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

Abstract

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.

Graphical abstract

Figure 1

Cas9 RNP Induces Site-Specific Indels or a Precise Single Nucleotide Change in the Presence of an ssODN in Primary Human B Cells (A) CD19+ B cells were isolated and activated in vitro for 2 days, mock treated or transfected with Cas9 RNPs targeting CCR5 or PRDM1 (CCR5g, PRDM1g-1, or PRDM1g-2), and cultured for 5 additional days. Total genomic DNA was isolated on day 5 and target regions were PCR amplified and analyzed using the T7 endonuclease 1 (T7E1) assay (one representative experiment shown) or Illumina sequencing for percentages of on-target indels (112,000 reads per experimental condition). (B) Diagram of wild-type PRDM1 locus, PRDM1g-2 target location, and the ssODN donor template containing a single nucleotide change. (C and D) B cells were activated for 2 days and mock treated and electroporated with Cas9 RNP-PRDM1g-2 alone or with the ssODN donor template at serially increasing doses from 7.5 pmol to 120 pmol. (C) Viabilities of B cells 2 days and 5 days after genome editing (n = 3, three donors). No significant difference in viability was observed between mock and up to 30 pmol ssODN plus RNP on either day 2 or day 5. Bar graph shows mean ± SEM. (D) Percentage of HDR, indel, and wild-type (WT) alleles in total genomic DNA extracted 5 days after genome editing as assessed by Illumina sequencing (greater than 1 million reads per experimental condition). n represents the number of independent experiments. We used one-way ANOVA with the Sidak correction for multiple comparisons; ***p < 0.001; ns, not significant.

Figure 2

Cas9-Mediated Disruption of PRDM1, IRF4, PAX5, or BACH2 Alters B Cell Phenotype (A) Experimental workflow of the plasma cell differentiation assay. This workflow includes a three-step differentiation culture that is divided by a B cell activation phase (phase 1), a plasmablast differentiation phase (phase 2), and a plasma cell differentiation phase (phase 3) using the different cocktails of soluble factors and cytokines specified. Cells were transfected with Cas9 RNP after 2 days of initial activation, indels were assessed in genomic DNA on day 5, and phenotypes and IgM/IgG secretion were quantified on day 11. (B) Schematic diagram of the roles of transcription factors PRDM1, IRF4, PAX5, and BACH2 as established by murine studies. (C–E) Primary B cells were activated and either mock treated or transfected with Cas9 RNP targeting CCR5, PRDM1, IRF4, PAX5, or BACH2 and were subsequently differentiated in vitro. (C) Top: representative flow plots showing CD19 and CD38 expression in live singlets and bar graph showing mean percentages of CD19lowCD38high plasmablasts 11 days post transfection. Bottom: representative flow plots showing CD27 and CD138 expression and a bar graph showing percentage of CD27+CD138+ plasma cells 11 days post transfection (n = 5, three donors). (D) Genomic DNA was isolated 5 days after RNP transfection for allelic indel analysis by Illumina sequencing (n = 5). (E) Amounts of IgM (left) and IgG (right) in each culture as measured by ELISA (n = 4, three donors). All bar graphs show mean ± SEM. n represents the number of independent experiments. Dotted lines mark mock levels. We used one-way ANOVA with the Sidak correction for multiple comparisons; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant. Statistical comparisons were made compared to mock. IFN-α, interferon-α; PB, plasmablast; PC, plasma cell.

Figure 3

HDR-Mediated GFP Addition at the PRDM1 Locus Using Cas9 RNP and an AAV6 Donor Template Leads to Progressively Increased GFP Expression in a Dose-Dependent Manner (A) B cells were activated for 2 days and were either mock treated or transduced with the GFP-expressing scAAV, which was packaged using a comprehensive panel of AAV serotypes (1, 2, 2.5, 5, 6, 8, 9, or D-J) at a MOI of 25,000. Shown are the percentages of GFP+ cells (left) and mean fluorescence intensities (right) (n = 3, three donors). (B) Schematic of wild-type PRDM1 locus, PRDM1g-2 target location, and an AAV GFP expression cassette with 400-bp flanking PRDM1 homology arms (AAV PRDM1-GFP). The 3-base PAM sequence is deleted from the AAV template homology sequence. (C) B cells were activated for 2 days and were either mock treated or transfected with Cas9 RNP-PRDM1g-2, with or without AAV transduction. Cells were subsequently cultured under the same activating condition for 11 days. Left: representative flow plots showing BFP and GFP expressions on day 2 and day 11 after genome editing. Green box highlights the GFP+ population in the gene-edited B cells. Right: bar graph showing percentages of GFP+ cells on day 2 and day 11 after gene editing (n = 4, four donors). All bar graphs show mean ± SEM. n represents the number of independent experiments. We used one-way ANOVA with the Sidak correction for multiple comparisons; *p < 0.05; ***p < 0.001; ns, not significant. HA, homology arm; MFI, mean fluorescence intensity; pA, SV40 poly-adenylation signal; PAM, protospacer adjacent motif; WT, wild-type.

