Materials and Methods for Improved Phosphotransferases

Abstract

Described herein are non-naturally occurring neomycin phosphotransferase (NPT) proteins and nucleic acid sequences encoding such NPT proteins. In certain embodiments, the non-naturally occurring NPT proteins have reduced activity compared to wild-type NPT. The non-naturally occurring NPT proteins provided herein are useful as selectable markers for screening transformed or transfected cells. Also provided herein are vectors and kits that include the nucleic acid sequences encoding the non-naturally occurring NPT proteins, as well as methods for producing non-naturally occurring NPT proteins and cells that express a protein of interest or a non-coding RNA sequence of interest.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application is a continuation of U.S. Patent Application No. 63 / 177,739 filed on April 21, 2021, U.S. Patent Application No. 63 / 177,744 filed on April 21, 2021, U.S. Patent Application No. 63 / 177,746 filed on April 21, 2021, U.S. Patent Application No. 63 / 177,749 filed on April 21, 2021, U.S. Patent Application No. This application claims the benefit of U.S. Patent Application No. 63 / 177,753, filed April 21, 2021, U.S. Patent Application No. 63 / 177,759, filed April 21, 2021, U.S. Patent Application No. 63 / 177,764, filed April 21, 2021, and U.S. Patent Application No. 63 / 177,767, filed April 21, 2021, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically via EFS-Web as an ASCII sequence listing with the filename "14620-686-228_SL.txt", created on April 9, 2022, and having a size of 118,113 bytes. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] (Field) Provided herein are non-naturally occurring neomycin phosphotransferase (NPT) proteins and nucleic acid sequences encoding such NPT proteins. In certain embodiments, the non-naturally occurring NPT proteins have reduced activity compared to wild-type NPT. The non-naturally occurring NPT proteins provided herein are useful as selectable markers for screening transformed or transfected cells. Also provided herein are vectors and kits comprising nucleic acid sequences encoding the non-naturally occurring NPT proteins, as well as methods for producing non-naturally occurring NPT proteins and cells expressing a protein of interest or a non-coding RNA sequence of interest. [Background technology]

[0004] In some instances, it has become more challenging to generate mammalian cell lines harboring exogenous transgenes stably integrated into the genome, while identifying clonal lines that express protein products at high levels and / or with high transgene copy numbers is challenging, e.g., inefficient and time-consuming. The sequence of the transgene integration site can significantly affect transgene expression (Lee et al., Trends Biotechnol., 37(9):931-942 (2019)), resulting in dramatically different expression levels in different clones. DNA regulatory elements, when placed between the transgene and host DNA, can be used to shield the transgene from chromosomal position effects (reviewed in Gupta et al., Biotechnol. Adv. 37(8):107415 (2019)). This approach can increase expression and expression stability, but significant screening may still be required to identify high-expressing clones. To develop virus-producing cell lines, generating lines with many copies of the packaged viral payload is likely more important than having high transgene expression. Methods for selecting for multicopy transgenes make cell line development more efficient.

[0005] One problem is that many constructs used to generate stable cell lines contain highly efficient selectable markers that confer a selective advantage to transformed cells, even when expressed at very low levels. Thus, there is no direct selection for high marker gene expression or multicopy transgenes. Several approaches have been described to reduce selectable marker expression or translation efficiency. These include the use of weak promoters to drive expression (Niwa et al., Gene 108(2):193-199 (1991); Fan et al., J Biotechnol 168(4):652-658 (2013); Zhou et al., BMC Biotechnol. 13:29 (2013)), initiation of translation from alternative codons (e.g., GTG or TTG instead of ATG) (van Blokland et al., J Biotechnol 128(2):237-245 (2007); Cairns et al., Biotechnol Bioeng 108(11):2611-2622 (2011)), and the use of internal ribosome entry sites (IRES) to initiate translation (Gurtu et al., Biochem Biophys Res Commun229(1):295-298(1996), Kwaks et al., Nat Biotechnol21(5):553-558(2003), Ho et al., J Biotechnol157(1):130-139(2012)).

[0006] Another approach to reducing selectable marker efficiency is to use mutant proteins with reduced activity. Mutations in the glutamine synthetase (GS) gene have been used to increase selection stringency in CHO cells (Lin et al., MAbs11(5):965-976(2019)). Neomycin phosphotransferase (NPT) from Tn5 (aminoglycoside phosphotransferase 3'-IIa) is one of the most commonly used selectable markers. It confers resistance to neomycin and kanamycin in bacteria and to G418 in mammalian and plant cells by phosphorylating these antibiotics (Shaw et al., Microbiol Rev57(1):138-163(1993)). Mutagenesis studies (Blázquez et al., Mol. Microbiol. 5(6):1511-1518 (1991); Kocabiyik et al., SAAS Bull Biochem Biotechnol 5:58-63 (1992); Kocabiyik and Perlin, Biochem Biophys Res Commun 185(3):925-931 (1992); Kocabiyik and Perlin, Int J Biochem 26(1):61-66 (1994)) and spontaneous mutational findings (Yenofsky et al., Proc Natl Acad Sci USA 87(9):3435-3439 (1990)) have identified key residues that reduce, but do not eliminate, the ability of this protein to confer antibiotic resistance in bacteria. When the mutant NPT gene was incorporated into a vector used to select stable antibody-producing cell lines in CHO cells, increasing the stringency of selection resulted in higher antibody expression and productivity compared to the use of the wild-type NPT gene (Sautter and Enenkel, Biotechnol Bioeng 89(5):530-538 (2005); Ho et al., J Biotechnol 157(1):130-139 (2012)).Using a two-vector system, NPT mutants with 2-16% enzymatic activity increased specific antibody productivity 5-10 fold compared to pools selected with the wild-type NPT gene (Sautter and Enenkel 2005). When a mutant NPT gene with 3% activity was used in a single tricistronic vector, specific productivity increased 17-fold compared to the use of the wild-type NPT gene (Ho et al. 2012). However, these approaches have limitations. Summary of the Invention

[0007] The present invention recognizes and addresses the identification of NPT mutants with significantly reduced activity that allow for more stringent selection of transformed cells, thereby reducing the screening required to identify and generate cell lines that express high levels of a transgene of interest. In one aspect, provided herein is a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises one, two, or more amino acid substitutions in wild-type NPT (e.g., one, two, or more of the amino acid substitutions disclosed in Table 1 or Table 2, or a combination thereof).

[0013] In certain embodiments, provided herein is a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (a) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (b) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine; (c) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (d) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine;Provided is a non-naturally occurring neomycin phosphotransferase (NPT) comprising the amino acid sequence of a wild-type neomycin phosphotransferase having: (a) an amino acid substitution at amino acid residue corresponding to amino acid residue 261 of SEQ ID NO:1 with asparagine; (b) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1 with methionine and serine; or (c) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1 with methionine and serine, respectively.

[0008] In some embodiments, the non-naturally occurring NPT comprises: (a) amino acid substitutions at positions 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at position 210 of SEQ ID NO:1 is with alanine; (b) amino acid substitutions at positions 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at position 182 of SEQ ID NO:1 is with aspartic acid; (c) amino acid substitutions at positions 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at position 218 of SEQ ID NO:1 is with phenylalanine. (d) amino acid substitutions at positions 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at position 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at position 261 of SEQ ID NO: 1 is a substitution with asparagine; (e) amino acid substitutions at positions 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 218 of SEQ ID NO: 1 is a substitution with serine; or (f) amino acid substitutions at positions 36 and 216 of SEQ ID NO: 1, where the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 216 of SEQ ID NO: 1 is a substitution with glycine.

[0009] In some embodiments, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0010] In some embodiments, wild-type NPT comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments, wild-type NPT comprises an amino acid sequence at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0011] In some embodiments, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to a wild-type NPT comprising the amino acid sequence of SEQ ID NO:1.

