Viral vectors encoding multiple highly homologous non-viral polypeptides and the use of same
The present invention relates to compositions and methods for creation of vector nucleic acid sequences (e.g., retroviral nucleic acid sequences) that comprise two or more exogenous nucleic acid sequences that encode highly homologous (e.g., identical) polypeptide sequences, yet wherein at least one of the exogenous nucleic acid sequences has been mutated using degenerate codons for purpose of reducing homology between the two or more exogenous nucleic acid sequences while maintaining the encoded polypeptide sequence. Preferred nucleic acid sequences include those encoding multi-chimeric immune receptor (CIR) genes. Specific nucleic acid sequences of such CIR genes are also disclosed.
1. A retroviral vector comprising:
a first nucleic acid sequence that encodes a first polypeptide sequence and a second nucleic acid sequence that encodes a second polypeptide sequence, wherein:
(a) the first polypeptide sequence and the second polypeptide sequence comprise an identical polypeptide sequence of greater than 15 amino acids in length; and
(b) the first nucleic acid sequence and the second nucleic acid sequence contain at least one non-identical codon within the codons that encode the identical polypeptide sequence of the first and second nucleic acid sequences; and
(c) one of the first or second nucleic acid sequences is selected from the group consisting of:
(1) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:1;
(2) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:4;
(3) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:7;
(4) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:16;
(5) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 20; and
(6) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:22.
2. A retroviral vector comprising:
a first nucleic acid sequence that encodes a first polypeptide sequence and a second nucleic acid sequence that encodes a second polypeptide sequence, wherein:
(a) the first polypeptide sequence comprises a polypeptide sequence of greater than 15 amino acids in length that is greater than 90% identical to the second polypeptide sequence;
(b) the first nucleic acid sequence and the second nucleic acid sequence contain at least one non-identical codon that encodes an identical amino acid residue, wherein the greater than 90% identical polypeptide sequence of the first and second nucleic acid sequences comprises the identical amino acid residue; and
(c) one of the first or second nucleic acid sequences is selected from the group consisting of :
(1) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:1;
(2) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:4;
(3) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:7;
(4) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:16;
(5) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO: 20; and
(6) a nucleic acid comprising at least 95% sequence identity to the polynucleotide sequence of SEQ ID NO:22.
3. The retroviral vector of claim 1 , wherein the retroviral vector is selected from the group consisting of avian sarcoma-leukosis virus (ASLV), murine leukemia virus (MLV), human-, simian-, feline-, and bovine immunodeficiency viruses (HIV, SIV, FIV, BIV), equine infectious anaemia virus (EIAV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukemia virus (Mo-MLV), MFG, FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukemia virus (A-MLV), Avian myelocytomatosis virus-29 (MC29), Avian erythroblastosis virus (AEV), AKR (endogenous) murine leukemia virus, Avian carcinoma, Mill Hill virus 2, Avian leukosis virus—RSA, Avian myeloblastosis virus, Avian myelocytomatosis virus 29, Bovine syncytial virus, Caprine arthritis encephalitis virus, Chick syncytial virus, Equine infectious anemia virus, Feline leukemia virus, Feline syncytial virus, Finkel-Biskis-Jinkins murine sarcoma virus, Friend murine leukemia virus, Fujinami sarcoma virus, Gardner-Arnstein feline sarcoma virus, Gibbon ape leukemia virus, Guinea pig type C oncovirus, Hardy-Auckerman feline sarcoma virus, Harvey murine sarcoma virus, Human foamy virus, Human spumavirus, Human T-lymphotropic virus 1, Human T-lymphotropic virus 2, Jaagsiekte virus, Kirsten murine sarcoma virus, Langur virus, Mason-Pfizer monkey virus, Mouse mammary tumor virus, Ovine pulmonary adenocarcinoma virus, Porcine type C oncovirus, Reticuloendotheliosis virus, Rous sarcoma virus, Simian foamy virus, Simian sarcoma virus, Simian T-lymphotropic virus, Simian type D virus 1, Snyder-Theilen feline sarcoma virus, Squirrel monkey retrovirus, Trager duck spleen necrosis virus, UR2 sarcoma virus, Viper retrovirus, Visna/maedi virus, Woolly monkey sarcoma virus, and Y73 sarcoma virus.
4. The retroviral vector of claim 1 , wherein at least 20% of the codons that encode the at least one identical polypeptide domain of the first and second nucleic acid sequences are non-identical.
5. The retroviral vector of claim 1 , wherein the first nucleic acid sequence and the second nucleic acid sequence are less than 80% identical within the codons that encode the at least one identical polypeptide domain of the first and second nucleic acid sequences.
6. The retroviral vector of claim 1 , wherein at least one of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence.
7. The retroviral vector of claim 6 , wherein at least one of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence comprising a signal sequence that directs the polypeptide to the cell surface.
8. The retroviral vector of claim 6 , wherein the signal sequence that directs the polypeptide to the cell surface is selected from the group consisting of TCR-α, TCR-β, TCR-.gamma., IgG-Δ, IgG, IgA, IgM, IgE, IgD, CD2, CD4, CD8, CD28, CD3λ, FcεRIγ, and LFA-1.
9. The retroviral vector of claim 6 , wherein at least one of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence comprising an extracellular binding domain selected from the group consisting of a surface membrane polypeptide that binds specifically to at least one ligand and a secreted polypeptide that binds specifically to at least one ligand.
10. The retroviral vector of claim 6 , wherein at least one of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence comprising a transmembrane domain selected from the group consisting of TCR-α, TCR-β, TCR-γ, TCR-Δ, IgG, IgA, IgM, IgE, IgD, CD2, CD4, CD8, CD28, CD3ζ, FcεRIγ, and LFA-1.
11. The retroviral vector of claim 6 , wherein both of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence.
12. The retroviral vector of claim 7 , wherein both of the first and second polypeptide sequences is a chimeric immune receptor polypeptide sequence.
13. The retroviral vector of claim 1 comprising a mutant nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:22.
14. A recombinant virus comprising the retroviral vector of claim 1 .
15. A cell comprising the retroviral vector of claim 1 .
16. The retroviral vector of claim 1 wherein: (a) the first polypeptide sequence and the second polypeptide sequence each comprises an identical single chain antibody polypeptide sequence or an antigen-binding fragment thereof and (b) the first nucleic acid sequence and the second nucleic acid sequence contain at least one non-identical codon within the codons that encode the identical single chain antibody polypeptide sequence or the antigen-binding fragment thereof.
17. The retroviral vector of claim 1 wherein: (a) the first polypeptide sequence and the second polypeptide sequence each comprises a TCR alpha chain and a TCR beta chain, respectively, or an antigen-binding fragment thereof and (b) the first nucleic acid sequence and the second nucleic acid sequence contain at least one non-identical codon within the codons that encode the first and the second polypeptide or the antigen-binding fragment thereof.
18. A pharmaceutical composition comprising the recombinant retrovirus of claim 1 , and a pharmaceutically acceptable carrier.
19. A kit comprising a virus comprising the retroviral vector of claim 1 , and instructions for use.