Non-vertebrate cells for performing v(d)j recombination
AssigneeGeorgia Tech Research Corporation
InventorsJohn James Blazeck, Andrew Paul Cazier
Life of this patent
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Abstract
The present disclosure relates to non-vertebrate cells, including but not limited to Saccharomyces cerevisiae, engineered to express recombination proteins which allows the non-vertebrate cells to perform V(D)J recombination. The present disclosure also relates to methods of using the non-vertebrate cells to develop diverse antibodies or fragments thereof, similar to the V(D)J recombination process performed in B cells of higher mammalian organisms.
Claims20
An engineered non-vertebrate cell comprising a nucleic acid encoding one or more recombination activating gene (RAG) proteins selected from RAG1, RAG2, or a variant thereof and a recombination signal sequence (RSS), wherein the engineered non-vertebrate cell is engineered to undergo variable-diversity-joining (V(D)J) recombination to generate an antibody or a fragment thereof.
A method of generating an antibody or a fragment thereof, the method comprising:
- a) obtaining a non-vertebrate cell,
- b) introducing into the non-vertebrate cell a nucleic acid encoding one or more recombination activating gene (RAG) proteins selected from RAGI, RAG2, or variants thereof and a recombination signal sequence (RSS), and
- c) culturing the non-vertebrate cell of step b) under cell culture conditions to allow V(D)J recombination activity,
- wherein the non-vertebrate cell expresses an antibody or a fragment thereof following V(D)J recombination.
A method of screening for disease severity caused by a mutation to a recombination activating gene (RAG) using an engineered non-vertebrate cell, the method comprising:
- a) obtaining a non-vertebrate cell,
- b) engineering the non-vertebrate cell to express a mutant RAG protein, wherein the mutant RAG protein is associated with an immunodeficiency disease, and wherein the non-vertebrate cell comprises a recombination signal sequence,
- c) culturing the non-vertebrate cell of step b) under cell culture conditions to allow V(D)J recombination activity, and
- d) measuring V(D)J recombination activity in the non-vertebrate cell of step c), wherein a decrease in V(D)J recombination activity relative to a control non-vertebrate cell indicates an increase in severity of the immunodeficiency disease.
Dependencies are inferred from the claim text.