[0012] In some embodiments, bacterial cells transfected or transformed with a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation compared to bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin. In some embodiments, the bacterial cells are E. coli. In some embodiments, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1.

[0013] In some embodiments, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a native NPT exhibit reduced colony formation, as assessed by colony formation assay, after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418), compared to mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells. In some embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 0.001% to 75% of the G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In certain embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 5.5% to 0.004% compared to G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0014] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine.

[0015] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid.

[0016] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with phenylalanine.

[0017] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine.

[0018] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with serine.

[0019] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0020] In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40. In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41. In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42. In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 43.

[0021] In another aspect, provided herein is a nucleic acid comprising a first nucleotide sequence encoding a non-naturally occurring NPT described herein. In some embodiments, the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:37.

[0022] In some embodiments, the nucleic acid sequence further comprises a second nucleotide sequence encoding a second protein or a non-coding RNA, hi some embodiments, the second nucleotide sequence encodes a second protein, and the second protein is a therapeutic protein.

[0023] In another aspect, provided herein is a vector comprising a nucleic acid sequence described herein.

[0024] In another aspect, provided herein is an in vitro or ex vivo host cell comprising a non-naturally occurring NPT. In some embodiments, the host cell comprises a nucleic acid comprising a first nucleotide sequence encoding the non-naturally occurring NPT. In some embodiments, the host cell comprises a nucleic acid comprising the nucleotide sequence of SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:37. In some embodiments, the nucleic acid sequence is stably integrated into the genome of the host cell. In some embodiments, the host cell comprises a vector. In certain embodiments, the host cell is a bacterium, a yeast cell, a mammalian cell, or a plant cell. In certain embodiments, the host cell is derived from a human cell line.

[0025] In another aspect, provided herein is a non-naturally occurring NPT, wherein the non-naturally occurring NPT is attenuated compared to wild-type neomycin phosphotransferase, the non-naturally occurring NPT comprising: (a) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (b) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid; (c) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and (d) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (e) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (f) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine;An in vitro or ex vivo host cell is provided that expresses a non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase having an amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1, wherein the amino acid substitution is a substitution of glycine.

[0026] In some embodiments, an in vitro or ex vivo host cell expresses a non-naturally occurring NPT that has attenuated activity compared to wild-type neomycin phosphotransferase, wherein the non-naturally occurring NPT comprises: (a) amino acid substitutions at amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (b) amino acid substitutions at amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (c) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (d) amino acid substitutions at amino acid residues 216 and 261 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine; (e) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and amino acid residue 218 of SEQ ID NO:1 is a substitution with serine; or (f) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0027] In some embodiments of the in vitro or ex vivo host cells described herein, the wild-type NPT comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments, the wild-type NPT comprises an amino acid sequence at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1. In some embodiments, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to a wild-type NPT comprising the amino acid sequence of SEQ ID NO: 1.

[0028] In some embodiments of the in vitro or ex vivo host cells described herein, bacterial cells transfected or transformed with a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin, compared to bacterial cells transfected or transformed with a nucleotide sequence encoding a wild-type NPT. In some embodiments, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bacterial cell is E. coli.

[0029] In some embodiments of the in vitro or ex vivo host cells described herein, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation, as assessed by a colony formation assay, after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418), compared to mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 0.001% to 75% of the G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In certain embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 5.5% to 0.004% compared to G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0030] In some embodiments of the in vitro or ex vivo host cells described herein, the expressed non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A). In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D). In some embodiments of the in vitro or ex vivo host cells described herein, the expressed non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F). In some embodiments of the in vitro or ex vivo host cells described herein, the expressed non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N). In some embodiments of the in vitro or ex vivo host cells described herein, the expressed non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S). In some embodiments of the in vitro or ex vivo host cells described herein, the expressed non-native NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0031] In some embodiments, the in vitro or ex vivo host cell further comprises a second nucleic acid sequence encoding a second protein or non-coding RNA. In some embodiments, the second nucleic acid sequence encodes a second protein, and the second protein is a therapeutic protein. In some embodiments, the second nucleic acid sequence encodes a non-coding RNA, and the non-coding RNA is an shRNA, miRNA, antisense RNA, Crispr nuclease guide RNA, catalytic RNA, ribosomal RNA, or tRNA. In certain embodiments, the host cell is a bacterium, yeast cell, mammalian cell, or plant cell.

[0032] In another aspect, provided herein is a method for selecting cells having a high copy number and / or high expression level of a transgene from a population of host cells into which the transgene has been introduced, the method comprising: a) introducing into the population of host cells a nucleic acid sequence comprising (i) a first nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein having neomycin phosphotransferase activity, and (ii) a second nucleotide sequence comprising the transgene; and b) selecting from the population of host cells into which the nucleic acid sequence has been introduced cells that grow in the presence of a neomycin phosphotransferase substrate.

[0033] In one embodiment, provided herein is a method for selecting cells having a high copy number and / or high expression level of a transgene from a population of host cells into which a transgene has been introduced, the method comprising: a) introducing into the host cell population a nucleic acid sequence comprising: (i) a first nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity; and (ii) a second nucleotide sequence comprising the transgene, wherein the non-naturally occurring NPT has (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is to alanine; (3) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine.or (6) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; and b) selecting cells from the population of host cells into which the nucleic acid sequence has been introduced that grow in the presence of a neomycin phosphotransferase substrate.

[0034] In certain embodiments, a method for selecting cells having a high copy number and / or high expression level of a transgene from a population of host cells into which a transgene has been introduced comprises: a) introducing into the host cell population a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity; and (ii) a second nucleotide sequence comprising the transgene, wherein the non-naturally occurring NPT has: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 21 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (4) amino acid substitutions at amino acid residues 216 and 261 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with serine; or (6) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine;b) selecting cells from the population of host cells into which the nucleic acid sequence has been introduced that grow in the presence of a neomycin phosphotransferase substrate;

[0035] In certain embodiments of the method for selecting cells with high copy numbers and / or high expression levels of the transgene, the non-native NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0036] In some embodiments of the methods for selecting cells with a high copy number of the transgene and / or a high expression level of the transgene, the high copy number of the transgene is 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the non-native or mutant NPT. In some embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of the transgene, the high expression levels of the transgene are 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 5-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the non-naturally occurring or mutant NPT.

[0037] In some embodiments of the methods for selecting cells with a high copy number and / or high expression level of the transgene, the wild-type NPT comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In certain embodiments of the methods for selecting cells with a high copy number and / or high expression level of the transgene, the wild-type NPT comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0038] In certain embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, bacterial cells transfected or transformed with a nucleotide sequence encoding the non-native NPT exhibit reduced colony formation compared to bacterial cells transfected or transformed with a nucleotide sequence encoding the wild-type NPT, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin. In some embodiments, the bacterial cells are E. coli. In certain embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding the non-native NPT exhibit reduced colony formation compared to mammalian cells transfected with the same expression vector but comprising the nucleotide sequence encoding the wild-type NPT, as assessed by a colony formation assay after 2 weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418). In certain embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 0.001% to 75% of the G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0039] In certain embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of the transgene, the non-native NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, bacterial cells transfected or transformed with a nucleotide sequence encoding the non-native NPT exhibit reduced colony formation, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin, compared to bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT comprising the amino acid sequence of SEQ ID NO: 1. In some embodiments, the bacterial cells are E. coli. In certain embodiments, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-naturally occurring NPT exhibit reduced colony formation, as assessed by a colony formation assay, after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418), compared to mammalian cells transfected with the same expression vector, except that the expression vector comprises a nucleotide sequence encoding wild-type NPT, wherein the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-naturally occurring NPT are generated at a frequency ranging from 5.5% to 0.004%, compared to G418-resistant colonies of mammalian cells transfected with the same expression vector, except that the expression vector comprises a nucleotide sequence encoding wild-type NPT, wherein the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mammalian cell is a HEK293 cell, a CHO cell, a PER.C6 cell, a mouse NS0 cell, a fibrosarcoma HT-1080 cell, a mouse Sp2 / 0 cell, a BHK cell, or a mouse C127 cell.

