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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)

1687: Peptide-Mediated Delivery of CRISPR Ribonucleoprotein (RNP) into Primary Human CD34+ Hematopoietic Stem Cells

Type: Poster Session

Poster Board Number: 1687
Presentation Details
Session Title: Friday Poster Session
Start Time: 5/19/2023 12:00
End Time: 5/19/2023 14:00

Following mobilization, cultured hematopoietic Stem and Progenitor Cells (HSPCs) can be edited by CRISPR-Cas9 ex vivo for cell therapy. Autologous transplantation of edited HSPCs has already demonstrated clinical success in addressing monogenic blood disorders such as β-hemoglobinopathies. Efficient and gentle intracellular delivery of the CRISPR ribonucleoprotein (RNP) complex is a remaining challenge, since the predominant technology is electroporation, which has substantial drawbacks including poor cell viability and dramatic impacts on cell state (which may, in turn, impair cells’ ability to engraft). Inspired by viruses, which rely on peptide sequences with cell-penetrating capacities to cross cell membranes, we have designed and tested a library of amphiphilic peptides with cell penetrating and endosomolytic capacities. When combined with different CRISPR enzymes: Cas9 nuclease, adenine base editor (ABE8e), and Cas12a (Cpf1), our novel peptides can deliver CRISPR RNP to the nucleus of primary human CD34+ HSPCs and perform efficient genome editing (up to 82% indels via NGS) while showing only limited toxicity (≤20% dead cells). Our delivery approach produces therapeutically relevant levels of editing using either nuclease or base editor, surpassing the clinical threshold (>20%) for therapeutic benefit of sickle cell disease patients (up to 60% and 55% editing at the BCL11A enhancer via Cas9 and ABE8e, respectively). This approach maintains cell viability, proliferation, and phenotype as well as their potential to differentiate and perform erythropoiesis. Our delivery technology involves applying the RNP-peptide complex directly to cells in suspension by simply mixing them into the culture, with no complicated manipulation or hardware requirements (for either synthesis or administration of the RNP-peptide formulation). Importantly, all components of our system can be readily manufactured: recombinant protein and synthetic RNA & peptide. Finding a reliable, efficient, gentle, convenient, and cost-effective delivery method is crucial to improve and deploy HSPC-based cell therapies, especially since β-hemoglobinopathies are most prevalent in developing countries. Our CRISPR delivery system is an appealing alternative to existing methodologies, and may facilitate a semi-automated, point-of-care manufacture pipeline for precision cell therapies.

Madalena Castro1,2,3, Lorena de Oñate1,2,3, John Hunsinger1,2,3, Ross C. Wilson1,2,3

1Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA,2Department of Molecular and Cell Biology, UC Berkeley, Berkeley, CA,3California Institute for Quantitative Biosciences (QB3), UC Berkeley, Berkeley, CA
 M. Castro: None.

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