Use of RNA trans-splicing for antibody gene transfer and antibody polypeptide production
View Patent ↗The present invention provides methods and compositions for generating novel nucleic acid molecules through RNA trans-splicing that target a highly expressed pre-mRNA and contain the coding sequence for antibody polypeptide(s). The compositions of the invention include pre-trans-splicing molecules (PTMs) designed to interact with the target precursor messenger RNA molecule (target pre-mRNA) that is abundantly expressed or tumor specific and mediate a trans-splicing reaction resulting in the generation of novel chimeric RNA molecule (chimeric RNA) capable of encoding an antibody polypeptide. The invention provides for the in vivo production of chimeric RNA molecules that encode and result in the production of an antibody polypeptide that is therapeutically effective against, for example, infectious agents, cancer cells, transplantation antigens, rheumatoid arthritis, etc.
1. An isolated cell comprising a chimeric RNA molecule that encodes an antibody polypeptide wherein said chimeric RNA molecule comprises:
a) one or more target binding domains that target binding of a nucleic acid molecule that encodes the antibody polypeptide to an albumin pre-mRNA within the cell;
b) a splice region;
c) a spacer region that separates the splice region from the target binding domain; and
d) a nucleotide sequence encoding the antibody polypeptide to be trans-spliced to the target albumin pre-mRNA;
wherein said chimeric RNA molecule is recognized by nuclear splicing components within the cell.
2. The isolated cell of claim 1 wherein the antibody polypeptide is selected from the group consisting of an Ig heavy chain, an Ig light chain, an Ig Fv fragment, an Ig Fab fragment, an Ig Fc fragment, a single chain antibody and combinations thereof.
3. The isolated cell of claim 1 wherein the antibody polypeptide is a single chain antibody.
4. The cell of claim 1 wherein the antibody polypeptide comprises an Ig heavy chain and an Ig light chain.
5. The isolated cell of claim 1 wherein the antibody polypeptide is specific for a tumor specific or tumor associated antigen.
6. The isolated cell of claim 1 wherein the antibody polypeptide is specific for a microbial or autoantigen associated antigen.
7. The isolated cell of claim 6 wherein the microbial associated antigen is selected from the group consisting of viral and yeast antigens.
8. The isolated cell of claim 1 wherein the nucleic acid molecule further comprises a sequence encoding a cytokine or a growth factor.
9. An isolated cell comprising a chimeric RNA molecule that encodes an antibody polypeptide wherein said chimeric RNA molecule comprises:
a) one or more target binding domains that target binding of a nucleic acid molecule that encodes the antibody polypeptide to an albumin pre-mRNA within the cell;
b) a splice region;
c) a spacer region that separates the splice region from the target binding domain; and
d) a nucleotide sequence encoding the antibody polypeptide to be trans-spliced to the albumin pre-mRNA;
wherein said chimeric RNA molecule is at an effective physiological and clinical level for production of antibody polypeptides and is recognized by nuclear splicing components within the cell.
10. The isolated cell of claim 9 wherein the antibody polypeptide is selected from the group consisting of an Ig heavy chain, an Ig light chain, an Ig Fv fragment, an Ig Fab fragment, an Ig Fc fragment, a single chain antibody and combinations thereof.
11. The isolated cell of claim 9 wherein the antibody polypeptide is a single chain antibody.
12. The isolated cell of claim 9 wherein the antibody polypeptide comprises an Ig heavy chain and an Ig light chain.
13. The isolated cell of claim 9 wherein the antibody polypeptide is specific for a tumor specific or tumor associated antigen.
14. The isolated cell of claim 9 wherein the antibody polypeptide is specific for a microbial or autoantigen associated antigen.
15. The isolated cell of claim 14 wherein the microbial associated antigen is selected from the group consisting of viral and yeast antigens.
16. The isolated cell of claim 9 wherein the nucleic acid molecule further comprises a sequence encoding a cytokine or a growth factor.