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PLoS One. 2012;7(11):e50438. doi: 10.1371/journal.pone.0050438. Epub 2012 Nov 28.

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

Author information

1
California Institute of Technology, Pasadena, California, United States of America.

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.

PMID:
23209743
PMCID:
PMC3508920
DOI:
10.1371/journal.pone.0050438
[Indexed for MEDLINE]
Free PMC Article

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