[0040] In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is to alanine. In certain embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine. In certain embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine.In certain embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0041] In certain embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A). In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D). In certain embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F). In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N). In certain embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S). In some embodiments, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0042] In certain embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of a transgene, the host cell is a bacterial, yeast, mammalian, or plant cell. In some embodiments, the host cell is a human cell. In certain embodiments, the host cell is derived from a mammalian cell line (e.g., a human cell line).

[0043] In certain embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of the transgene, the nucleic acid sequence is stably integrated into the genome of the selected cells. In some embodiments, the selected cells have 5 to 100 copies of the transgene integrated into their genomic DNA. In certain embodiments, the selected cells have 1 to 5 copies of the transgene integrated into their genomic DNA.

[0044] In certain embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells have a high copy number of the transgene. In some embodiments, the high copy number of the transgene is 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the NPT variant or non-native NPT. In certain embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells have a high level of expression of the transgene. In some embodiments, the high expression level of the transgene is 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 5-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the non-native or mutant NPT. In certain embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells have a high copy number of the transgene and a high level of expression of the transgene. In some embodiments, the high copy number of the transgene is 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, or 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of an NPT mutant or non-naturally occurring NPT.In some embodiments, the high expression levels of the transgene are 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1,000 fold, or 5-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the non-naturally occurring or mutant NPT.

[0045] In certain embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of a transgene, the transgene comprises a viral gene. In some embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of a transgene, the transgene comprises a human growth factor gene.

[0046] In certain embodiments of the method for selecting cells with high copy numbers and / or high expression levels of the transgene, the neomycin phosphotransferase substrate is neomycin, kanamycin, or G418.

[0047] In some embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells comprise a 10-1000 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells comprise a 100-1000 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells comprise a 500-1000-fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells comprise a 750-1000-fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene.In some embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells comprise a 100-500 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells comprise a 10-100 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells comprise a 10-50 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells comprise a 10-25 fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene.In some embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells contain a 2- to 10-fold higher copy number of the transgene compared to the copy number of the transgene in a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene.

[0048] In some embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells achieve a 10-1000 fold higher level of expression of the transgene compared to the level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the methods for selecting cells having a high copy number of the transgene and / or a high expression level of the transgene, the selected cells achieve a 100-1000 fold higher level of expression of the transgene compared to the level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 500-1000 fold higher level of expression of the transgene compared to the level of expression of the transgene by the second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 750-1000 fold higher level of expression of the transgene compared to the level of expression of the transgene by the second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene.In some embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 10-100 fold higher level of expression of the transgene compared to the level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the methods for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 10-50 fold higher level of expression of the transgene compared to the level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 5-25 fold higher level of expression of the transgene compared to the level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In some embodiments of the method for selecting cells having a high copy number and / or high expression level of the transgene, the selected cells achieve a 5-10 fold higher level of expression of the transgene by a second set of cells after selection of a second population of host cells grown in the presence of a neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene.In some embodiments of the methods for selecting cells with high copy numbers and / or high expression levels of the transgene, the selected cells achieve 2-10 fold higher levels of expression of the transgene by a second set of cells following selection of a second population of host cells grown in the presence of neomycin phosphotransferase substrate, the second population of host cells having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene. In certain embodiments, the population of host cells is the same and the conditions used are the same.

[0049] In certain embodiments of the method for selecting cells with a high copy number and / or high expression level of a transgene, the transgene encodes a protein or a non-coding RNA. In some embodiments, the non-coding RNA is selected from the group consisting of antisense RNA, miRNA, shRNA, long non-coding RNA, catalytic RNA, ribosomal RNA, tRNA, or guide RNA for a CRISPR nuclease. In certain embodiments, the protein is a therapeutic protein or antigen. The therapeutic protein or antigen may be one described herein or known to those of skill in the art. In certain embodiments, the protein is a viral protein. The viral protein may be one described herein or known to those of skill in the art.

[0050] In some embodiments, the use of a non-native NPT described herein reduces the need to screen cells transfected or transformed with a transgene by 7,500-10,000 fold. In some embodiments, the use of a non-native NPT described herein reduces the need to screen cells transfected or transformed with a transgene by 5,000-10,000 fold. In some embodiments, the use of a non-native NPT described herein reduces the need to screen cells transfected or transformed with a transgene by 2,500-10,000 fold. In some embodiments, the use of a non-native NPT described herein reduces the need to screen cells transfected or transformed with a transgene by 1,000-10,000 fold. In some embodiments, the use of a non-native NPT described herein reduces the need to screen cells transfected or transformed with a transgene by 5,000-7,500 fold. In some embodiments, the use of non-naturally occurring NPTs described herein reduces the need to screen cells transfected or transformed with a transgene by 1,000-5,000 fold. In some embodiments, the use of non-naturally occurring NPTs described herein reduces the need to screen cells transfected or transformed with a transgene by 500-1,000 fold.

[0051] In another aspect, provided herein are methods of using a plasmid or transposon comprising a nucleic acid sequence encoding a non-native NPT described herein that has attenuated neomycin phosphotransferase activity relative to wild-type NPT as a selectable marker, the method comprising: a) introducing the plasmid or transposon comprising the nucleic acid sequence into a host cell; and b) growing the cells in the presence of a neomycin phosphotransferase substrate. In some embodiments, the method further comprises selecting host cells that grow in the presence of the neomycin phosphotransferase substrate.

[0052] In one embodiment, provided herein is a method of using a plasmid or transposon comprising a nucleic acid sequence encoding a non-naturally occurring NPT having attenuated neomycin phosphotransferase activity relative to wild-type NPT as a selectable marker, the method comprising: a) introducing into a host cell the plasmid or transposon comprising a nucleic acid sequence encoding the non-naturally occurring NPT, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) an amino acid substitution at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) introducing into a host cell a mutant comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; and b) growing the cell in the presence of a neomycin phosphotransferase substrate. In some embodiments, the method further comprises selecting host cells that grow in the presence of the neomycin phosphotransferase substrate.

[0053] In some embodiments, provided herein are methods for using a plasmid or transposon comprising a nucleic acid sequence encoding a non-naturally occurring NPT having attenuated neomycin phosphotransferase activity relative to wild-type NPT as a selectable marker, the method comprising: a) introducing into a host cell a plasmid or transposon comprising a nucleic acid sequence encoding the non-naturally occurring NPT, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine; (4) amino acid substitutions at amino acid residues 216 and 261 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is with asparagine; (5) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is with serine; or (6) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is with glycine; and b) growing the cell in the presence of a neomycin phosphotransferase substrate.In some embodiments, the method further comprises selecting host cells that grow in the presence of a neomycin phosphotransferase substrate.

[0054] In some embodiments of plasmid- or transposon-based methods, the wild-type NPT comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In certain embodiments of plasmid- or transposon-based methods, the wild-type NPT comprises an amino acid sequence at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0055] In certain embodiments of the methods that use a plasmid or a transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is a substitution with alanine. In some embodiments of the methods that use a plasmid or a transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is a substitution with aspartic acid. In certain embodiments of the methods that use a plasmid or a transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine. In some embodiments of the methods that use a plasmid or a transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine. In certain embodiments of the methods using a plasmid or transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with serine.In some embodiments of the methods using a plasmid or transposon, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0056] In certain embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A). In some embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D). In certain embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F). In some embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N). In certain embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S). In some embodiments of the methods that use a plasmid or transposon, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0057] In certain embodiments of methods using plasmids or transposons, the host cell is a bacterial, yeast, mammalian, or plant cell. In some embodiments, the host cell is a human cell.

