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 of a protein or polypeptide of interest. 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, and mediate a trans-splicing reaction resulting in the generation of novel chimeric RNA molecule (chimeric RNA) capable of encoding a protein or polypeptide of interest. The invention provides for the in vivo production of chimeric RNA molecules that encode and result in the production of a protein or polypeptide of interest.
1. An isolated cell comprising a nucleic acid molecule that encodes a protein or polypeptide of interest wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule that encodes the protein or polypeptide of interest to an abundantly expressed target pre-mRNA within the cell, wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin;
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 a protein or polypeptide of interest to be trans-spliced to the target pre-mRNA;
wherein said nucleic acid molecule encodes a polypeptide of interest heterologous to the target pre-mRNA.
2. The isolated cell of claim 1 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.
3. The isolated cell of claim 1 wherein the protein or polypeptide of interest is selected from the group consisting of ApoA1 and ApoA1 milano variant.
4. The isolated cell of claim 1 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.
5. The isolated cell of claim 1 wherein the protein or polypeptide of interest comprises Factor VIII protein.
6. An isolated nucleic acid molecule that encodes a protein or polypeptide of interest wherein said nucleic acid molecule comprises:
a) one or more target binding domains that target binding of the nucleic acid molecule that encodes the protein or polypeptide of interest to an abundantly expressed target pre-mRNA within the cell, wherein said nucleic acid molecule is recognized by nuclear splicing components within the cell and wherein the abundantly expressed target pre-mRNA is selected from the group consisting of pre-mRNAs encoding albumin, casein, myosin and fibroin;
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 protein or polypeptide of interest to be trans-spliced to the target pre-mRNA;
wherein said isolated nucleic acid molecule encodes a polypeptide of interest heterologous to the target pre-mRNA.
7. The isolated nucleic acid molecule of claim 6 wherein the protein or polypeptide of interest is selected from the group consisting of cytokines, growth factors, insulin, hormones, enzymes and antibody polypeptides.
8. The isolated nucleic acid molecule of claim 6 wherein the protein or polypeptide of interest is selected from the group consisting of ApoA1 and ApoA1 milano variant.
9. The isolated nucleic acid molecule of claim 6 wherein the protein or polypeptide of interest comprises a single chain antibody polypeptide.
10. The isolated nucleic acid molecule of claim 6 wherein the protein or polypeptide of interest comprises Factor VIII protein.