Use of mutated self-cleaving 2A peptides as a molecular rheostat to direct simultaneous formation of membrane and secreted anti-HIV immunoglobulins

• Published in: PloS one 2012-11, Vol.7 (11), p.e50438-e50438
• Main Authors: Yu, Kenneth K, Aguilar, Kiefer, Tsai, Jonathan, Galimidi, Rachel, Gnanapragasam, Priyanthi, Yang, Lili, Baltimore, David
• Format: Article
• Language: English
• Subjects: Alternative splicing; Antibodies; Antigens; B cells; Biology; Bone marrow; Calcium - metabolism; Cell lines; Cell Separation; Enzyme-Linked Immunosorbent Assay - methods; Flow Cytometry - methods; Gene loci; Gene therapy; Genetic engineering; Genetic Vectors; HEK293 Cells; HIV; HIV - chemistry; HIV Envelope Protein gp120 - metabolism; HIV Infections - immunology; HIV-1 - metabolism; Human immunodeficiency virus; Humans; Immunoglobulin G; Immunoglobulin G - chemistry; Immunoglobulin M; Immunoglobulin M - chemistry; Immunoglobulins; Immunoglobulins - chemistry; Immunologic Techniques - methods; Lentivirus - genetics; Lymphocytes B; Lymphopoiesis; Medicine; Models, Biological; mRNA; Mutation; Neutralization Tests; Peptides; Peptides - chemistry; Polypeptides; Protein Binding; Receptors, Antigen, B-Cell - chemistry; Ribonucleic acid; RNA; Rodents; Surface Plasmon Resonance - methods
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0050438

In nature, B cells produce surface immunoglobulin and secreted antibody from the same immunoglobulin gene via alternative splicing of the pre-messenger RNA. Here we present a novel system for genetically programming B cells to direct the simultaneous formation of membrane-bound and secreted immunoglobulins that we term a "Molecular Rheostat", based on the use of mutated "self-cleaving" 2A peptides. The Molecular Rheostat is designed so that the ratio of secreted to membrane-bound immunoglobulins can be controlled by selecting appropriate mutations in the 2A peptide. Lentiviral transgenesis of Molecular Rheostat constructs into B cell lines enables the simultaneous expression of functional b12-based IgM-like BCRs that signal to the cells and mediate the secretion of b12 IgG broadly neutralizing antibodies that can bind and neutralize HIV-1 pseudovirus. We show that these b12-based Molecular Rheostat constructs promote the maturation of EU12 B cells in an in vitro model of B lymphopoiesis. The Molecular Rheostat offers a novel tool for genetically manipulating B cell specificity for B-cell based gene therapy.