[0058] In certain embodiments of methods using a plasmid or transposon, the plasmid or transposon further comprises a second nucleotide sequence encoding a protein or a non-coding RNA. In some embodiments, the protein is a viral protein. In certain embodiments, the protein is a therapeutic protein.

[0059] In certain embodiments of the plasmid or transposon-based methods, the neomycin phosphotransferase substrate is neomycin, kanamycin, or G418.

[0060] In another aspect, provided herein are methods of producing a host cell, the method comprising: a) introducing into a population of host cells a first nucleic acid sequence comprising (i) a first nucleotide sequence encoding a non-naturally occurring NPT as described herein and (ii) a second nucleotide sequence comprising a transgene encoding a second protein or non-coding RNA; b) growing the host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies; and c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate. In some embodiments, the method further comprises culturing the selected cell colonies.

[0061]

[0013] In one embodiment, provided herein is a method of making a host cell comprising a second nucleotide sequence, the method comprising: a) introducing a host cell population having a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding a non-naturally occurring NPT; and (ii) a second nucleotide sequence comprising a transgene encoding a second protein or non-coding RNA, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (6) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1,a) introducing a host cell population comprising the amino acid sequence of a wild-type neomycin phosphotransferase having an amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 with a methionine substitution and at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 with a glycine substitution; b) growing the host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies; and c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate.

[0062] In another aspect, provided herein are methods of producing host cells comprising a second nucleotide sequence, the method comprising: a) co-introducing into a host cell population (i) a first nucleic acid sequence comprising a first nucleotide sequence encoding a non-naturally occurring NPT as described herein, and (ii) a second nucleic acid sequence comprising a transgene encoding a second protein or non-coding RNA; b) growing the host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies; and c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate. In some embodiments, the method further comprises culturing the selected cell colonies.

[0063]

[0013] In some embodiments, provided herein are methods of generating a host cell comprising a second nucleotide sequence, the method comprising: a) co-introducing into a host cell population (i) a first nucleic acid sequence comprising a first nucleotide sequence encoding a non-naturally occurring NPT as described herein; and (ii) a second nucleic acid sequence comprising a transgene encoding a second protein or non-coding RNA, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) an amino acid substitution at amino acid residue 36 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (6) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1,a) co-introducing a host cell population comprising an amino acid sequence of a wild-type neomycin phosphotransferase having an amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 with a methionine substitution and at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 with a glycine substitution; b) growing the host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies; and c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate.

[0064]

[0013] In some embodiments, provided herein are methods of generating a host cell comprising a second nucleotide sequence, the method comprising: a) co-introducing into a host cell population (i) a first nucleic acid sequence comprising a first nucleotide sequence encoding a non-naturally occurring NPT; and (ii) a second nucleic acid sequence comprising a transgene encoding a second protein or non-coding RNA, wherein the non-naturally occurring NPT comprises (1) amino acid substitutions at positions 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is a substitution of methionine and the amino acid substitution at position 36 of SEQ ID NO:1 is a substitution of methionine and the amino acid substitution at position 210 ... (2) amino acid substitutions at positions 36 and 182 of SEQ ID NO: 1, where the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 182 of SEQ ID NO: 1 is a substitution with aspartic acid; (3) amino acid substitutions at positions 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 218 of SEQ ID NO: 1 is a substitution with phenylalanine. (4) amino acid substitutions at positions 216 and 261 of SEQ ID NO:1, where the amino acid substitution at position 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at position 261 of SEQ ID NO:1 is a substitution with asparagine; (5) amino acid substitutions at positions 36 and 218 of SEQ ID NO:1, where the amino acid substitution at position 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at position 218 of SEQ ID NO:1 is a substitution with serine; or (6) amino acid substitutions at positions 36 and 216 of SEQ ID NO:1, where the amino acid substitution at position 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at position 216 of SEQ ID NO:1 is a substitution with glycine; b) growing the host cell population in the presence of neomycin phosphotransferase substrate to generate colonies; and c) selecting cell colonies that grow in the presence of neomycin phosphotransferase substrate.

[0065] In another aspect, provided herein are methods of producing host cells comprising a second nucleotide sequence, the method comprising: a) growing a host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies, wherein the host cell population comprises a first nucleic acid sequence comprising (i) a first nucleotide sequence encoding a non-naturally occurring NPT described herein, and (ii) a second nucleotide sequence comprising a transgene encoding a second protein or non-coding RNA; and b) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate. In some embodiments, the method further comprises culturing the selected cell colonies.

[0066]

[0013] In one embodiment, provided herein is a method of making a host cell comprising a second nucleotide sequence, the method comprising: a) growing a host cell population in the presence of a substrate for neomycin phosphotransferase to produce colonies, wherein the host cell population comprises a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding a non-naturally occurring NPT; and (ii) a second nucleotide sequence comprising a transgene encoding a second protein or non-coding RNA, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is a substitution with alanine; (3) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) an amino acid substitution at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) an amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; and b) selecting cell colonies that grow in the presence of a neomycin phosphotransferase substrate. In some embodiments, the method further comprises culturing the selected cell colonies.

[0067] In some embodiments, a method of producing a host cell comprising a second nucleotide sequence comprises a) growing a host cell population in the presence of a substrate for neomycin phosphotransferase to produce colonies, wherein the host cell population comprises (i) a first nucleic acid sequence comprising a first nucleotide sequence encoding a non-naturally occurring NPT; and (ii) a second nucleic acid sequence comprising a transgene encoding a second protein or non-coding RNA, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at positions 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at position 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at positions 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at position 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at positions 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at position 182 of SEQ ID NO:1 is to aspartic acid; (4) amino acid substitutions at positions 216 and 261 of SEQ ID NO:1, where the amino acid substitution at position 216 of SEQ ID NO:1 is to glycine and the amino acid substitution at position 261 of SEQ ID NO:1 is to asparagine; (5) amino acid substitutions at positions 36 and 218 of SEQ ID NO:1, where the amino acid substitution at position 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at position 218 of SEQ ID NO:1 is to serine; or (6) amino acid substitutions at positions 36 and 216 of SEQ ID NO:1, where the amino acid substitution at position 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at position 216 of SEQ ID NO:1 is to glycine; and b) selecting cell colonies that grow in the presence of neomycin phosphotransferase substrate. In some embodiments, the method further comprises culturing the selected cell colonies.

[0068] In another aspect, provided herein is a host cell comprising a second nucleotide sequence produced by the methods described herein.

[0069] In another aspect, provided herein is a method for producing a stable cell line that expresses a therapeutic protein or enzyme, the method comprising: a) introducing one or more nucleic acid sequences into a host cell population, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein that has neomycin phosphotransferase activity; b) selecting, from the cell population of step (a), cells that grow in the presence of G418; and c) culturing the selected cells to produce a stable cell line that expresses the therapeutic protein or enzyme.

[0070]

[0013] In one embodiment, provided herein is a method for producing a stable cell line that expresses a therapeutic protein or enzyme, comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid; (3) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine, and (ii) a second nucleic acid sequence encoding a therapeutic protein or enzyme; b) selecting cells that grow in the presence of G418 from the cell population of step (a); and c) culturing the selected cells to produce a stable cell line expressing the therapeutic protein or enzyme.

