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Genome Engineering of Primary Human B Cells Using CRISPR/Cas9
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Multifunctional CRISPR-Cas9 with engineered immunosilenced human T cell epitopes.

Shayesteh R Ferdosi1,2, Radwa Ewaisha1,3, Farzaneh Moghadam4

  • 1Center for Personalized Diagnostics, Biodesign Institute, Arizona State University, Tempe, AZ, 85287, USA.

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Pre-existing immunity to CRISPR-Cas9 (SpCas9) is common in healthy individuals, posing challenges for gene therapies. This study identifies key immune epitopes and demonstrates a method to modify SpCas9 to reduce immune responses.

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Area of Science:

  • Immunology
  • Gene Therapy
  • Molecular Biology

Background:

  • CRISPR-Cas9 gene editing holds promise for personalized therapies.
  • The bacterial Cas9 protein's immunogenicity is a significant concern for human applications.
  • Pre-existing immunity can limit the efficacy of CRISPR-based treatments.

Purpose of the Study:

  • To investigate pre-existing immunity to Streptococcus pyogenes Cas9 (SpCas9) in healthy individuals.
  • To identify immunodominant T cell epitopes within SpCas9.
  • To develop strategies for mitigating SpCas9 immunogenicity in gene therapy.

Main Methods:

  • Serological screening for antibodies against SpCas9.
  • T cell assays using peripheral blood mononuclear cells (PBMCs) from healthy donors.
  • Epitope prediction algorithms incorporating T cell receptor contact residue hydrophobicity and HLA binding.
  • Functional and specificity assays of modified SpCas9 variants.

Main Results:

  • Antibodies to SpCas9 were detected in at least 5% of 143 healthy individuals.
  • The majority of screened healthy individuals exhibited pre-existing CD8+ T cell immunity to SpCas9.
  • Two immunodominant SpCas9 T cell epitopes for HLA-A*02:01 were identified.
  • Cas9 protein was successfully modified to eliminate identified epitopes while retaining function and specificity.

Conclusions:

  • Pre-existing immunity to CRISPR-associated nucleases like SpCas9 is prevalent.
  • Targeted modification of SpCas9 can reduce T cell immunogenicity.
  • This approach offers a potential strategy to overcome immune barriers in CRISPR-based gene therapies.