Figure 4

HDR-Mediated Integration of FIX Coding Sequence at the CCR5 Locus Leads to High Levels of FIX Secretion by Gene-Edited Plasma Cells Ex Vivo (A) Schematic of wild-type CCR5 locus, CCR5g target location, and a FIX-expressing AAV construct with 800-bp flanking CCR5 homology arms (AAV CCR5-FIX). (B–D) Primary B cells were gene edited using the AAV CCR5-FIX donor template and CCR5-targeting RNP (CCR5g RNP), with or without PAX5-targeting RNP (PAX5g RNP). B cells were subsequently differentiated in vitro. (B) Frequency of on-target FIX integration in total alleles on day 11 after genome editing as assessed by digital droplet PCR (n = 2, two donors). (C) CD19 and CD38 expression on day 11. Left: representative flow plots; right: mean percentages of CD19lowCD38high plasmablasts (n = 3, two donors). (D) FIX production at day 11 after genome editing as measured by ELISA (n = 3, two donors). (E and F) Gene-edited B cells were differentiated in vitro as before; at day 8 post genome editing, FBS content was reduced to 2% (unless otherwise specified) and cells were cultured with or without vitamin K1. At day 11, supernatants were collected for FIX chromogenic activity assay and ELISA (n = 3, three donors). Shown are the total FIX activity (E) and FIX-specific activity (F) (specific activity in IU/mg was obtained by dividing net activity values by antigen concentrations). AAV was added at 20,000 MOI. Bar graphs show mean ± SEM. n represents the number of independent experiments. We used one-way ANOVA with the Sidak correction for multiple comparisons (C–E) or paired two-tailed t test for comparison of two groups (F); **p < 0.01; ***p < 0.001. HA, homology arm; pA, SV40 poly-adenylation signal; PAM, protospacer adjacent motif; WPRE3, a shortened woodchuck hepatitis virus posttranscriptional regulatory element 55; WT, wild-type.

Figure 5

HDR-Mediated Integration of BAFF Coding Sequence at the CCR5 Locus Results in Persistent BAFF Secretion by Gene-Edited Plasma Cells and Increases Plasma Cell Differentiation and Viability (A) Schematic of wild-type CCR5 locus, CCR5g target location, an AAV construct that co-expresses GFP and BAFF via T2A linkage (AAV CCR5-GFP-BAFF), and a BAFF-expressing AAV construct (AAV CCR5-BAFF) with identical 800-bp flanking CCR5 homology arms. (B–F) B cells were gene edited after 2 days of in vitro activation, and were subsequently differentiated into plasma cells using the three-step culture system. (B) Upper: representative flow plots showing GFP expression on day 2 and day 11 post gene editing in mock, CCR5-GFP-BAFF transduced cells, with or without Cas9 RNP. Lower: bar graph summarizing percentages of GFP+ cells on day 2 and day 11 post gene editing (n = 4, three donors). (C) Frequency of on-target donor template integration in total alleles on day 11 after genome editing as assessed by digital droplet PCR (n = 3, two donors). (D) Bar graph shows BAFF production as measured by ELISA at day 11 (n = 4, three donors). (E) Left: cells were counted at day 5 and day 11 post genome editing. Bar graph shows fold changes in cell numbers. Right: viabilities at day 11 by flow cytometry (n = 5, four donors). (F) Left: representative flow plots showing CD19 and CD38 expression at day 11 after genome editing using the AAV CCR5-BAFF vector. Right: bar graph summarizing percentages of CD19lowCD38high plasmablasts/plasma cells at day 11 (n = 5, four donors). Both AAV CCR5-GFP-BAFF and CCR5-BAFF were added at 20,000 MOI. All bar graphs show mean ± SEM. n represents the number of independent experiments. We used one-way ANOVA with the Sidak correction for multiple comparisons; **p < 0.01; ***p < 0.001. HA, homology arm; IL-2ss, IL-2 signal sequence; pA, SV40 poly-adenylation signal; PAM, protospacer adjacent motif; WT, wild-type.

Figure 6

Gene-Edited, BAFF-Expressing Human Plasma Cells Stably Secrete BAFF and Immunoglobulins in NSG Mice (A) Experimental layout of NSG mouse transplant. Gene-edited B cells expressing human BAFF were generated as before after 2 days of in vitro B cell activation and were subsequently differentiated into plasma cells using the three-step culture system. 11 days post genome editing, edited or control cells (10 million cells/animal) were delivered intravenously into busulfan-conditioned, NSG mice. A control animal cohort (no PCs) was also evaluated as noted. Blood samples were collected at day 10 and at day 21 when mice were sacrificed. (B and C) Serum proteins were quantified by ELISA (n = 3/cohort). Shown are serum BAFF levels (B) and serum human IgM and IgG levels (C). All graphs represent mean ± SEM. We used one-way ANOVA with the Sidak correction for multiple comparisons between groups and paired two-tailed t test for comparisons between two time points (day 10 versus day 21); *p < 0.05; **p < 0.01; ***p < 0.001. IV, intravenous; NSG, NOD/SCID/gamma-c null; PCs, plasma cells.