[0071] In some embodiments, provided herein are methods for producing a stable cell line that expresses a therapeutic protein or enzyme, comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine; introducing (ii) a first nucleic acid sequence comprising the amino acid sequence of SEQ ID NO: 1 having (i) an amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 with methionine and at amino acid residue 218 of SEQ ID NO: 1 with phenylalanine, (ii) an amino acid substitution at amino acid residues 216 and 261 of SEQ ID NO: 1 with glycine and at amino acid residue 261 of SEQ ID NO: 1 with asparagine, (iii) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO: 1 with serine, or (iv) an amino acid substitution at amino acid residues 36 and 216 of SEQ ID NO: 1 with methionine and at amino acid residue 216 of SEQ ID NO: 1 with glycine;b) selecting cells from the cell population of step (a) that grow in the presence of G418; and c) culturing the selected cells to produce a stable cell line that expresses the therapeutic protein or enzyme.

[0072] In some embodiments of the methods provided herein, the stable cell line is a mammalian cell line. In some embodiments of the methods provided herein, the stable cell line is a human cell line. In some embodiments, the stable cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line. In some embodiments of the methods provided herein, the stable cell line expresses a therapeutic protein. In some embodiments of the methods provided herein, the therapeutic protein is an antibody or antibody fragment. In some embodiments, the stable cell line expresses an enzyme.

[0073] In another aspect, provided herein are stable cell lines produced by the methods described herein. In some embodiments, the stability of the cell line can be determined by measuring the copy number of the transgene by a quantitative method, such as, for example, qPCR or hybridization.

[0074] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0075] In some embodiments of the methods provided herein, the wild-type NPT comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments of the methods provided, the wild-type NPT comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0076] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to a wild-type NPT comprising the amino acid sequence of SEQ ID NO:1.

[0077] In some embodiments of the methods provided herein, bacterial cells transfected or transformed with a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation compared to bacterial cells transfected or transformed with a nucleotide sequence encoding a wild-type NPT, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin. In some embodiments, the bacterial cells are E. coli.

[0078] In some embodiments of the methods provided herein, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation, as assessed by a colony formation assay, after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418), compared to mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0079] In some embodiments of the methods provided herein, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 0.001% to 75% compared to G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-native NPT are generated at a frequency ranging from 5.5% to 0.004% compared to G418-resistant colonies of mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT. In some embodiments, the mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0080] In some embodiments of the methods provided herein, bacterial cells transfected or transformed with a nucleotide sequence encoding a non-native NPT exhibit reduced colony formation, as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin, compared to bacterial cells transfected or transformed with a nucleotide sequence encoding a wild-type NPT, wherein the wild-type NPT comprises the amino acid sequence of SEQ ID NO:1.

[0081] In some embodiments of the methods provided herein, mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-naturally occurring NPT exhibit reduced colony formation, as assessed by a colony formation assay, after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418), compared to mammalian cells transfected with the same expression vector except comprising a nucleotide sequence encoding wild-type NPT, wherein the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, G418-resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding a non-naturally occurring NPT are generated at a frequency ranging from 5.5% to 0.004%, compared to G418-resistant colonies of mammalian cells transfected with the same expression vector except comprising a nucleotide sequence encoding wild-type NPT, wherein the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the mammalian cell is a HEK293 cell, a CHO cell, a PER.C6 cell, a mouse NS0 cell, a fibrosarcoma HT-1080 cell, a mouse Sp2 / 0 cell, a BHK cell, or a mouse C127 cell.

[0082] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is to alanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is with asparagine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is with serine.In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0083] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A).

[0084] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D).

[0085] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F).

[0086] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N).

[0087] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S).

[0088] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0089] In some embodiments of the methods provided herein, a host cell population is transfected or transformed, and the host cells of the host cell population produce fewer colonies than a second host cell population transfected or transformed with a second nucleic acid sequence and grown in the presence of a neomycin phosphotransferase substrate, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence.

[0090] The host cell can be, for example, a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell, a CHO cell, a PER.C6 cell, a mouse NS0 cell, a fibrosarcoma HT-1080 cell, a mouse Sp2 / 0 cell, a BHK cell, or a mouse C127 cell. In some embodiments, the cell is a human cell.

[0091] In some embodiments of the methods provided herein, the neomycin phosphotransferase substrate is neomycin, kanamycin, or G418.

[0092] In some embodiments of the methods provided herein, the protein is a therapeutic protein or an antigen.

[0093] In some embodiments of the methods provided herein, the non-coding RNA is an shRNA, miRNA, antisense RNA, guide RNA for Crispr nuclease, catalytic RNA, ribosomal RNA, or tRNA.

[0094] In another aspect, provided herein is a method of generating a virus-producing cell line, the method comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein that has neomycin phosphotransferase activity; b) selecting from the cell population cells that grow in the presence of a neomycin phosphotransferase substrate; and c) growing the selected cells to generate a virus-producing cell line. The virus-producing cell line can be used, for example, to generate viruses for gene therapy or cancer therapy.

[0095]

[0013] In one embodiment, provided herein is a method of generating a virus-producing cell line, comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) an amino acid substitution at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; and (ii) a second nucleic acid sequence encoding one or more viral proteins; b) selecting from the cell population cells that grow in the presence of a neomycin phosphotransferase substrate; and c) growing the selected cells to generate a virus-producing cell line. In some embodiments, the one or more viral proteins include a capsid protein, an envelope protein, a viral protein required for replication, or a combination thereof.

[0096] In some embodiments, a method of generating a virus producer cell line comprises: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (4) amino acid substitutions at amino acid residues 216 and 261 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine, (5) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with serine, or (6) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine, and (ii) a second nucleic acid sequence encoding one or more viral proteins; b) introducing from the cell populationand c) selecting cells that grow in the presence of a neomycin phosphotransferase substrate; and c) growing the selected cells to generate a virus-producing cell line. In some embodiments, the one or more viral proteins include a capsid protein, an envelope protein, a viral protein required for replication, or a combination thereof.

[0097] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0098] In some embodiments of the methods provided herein, the wild-type NPT comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments of the methods provided, the wild-type NPT comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0099] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to a wild-type NPT comprising the amino acid sequence of SEQ ID NO:1.

[0100] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is to alanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine.In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0101] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A).

[0102] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D).

[0103] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F).

[0104] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N).

[0105] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S).

[0106] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0107] In some embodiments of the methods provided herein, the cell line is a mammalian cell line. In some embodiments of the methods provided herein, the cell line is a human cell line. In some embodiments of the methods provided herein, the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

[0108] In some embodiments of the methods provided herein, the one or more viral proteins include an AAV capsid protein.

[0109] In some embodiments of the methods provided herein, the one or more viral proteins include an AAV capsid protein and an AAV rep protein.

[0110] In some embodiments of the methods provided herein, the one or more viral proteins include an envelope protein.

[0111] In some embodiments of the methods provided herein, the one or more viral proteins include an adenovirus E1 region protein necessary for adenovirus replication.

[0112] In some embodiments of the methods provided herein, the one or more viral proteins comprise a retroviral envelope protein.

[0113] In some embodiments of the methods provided herein, the one or more viral proteins include a retroviral gag protein.

[0114] In some embodiments of the methods provided herein, the one or more viral proteins include a retroviral reverse transcriptase.

[0115] In some embodiments of the methods provided herein, the one or more viral proteins include a retroviral envelope protein, a gag protein, and a reverse transcriptase.

[0116] In another aspect, provided herein is a viral producer cell line comprising one or more nucleic acid sequences, the one or more nucleic acid sequences comprising: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein having neomycin phosphotransferase activity; and (ii) a second nucleic acid sequence encoding one or more viral proteins. In some embodiments, the one or more viral proteins comprise a capsid protein, an envelope protein, a viral protein required for replication, or a combination thereof.

