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. Author manuscript; available in PMC: 2023 Jun 1.
Published in final edited form as: Trends Mol Med. 2022 Apr 13;28(6):528–529. doi: 10.1016/j.molmed.2022.03.007

Primary B cell Engineering for Therapeutic Research

Evan Kleinboehl 1, Kanut Laoharawee 1, Branden Moriarity 1
PMCID: PMC9936666  NIHMSID: NIHMS1866417  PMID: 35430120

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Peripheral blood mononuclear cells (PBMCs) are harvested from a patient and processed further to purify CD19 + B cells. Alternatively, B cells can be derived from induced pluripotent stem cells (iPSC). Purified B cells are then activated/expanded in culture media in order to prime them for efficient genome engineering. Lentivirus, transposons, and CRISPR/Cas9, together with recombinant adeno-associated viral vector (rAAV) carrying DNA donor template for homology directed repair (HDR), can be used to integrate a transgene of interest into the B-cell genome. CRISPR-based engineering tools can also be used for gene modification and gene knockout. The engineered B cells may be further expanded in culture and used for downstream applications.

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Engineered B cells have multiple applications in therapeutics, industry, and basic research. For example, B cells can be used in the clinical setting to express antibodies and/or cytokines to treat infections, cancers or autoimmune diseases. In addition, B cells may be engineered to express proteins for novel cell-based protein replacement therapy for protein deficiencies, such as enzymopathies. Engineered B cells can be used to produce industrial scale quantities of monoclonal antibodies. Alternatively, genetically manipulated B cells can be used to deepen understanding of B cell functions and basic biology.

Advantages:

B cells are abundant and can be easily isolated from peripheral blood. These cells can then be expanded ex vivo to large numbers prior to use.

Physiologically, B cells can differentiate into long-lived plasma cells that live for many years and even decades.

CRISPR/Cas9 and rAAV can be used for efficient site-specific HDR in human B cells.

Insertion of scFv of a novel antibody at Immunoglobulin heavy chain locus allows for the production of various isotypes of Immunoglobulins.

Challenges:

Ex vivo B cell stimulation/expansion protocols have not become standardized, as compared to T cell activation with CD3/CD28 stimulation, for example. There are multiple formulations that result in differing B cell sub-population outgrowth.

Ex vivo differentiation is not efficient and not completely understood. Long-live plasma cell markers are unclear and remain ambiguous.

Human engraftment protocols for engineered B cells have not been established. It remains unclear if B cell differentiation is necessary for successful engraftment and cell persistence. It is also unclear if pre-conditioning will be required for successful B cell engraftment in humans. It is unknown if allogeneic B cell transplantation is possible.

Application:

Clinical applications: targeting infections, cancers, autoimmune diseases, and production of proteins/enzymes.

Industrial applications: monoclonal antibody production, protein production.

Research applications: deepened understanding of biology and functions of human B cells.

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