title: Use of mutated self-cleaving 2A peptides as a molecular rheostat to direct simultaneous formation of membrane and secreted anti-HIV immunoglobulins.
abstract: 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.
authors.affiliation: California Institute of Technology, Pasadena, California, United States of America.
authors.forename: Kenneth K
authors.initials: KK
authors.lastname: Yu
authors.forename: Kiefer
authors.initials: K
authors.lastname: Aguilar
authors.forename: Jonathan
authors.initials: J
authors.lastname: Tsai
authors.forename: Rachel
authors.initials: R
authors.lastname: Galimidi
authors.forename: Priyanthi
authors.initials: P
authors.lastname: Gnanapragasam
authors.forename: Lili
authors.initials: L
authors.lastname: Yang
authors.forename: David
authors.initials: D
authors.lastname: Baltimore
mesh_terms: D002118:Calcium / Q000378:metabolism; D002469:Cell Separation; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D005822:Genetic Vectors; D057809:HEK293 Cells; D006678:HIV / Q000737:chemistry; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015658:HIV Infections / Q000276:immunology*; D015497:HIV-1 / Q000378:metabolism; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D007075:Immunoglobulin M / Q000737:chemistry*; D007136:Immunoglobulins / Q000737:chemistry*; D007158:Immunologic Techniques / Q000379:methods; D016086:Lentivirus / Q000235:genetics; D008954:Models, Biological; D009500:Neutralization Tests; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011947:Receptors, Antigen, B-Cell / Q000737:chemistry; D020349:Surface Plasmon Resonance / Q000379:methods
chemical_list: D015699:HIV Envelope Protein gp120; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D007136:Immunoglobulins; D010455:Peptides; D011947:Receptors, Antigen, B-Cell; D002118:Calcium