[0117]

[0013] In one embodiment, provided herein is a virus-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences include: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (4) amino acid substitutions at corresponding amino acid residues, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (6) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1,Provided is a virus-producing cell line comprising: (i) a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1, where the amino acid substitution is a substitution with methionine and at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1, where the amino acid substitution is a substitution with glycine; and (ii) a second nucleic acid sequence encoding one or more viral proteins. In some embodiments, the one or more viral proteins comprise a capsid protein, an envelope protein, a viral protein required for replication, or a combination thereof.

[0118]

[0013] In some embodiments, provided herein is a virus-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences include: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is to aspartic acid; and (ii) a second nucleic acid sequence encoding one or more viral proteins.In some embodiments, the one or more viral proteins comprise a capsid protein, an envelope protein, a viral protein required for replication, or a combination thereof.

[0119] In some embodiments of the virus-producing cell line, the non-native NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0120] In some embodiments of the virus-producing cell line, the wild-type NPT comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments of the virus-producing cell line, the wild-type NPT comprises an amino acid sequence at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1. In some embodiments of the virus-producing cell line, the non-native NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT comprising the amino acid sequence of SEQ ID NO: 1.

[0121] In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is to alanine. In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine. In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with serine.In some embodiments of the virus-producing cell line, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0122] In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A). In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D). In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F). In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N). In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S). In some embodiments of the virus-producing cell line, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0123] In some embodiments of the virus-producing cell line, the cell line is a mammalian cell line. In some embodiments of the virus-producing cell line, the cell line is a human cell line. In some embodiments of the virus-producing cell line, the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

[0124] In some embodiments of the virus producer cell line, the one or more viral proteins comprise an AAV capsid protein. In some embodiments of the virus producer cell line, the one or more viral proteins comprise an AAV capsid protein and an AAV rep protein. In some embodiments of the virus producer cell line, the one or more viral proteins comprise an envelope protein. In some embodiments of the virus producer cell line, the one or more viral proteins comprise an adenoviral E1 region protein required for adenoviral replication. In some embodiments of the virus producer cell line, the one or more viral proteins comprise a retroviral envelope protein. In some embodiments of the virus producer cell line, the one or more viral proteins comprise a retroviral gag protein. In some embodiments of the virus producer cell line, the one or more viral proteins comprise a retroviral reverse transcriptase. In some embodiments of the virus producer cell line, the one or more viral proteins comprise a retroviral envelope protein, a gag protein, and a reverse transcriptase.

[0125] In one aspect, provided herein is a method for producing a mammalian cell line that expresses an antigen, the method comprising: a) introducing one or more nucleic acid sequences into a host cell population, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein having neomycin phosphotransferase activity; and (ii) a second nucleic acid sequence encoding an antigen; b) selecting cells from the cell population of step (a) that grow in the presence of G418; and c) culturing the selected cells to generate a cell line that expresses the antigen. In some embodiments, the antigen is used to immunize a mammalian subject (e.g., a human) or to induce an immune response in a mammalian subject (e.g., a human). The antigen can also be used in vitro or ex vivo.

[0126] In one embodiment, provided herein is a method for producing a mammalian cell line that expresses an antigen, comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences include: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring neomycin NPT comprises: (1) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with phenylalanine; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine, and (ii) a second nucleic acid sequence encoding an antigen; b) selecting cells that grow in the presence of G418 from the cell population of step (a); and c) culturing the selected cells to generate a cell line that expresses the antigen.

[0127] In some embodiments, provided herein are methods for producing a mammalian cell line that expresses an antigen, the method comprising: a) introducing into a host cell population one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring neomycin NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution of methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is a substitution of alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution of methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is a substitution of aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution of methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is a substitution of aspartic acid; (4) amino acid substitutions at amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution of glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution of asparagine; (5) amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution of methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution of serine; or (6) amino acid substitutions at amino acid residues 36 and 216 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution of methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution of glycine; and (ii) a second nucleic acid sequence encoding an antigen.b) selecting from the cell population of step (a) cells that grow in the presence of G418; and c) culturing the selected cells to generate a cell line that expresses the antigen.

[0128] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0129] In some embodiments of the methods provided herein, the wild-type NPT comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to SEQ ID NO: 1. In some embodiments of the methods provided herein, the wild-type NPT comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO: 1.

[0130] In some embodiments of the methods provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to a wild-type NPT comprising the amino acid sequence of SEQ ID NO:1.

[0131] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO: 1 is to alanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine. In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine.In some embodiments, the non-naturally occurring NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0132] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A).

[0133] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D).

[0134] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F).

[0135] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N).

[0136] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S).

[0137] In some embodiments of the methods provided herein, the non-naturally occurring NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0138] In some embodiments of the method for producing a mammalian cell line, the cell line is a mammalian cell line. In some embodiments of the method for producing a mammalian cell line, the cell line is a human cell line. In some embodiments of the method for producing a mammalian cell line, the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

[0139] In some embodiments of the method for producing a mammalian cell line, the antigen is a viral antigen, a bacterial antigen, or a fungal antigen. In some embodiments of the method for producing a mammalian cell line, the antigen is a cancer antigen.

[0140] In another aspect, provided herein is an antigen-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) described herein having neomycin phosphotransferase activity; and (ii) a second nucleic acid sequence encoding one or more antigens.

[0141] In another aspect, provided herein is an antigen-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) an antigen-producing cell line comprising a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine; and (ii) a second nucleic acid sequence encoding one or more antigens.

[0142]

[0013] In some embodiments, provided herein is an antigen-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences include: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is to alanine; (2) an amino acid substitution at amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is to aspartic acid; (3) an amino acid substitution at amino acid residues 36 and 218 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is to methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is to aspartic acid; and (ii) a second nucleic acid sequence encoding one or more antigens.

[0143] In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

[0144] In some embodiments of the antigen-producing cell lines provided herein, the wild-type NPT comprises an amino acid sequence that is at least 80%, at least 90%, or at least 98% identical to SEQ ID NO:1.

[0145] In some embodiments of the antigen-producing cell lines provided herein, the wild-type NPT comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, or at least 65% identical to SEQ ID NO:1.

[0146] In some embodiments of the antigen-producing cell lines provided herein, the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to the wild-type NPT comprising the amino acid sequence of SEQ ID NO:1.

[0147] In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO: 1 is to alanine. In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO: 1 is to aspartic acid. In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is to methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is to phenylalanine. In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine. In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine.In some embodiments, the NPT comprises amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine.

[0148] In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 38 (V36M, G210A). In some embodiments, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 39 (V36M, E182D). In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 40 (V36M, Y218F). In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 41 (D216G, D261N). In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 42 (V36M, Y218S). In some embodiments of the antigen-producing cell lines provided herein, the non-native NPT comprises the amino acid sequence of SEQ ID NO: 43 (V36M, D216G).

[0149] In some embodiments of the antigen-producing cell lines provided herein, the cell line is a mammalian cell line. In some embodiments of the antigen-producing cell lines provided herein, the cell line is a human cell line. In some embodiments of the antigen-producing cell lines provided herein, the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

[0150] In some embodiments of the antigen-producing cell lines provided herein, the one or more antigens are viral antigens, bacterial antigens, or fungal antigens. In some embodiments of the antigen-producing cell lines provided herein, the one or more antigens are cancer antigens.

[0151] In another aspect, provided herein are selectable marker means for conferring resistance to kanamycin when introduced into a bacterial cell and for conferring resistance to G418 when introduced into a mammalian cell. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 20. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 32. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 33. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 34. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 36. In some embodiments, the selectable marker means comprises the nucleic acid sequence of SEQ ID NO: 37.

