ENHANCED NUCLEIC ACID CONSTRUCTS FOR EUKARYOTIC GENE EXPRESSION
The present invention provides polynucleotide vectors for high expression of heterologous genes, and methods for constructing such vectors. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, but not limited to, gene expression, gene therapy, insertional mutagenesis, or gene discovery.
1 - 41 . (canceled)
42 . A polynucleotide comprising a transposon comprising inverted terminal repeats of a piggyBac-like transposon flanking a heterologous polynucleotide, the inverted repeats being flanked by copies of a target sequence, where the transposon is capable of transposition by a transposase identical to SEQ ID NO: 45 fused to a heterologous nuclear localization signal (NLS).
43 . The polynucleotide of claim 42 , wherein the transposon comprises at least 14 contiguous nucleotides from SEQ ID NO: 5 or 7 or 9 to provide a copy of the target sequence and one inverted repeat, and at least 14 contiguous nucleotides from SEQ ID NO: 6 or 8 to provide the other copy of the target sequence and the other inverted repeat.
44 . The polynucleotide of claim 42 , wherein SEQ ID NO: 124 provides the sequence of each inverted terminal repeat (ITR).
45 . The polynucleotide of claim 42 , wherein the transposon comprises SEQ ID NO: 37 or 38 or 39 to provide a copy of the target sequence and one inverted repeat, and SEQ ID NO: 40 or 41 to provide the other copy of the target sequence and the other inverted repeat.
46 . The polynucleotide of claim 42 , wherein the transposon comprises a sequence that is at least 90% identical to SEQ ID NO: 5 and a sequence that is at least 90% identical to SEQ ID NO: 6.
47 . The polynucleotide of claim 42 , wherein the heterologous nucleic acid comprises a promoter.
48 . The polynucleotide of claim 47 , wherein the promoter is an EF1a promoter, a CMV promoter, a GAPDH promoter, a Herpes Simplex Virus thymidine kinase (HSV-TK) promoter, an actin promoter, a PGK promoter, and an ubiquitin promoter.
49 . The polynucleotide of claim 47 , wherein the heterologous polynucleotide further comprises a second promoter, and wherein the transcription directions from the first and second promoters are different.
50 . The polynucleotide of claim 47 , wherein the promoter is operably linked to one or more of: i) an open reading frame; ii) a selectable marker; iii) a counter-selectable marker; iii) a nucleic acid encoding a regulatory protein; iv) a nucleic acid encoding an inhibitory RNA.
51 . The polynucleotide of claim 50 , wherein the selectable marker is glutamine synthetase (GS) or dihydrofolate reductase (DHFR).
52 . The polynucleotide of claim 42 , wherein the heterologous nucleic acid comprises one or more sequence elements that increase expression by enhancing RNA processing or export from the nucleus.
53 . The polynucleotide of claim 52 , wherein one or more sequence elements are selected from WPRE, HPRE (SEQ ID NO: 104-105), SAR (SEQ ID NOS: 108-111), AGS (SEQ ID NOS: 106-107).
54 . The polynucleotide of claim 42 , wherein the heterologous nucleic acid comprises a pair of insulators.
55 . The polynucleotide of claim 54 , wherein the insulators are selected from SEQ ID NOS: 112-113.
56 . The polynucleotide of claim 42 , further comprising one or more viral replication sequences positioned outside the transposon such that said replication sequences are not capable of transposition by the transposase.
57 . The polynucleotide of claim 56 , wherein the viral replication sequences are selected from the SV40ori, SV40 large T antigen, EBVoriP and EBNA.
58 . The polynucleotide of claim 47 , wherein the promoter is active in a eukaryotic cell.
59 . The polynucleotide of claim 42 , wherein the heterologous DNA sequence comprises two open reading frames operably linked to a promoter, wherein the two open reading frames are linked by translational coupling elements selected from IRES or CHYSEL.
60 . The polynucleotide of claim 42 , wherein the heterologous DNA encodes an antibody heavy chain and/or an antibody light chain.
61 - 71 . (canceled)
72 . A method for producing an antibody from a cell, the method comprising:
i. integrating a transposon according to claim 42 , wherein the heterologous nucleic acid encodes heavy and light chains of the antibody linked by coupling elements selected from IRES or CHYSEL
ii. obtaining antibody from the cell.
73 . A cell line comprising the transposon of claim 42 .
74 - 77 . (canceled)
78 . A transgenic animal comprising the transposon of claim 42 .
79 . A pharmaceutical composition comprising a transposon according to claim 42 and transposase encoding a transposase fused to a heterologous NLS and operably linked to a heterologous promoter wherein the transposase is at least 90% identical to SEQ ID NO: 45 together with a pharmaceutically acceptable carrier, adjuvant or vehicle.
80 . (canceled)
81 . The polynucleotide of claim 59 , wherein two open reading frames are linked by an IRES element, which is any of SEQ ID NOS: 60-100.
82 - 153 . (canceled)