Engineering Protein-Secreting Plasma Cells by Homology-Directed Repair in Primary Human B Cells

Mol Ther. 2018 Feb 7;26(2):456-467. doi: 10.1016/j.ymthe.2017.11.012. Epub 2017 Nov 22.


The ability to engineer primary human B cells to differentiate into long-lived plasma cells and secrete a de novo protein may allow the creation of novel plasma cell therapies for protein deficiency diseases and other clinical applications. We initially developed methods for efficient genome editing of primary B cells isolated from peripheral blood. By delivering CRISPR/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complexes under conditions of rapid B cell expansion, we achieved site-specific gene disruption at multiple loci in primary human B cells (with editing rates of up to 94%). We used this method to alter ex vivo plasma cell differentiation by disrupting developmental regulatory genes. Next, we co-delivered RNPs with either a single-stranded DNA oligonucleotide or adeno-associated viruses containing homologous repair templates. Using either delivery method, we achieved targeted sequence integration at high efficiency (up to 40%) via homology-directed repair. This method enabled us to engineer plasma cells to secrete factor IX (FIX) or B cell activating factor (BAFF) at high levels. Finally, we show that introduction of BAFF into plasma cells promotes their engraftment into immunodeficient mice. Our results highlight the utility of genome editing in studying human B cell biology and demonstrate a novel strategy for modifying human plasma cells to secrete therapeutic proteins.

Keywords: AAV; B cells; RNP; antibody secreting cells; engraftment; gene editing; genome engineering; immunotherapy; plasma cells; protein therapy.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • B-Lymphocytes / immunology*
  • B-Lymphocytes / metabolism*
  • Biomarkers
  • CRISPR-Associated Protein 9
  • Cytokines / metabolism
  • Dependovirus / genetics
  • Gene Editing*
  • Genetic Engineering*
  • Genetic Loci
  • Genetic Vectors / genetics
  • Humans
  • Immunotherapy
  • Mice
  • Phenotype
  • Plasma Cells / immunology*
  • Plasma Cells / metabolism*
  • Polymorphism, Single Nucleotide
  • Positive Regulatory Domain I-Binding Factor 1 / genetics
  • Receptors, CCR5 / genetics
  • Recombinational DNA Repair*
  • Transduction, Genetic


  • Biomarkers
  • CCR5 protein, human
  • Cytokines
  • Receptors, CCR5
  • Positive Regulatory Domain I-Binding Factor 1
  • CRISPR-Associated Protein 9