[0152] In another aspect, provided herein is a method for making a producer cell line, the method comprising: a) transforming a bacterial cell or a mammalian cell with an expression vector to produce a transformed cell, wherein the expression vector comprises a nucleic acid sequence encoding one or more viral proteins, and a means for growing the transformed cell in the presence of kanamycin if the transformed cell is a bacterial cell, or a means for growing the transformed cell in the presence of G418 if the transformed cell is a mammalian cell; and b) culturing the transformed cell in the presence of kanamycin or G418 to obtain a producer cell line, wherein the producer cell line expresses one or more viral proteins from AAV, adenovirus, retrovirus, lentivirus, herpes simplex virus, vaccinia virus, or baculovirus.

[0153] In another aspect, provided herein is a method for selecting cells with stable chromosomal integration of an exogenous nucleic acid sequence, the method comprising: a) transforming a population of eukaryotic cells with the exogenous nucleic acid sequence comprising a means for growth in the presence of G418; b) culturing the transformed cell population in the presence of G418 to generate transformed cell colonies capable of growth in the presence of G418; and c) selecting cells from the colonies generated in step (b) to obtain cells with stable chromosomal integration of the exogenous nucleic acid.

[0154] In some embodiments, the exogenous nucleic acid sequence further comprises a transgene, and the selected cells express the transgene.

[0155] In some embodiments, the exogenous nucleic acid sequence disrupts expression of a gene endogenous to the selected cell.

[0156] In another aspect, provided herein is a method for selecting mammalian cells having a stable episome, the method comprising: a) transforming a population of mammalian cells with a plasmid comprising a means for growth in the presence of G418; b) culturing the population of transformed cells in the presence of G418 to generate colonies of transformed cells capable of growth in the presence of G418; and c) selecting cells from the colonies generated in step (b) to obtain cells having a stable episome comprising the plasmid.

[0157] In some embodiments, the plasmid further comprises an EBNA1 OriP nucleic acid sequence, and the selected cells express EBNA1.

[0158] In one aspect, provided herein is a method for selecting mammalian cells that transiently express a transgene, the method comprising: a) introducing into a population of mammalian cells a nucleic acid encoding the transgene and a means for growth in the presence of G418; b) culturing the population of mammalian cells in the presence of G418 for 48 to 72 hours; and c) selecting from the cultured population of mammalian cells those mammalian cells that grow in the presence of G418, wherein the selected mammalian cells transiently express the transgene.

[0159] In some embodiments, the transgene comprises a nucleic acid sequence encoding a Crispr endonuclease or a Crispr guide RNA.

[0160] In some embodiments, the means is a nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase comprising an amino acid sequence selected from the group of SEQ ID NOs: 38, 39, 40, 41, 42, or 43. [Brief explanation of the drawings]

[0161] The foregoing summary, as well as the following detailed description of certain embodiments of the present application, will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the present application is not limited to the precise embodiments shown in the drawings. [Figure 1] A representative expression vector (plasmid P313) described herein is shown. [Figure 2] Constructs containing transposon elements ("Leapin Left" and "Leapin Right"), the human elongation factor alpha promoter ("EF1α"), the mCherry coding region with polyadenylation signal ("pA"), the NPT coding region ("Kan / NEO"), and the origin of replication ("pMB1 Ori") are shown. [Figure 3] 1 shows the results from the colony formation assay described herein. [Figure 4]mCherry expression in a stable pool of HEK293 cells transformed with constructs expressing mCherry and NPT proteins (labeled "NEO") is shown compared to uncolored untransformed cells (leftmost tube). [Figure 5] 1 shows a graph of transgene (mCherry) copy number in HEK293 cells transformed with constructs P724 encoding wild-type NPT, P725 (V36M, G210A) encoding NPT mutant #1, or P726 (V36M, E182D) encoding NPT mutant #2, where the constructs either contain (+) or do not contain (−) the transposase element. [Figure 6A-1] 6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. [Figure 6A-2] 6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. [Figure 6A-3] 6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. [Figure 6B-1] 6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. [Figure 6B-2]6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. [Figure 6B-3] 6A-6B show an alignment of aminoglycoside phosphotransferases adapted from Shaw et al., Microbiological Reviews 57:138-163 (1993). SEQ ID NOs: 18, 19, and 45-62 are assigned to the sequences shown in Figures 6A-6B. Detailed Description of the Invention

[0162] The present disclosure is based, in part, on the surprising discovery of NPTs with specific amino acid substitutions that have significantly reduced phosphotransferase activity compared to wild-type NPT. The use of the nucleic acid sequences encoding NPT described herein provides significant advantages as selectable markers for the selection and generation of transformed cell lines that express, in addition to a gene of interest, the mutated NPT, which confers a selective advantage to the transformed cells over non-transformed cells.

[0163] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0164] The term "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of the same amino acid residues or nucleotides when compared and aligned for maximum correspondence, as determined using one of the following sequence comparison algorithms or by visual inspection.

[0165] For sequence comparison, typically one sequence serves as a reference sequence, with which test sequences are compared.When using sequence comparison algorithm, test and reference sequences are input into computer, and if necessary, subsequence coordinates are designated, and sequence algorithm program parameters are designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequences to reference sequences based on designated program parameters.

[0166] Optimal alignment of sequences for comparison can be determined, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or visual inspection (see generally, Current Protocols in Molecular Biology, F.M.A.usubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995)). This can be done by the method described in the Supplement (see Ausubel).

[0167] Examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match when aligned with words of the same length in a database sequence or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased.

[0168] For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, always >0) and N (penalty score for mismatching residues, always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops below its maximum achieved value by the amount X, when the accumulation of one or more alignments of negative-scoring residues causes the cumulative score to fall below zero, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0169] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide sequences or two amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid with the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

[0170] A further indication that two nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, as described below. Thus, a polypeptide is typically substantially identical to a second polypeptide; for example, the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

[0171] The terms "wild-type NPT" and "wild-type neomycin phosphotransferase" are used interchangeably herein and are understood by those of skill in the art. Generally, wild-type NPT refers to the neomycin phosphotransferase that is predominant among organisms in nature. In some embodiments, the wild-type NPT is aminoglycoside phosphotransferase 3'-II. In certain embodiments, the wild-type NPT is aminoglycoside phosphotransferase 3'-IIa. In some embodiments, the wild-type NPT is neomycin phosphotransferase (aminoglycoside phosphotransferase 3'-IIa) from Tn5. In certain embodiments, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 1. In another specific embodiment, the wild-type NPT comprises the amino acid sequence of SEQ ID NO: 44. In other embodiments, the wild-type NPT comprises an amino acid sequence other than SEQ ID NO: 1 or SEQ ID NO: 44.

[0172] The descriptions of amino acid positions of substitutions in NPT described herein are relative to the amino acid positions in SEQ ID NO: 1. For example, an amino acid substitution in wild-type NPT at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1 refers to a wild-type NPT having an amino acid substitution at the amino acid residues in wild-type NPT corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1 in the alignment as provided in Figures 6A-6B. In Figures 6A-6B, the sequence of APH(3')-IIa is the reference sequence (i.e., the amino acid sequence corresponding to SEQ ID NO: 1) to which other wild-type NPTs are compared. An exemplary nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 1 is provided as SEQ ID NO: 6.

[0173] As used herein, the phrase "selectable marker means" refers to an NPT mutant or non-native NPT described herein, or a nucleotide sequence encoding an NPT mutant or non-native NPT described herein, that enables growth of a host cell in the presence of a neomycin phosphotransferase substrate (e.g., neomycin, kanamycin, or G418, or a derivative thereof).

[0174] As used herein, the phrase "means for growing in the presence of" a neomycin phosphotransferase substrate (e.g., neomycin, kanamycin, or G418, or a derivative thereof) refers to an NPT mutant or non-naturally occurring NPT described herein, or a nucleotide sequence encoding an NPT mutant or non-naturally occurring NPT described herein, that allows a host cell to grow in the presence of the neomycin phosphotransferase substrate.

