e.g. Finn, J. D. et al. Cell Reports 22, 2227-2235 (2018)). However, partially protected RNAs have a short half-life in vivo unless protected (e.g. via lipid nanoparticles). In addition, selective targeting of many tissues remains challenging with existing delivery modalities and unmodified RNA segments can stimulate counterproductive immune responses. Our strategy to improve the safety and efficiency of genome editing in vivo relies on the development of fully modified (FM), conjugated guide RNAs that can be delivered in a tissue-specific fashion as RNPs with the effector Cas9 protein, or co-delivered with an AAV vector expressing the effector protein and the tracrRNA. Chemical modification of the guide RNA will increase guide stability and potency, improve biodistribution, reduce immune responses, and eliminate the need for lipid nanoparticle formulation. We previously described our first generation of heavily modified (HM) crRNAs and tracrRNAs that can support effective SpyCas9 activity in mammalian cells. We have now demonstrated that our HM crRNA/tracrRNA can support efficient editing in vivo when directly injected into the mouse CNS as SpyCas9 RNP. We have also extended these approaches to base editors and to additional Cas9 homologues with distinct PAM specificities. Moreover, we defined new variants of HM and FM crRNA frameworks with improved Cas9 genome editing in mammalian cells and validated these guides for self-delivery in conjunction with AAV vectors that encode Cas9 and tracrRNA. This strategy promises to be particularly useful for multiplex targeting and inactivation of the AAV episome after editing is achieved, thereby minimizing genotoxicity and immune responses that could be caused by prolonged Cas9 expression.

Scot A. Wolfe1, Nadia Amrani1, Nathan Bamidele1, Julia Alterman1, Zexiang Chen1, Gitali Devi1, Dimas Echeverria-Moreno1, Hailey Feinzig1, Nicholas Gaston1, Karin Kelly1, Zachary Kennedy1, Pengpeng Liu1, Socheata Ly1, Stacy Maitland1, Karthikeyan Ponnienselvan1, Julia Rembetsy-Brown1, Kathy Snow2, Han Zhang1, Cathleen Lutz2, Stephen Murray2, Anastasia Khvorova1, Erik Sontheimer1, Jonathan K. Watts1

1UMass Chan Medical School, Worcester, MA,2The Jackson Laboratory, Bar Harbor, ME
 S.A. Wolfe: None.

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