[0175] 7.1 Neomycin phosphotransferase (NPT) protein In one aspect, provided herein are NPT variants that differ in amino acid sequence from wild-type NPT and have altered phosphotransferase activity (e.g., decreased phosphotransferase activity) compared to wild-type NPT. In one embodiment, the NPT variants comprise one, two, or more amino acid substitutions described herein in wild-type NPT (e.g., in Table 1 or Table 2), or a combination thereof. In certain embodiments, the NPT variants provided herein are non-naturally occurring NPT proteins. In certain embodiments, the NPT variants provided herein are isolated NPT proteins. In certain embodiments, the NPT variants provided herein have attenuated activity as a selectable marker compared to wild-type NPT. In certain embodiments, the NPT variants have decreased enzymatic activity compared to the corresponding wild-type NPT in an assay described herein or known to one of skill in the art. For example, the enzymatic activity of NPT can be measured in an in vitro kinase assay, such as that described in Kocabiyik and Perlin, Biochem Biophys Res Commun 185(3):925-931 (1992). The enzymatic activity of the NPT variants is compared to the corresponding wild-type NPT under the same conditions. Alternatively, or additionally, enzymatic activity NPT may be measured indirectly by assessing colony formation by bacteria (e.g., E. coli) transformed with a plasmid encoding the NPT variant after a period of time (e.g., 36, 48, 72 hours, or more) on plates containing a fixed amount of kanamycin (e.g., 25 μg / ml, 75 μg / ml, or 100 μg / ml) and appropriate nutrients and conditions for bacterial growth (e.g., temperature, etc.).The colony formation of bacteria transformed with a nucleotide sequence encoding an NPT variant is compared to the colony formation of bacteria of the same species transformed with a nucleotide sequence encoding the corresponding wild-type NPT grown under the same growth conditions as the bacteria transformed with the nucleotide sequence encoding the NPT variant, and a lower number and / or smaller colonies formed by bacteria transformed with a nucleotide sequence encoding an NPT variant compared to colonies formed by bacteria transformed with a plasmid encoding the wild-type NPT indicates diminished enzymatic activity and / or protein stability of the NPT variant. Another example of an indirect assay for assessing enzymatic activity of NPT mutants involves comparing colony formation by mammalian cells transfected or transformed with DNA encoding an NPT mutant protein with colony formation by mammalian cells transfected with DNA encoding the corresponding wild-type NPT, where both populations of mammalian cells are grown on plates or another suitable type of container containing a medium necessary for growth and a concentration of G418 (e.g., 500 μg / ml) under the same conditions (e.g., same temperature, CO2, etc.) for a period of time (e.g., 2 weeks, 2.5 weeks, 3 weeks, or more), and a decrease in colony formation by mammalian cells transfected with the NPT mutant compared to colony formation by mammalian cells transfected with wild-type NPT indicates that the NPT mutant has attenuated enzymatic activity.

[0176] Another example of an indirect assay for assessing the enzymatic activity of the NPT gene involves measuring the percentage of cells transfected with a mammalian expression construct that stably integrate the construct into the host chromosome and form colonies when diluted and plated in tissue culture dishes containing a selection agent. For example, HEK293 cells transfected with a plasmid designed to express a wild-type or mutant NPT isoform are plated at 2E6 cells or less in 150 mm tissue culture dishes in DMEM medium containing 10% fetal bovine serum and 600 μg / ml G418 and cultured at 37°C and 8% CO2 for 2 weeks. The medium is removed, and the cells are stained with 10 ml of 0.4% methylene blue in 50% methanol by incubating at room temperature for 10 minutes. The stain is removed, and the cells are washed with 100% methanol, air-dried, and photographed. A decreased colony:cell ratio plated using a mutant NPT expression construct compared to a wild-type NPT expression construct indicates that the mutant has attenuated enzymatic activity.

[0177] In certain embodiments, NPT variants with reduced activity compared to wild-type NPT exhibit 0.001% to 10% of the phosphotransferase activity of wild-type NPT (e.g., SEQ ID NO: 1 or SEQ ID NO: 44), as determined in a suitable assay. In some embodiments, NPT variants with reduced activity compared to wild-type NPT exhibit 0.001% to 8% of the phosphotransferase activity of wild-type NPT (e.g., SEQ ID NO: 1 or SEQ ID NO: 44), as determined in a suitable assay. In certain embodiments, NPT variants with reduced activity compared to wild-type NPT exhibit 0.01% to 6% of the phosphotransferase activity of wild-type NPT (e.g., SEQ ID NO: 1 or SEQ ID NO: 44), as determined in a suitable assay. NPT phosphotransferase activity can be measured using any of the assays known in the art (see, e.g., Kocabiyik and Perlin, Biochem Biophys Res Commun 185(3):925-931 (1992) and the references cited therein for exemplary methods of assaying phosphotransferase activity) or the assays described herein (e.g., colony formation). In certain embodiments, the NPT variants have one or two amino acid substitutions in the amino acid sequence of wild-type NPT, where the amino acid substitutions at the amino acid residues of wild-type NPT correspond to one or two of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In some embodiments, the NPT variants have one amino acid substitution in the amino acid sequence of wild-type NPT, where the amino acid substitution is at the amino acid residue of wild-type NPT that corresponds to one of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In certain embodiments, the NPT variants have reduced activity as assessed by techniques known to those of skill in the art or described herein. In certain embodiments, the NPT variant has two amino acid substitutions in the amino acid sequence of wild-type NPT, where the amino acid substitutions are at two of the amino acid residues in wild-type NPT that correspond to two of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In certain embodiments, the NPT variant has reduced activity as assessed by techniques known to those of skill in the art or described herein.

[0178] In certain embodiments, the NPT mutant has one or two amino acid substitutions in the amino acid sequence of a neomycin phosphotransferase variant that has wild-type neomycin phosphotransferase activity, where the amino acid substitutions at amino acid residues of the variant correspond to one or two of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In some embodiments, the NPT mutant has one amino acid substitution in the amino acid sequence of a neomycin phosphotransferase variant that has wild-type neomycin phosphotransferase activity, where the amino acid substitution is at an amino acid residue of the variant that corresponds to one of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In certain embodiments, the NPT mutant has reduced activity as assessed by techniques known to those of skill in the art or described herein. In certain embodiments, the NPT mutant has two amino acid substitutions in the amino acid sequence of a neomycin phosphotransferase variant that has wild-type neomycin phosphotransferase activity, where the amino acid substitutions are at two of the amino acid residues of the variant that correspond to two of the amino acid residues of SEQ ID NO: 1 listed in Table 1 or Table 2. In certain embodiments, NPT variants have reduced activity as assessed by techniques known to those of skill in the art or described herein.

[0179] In certain embodiments, NPT variants provided herein differ from wild-type NPT in having a methionine at the position corresponding to amino acid 36 of SEQ ID NO:1 and an alanine at the position corresponding to amino acid 210 of SEQ ID NO:1. In certain embodiments, NPT variants differ from wild-type NPT in having a methionine at the position corresponding to amino acid 36 of SEQ ID NO:1 and an aspartic acid at the position corresponding to amino acid 182 of SEQ ID NO:1. In certain embodiments, NPT variants differ from wild-type NPT in having a methionine at the position corresponding to amino acid 36 of SEQ ID NO:1 and a phenylalanine at the position corresponding to amino acid 218 of SEQ ID NO:1. In certain embodiments, NPT variants differ from wild-type NPT in having a glycine at the position corresponding to amino acid 216 of SEQ ID NO:1 and an asparagine at the position corresponding to amino acid 261 of SEQ ID NO:1. In certain embodiments, NPT variants differ from wild-type NPT in having a methionine at the position corresponding to amino acid 36 of SEQ ID NO:1 and a serine at the position corresponding to amino acid 218 of SEQ ID NO:1. In certain embodiments, the NPT variants differ from wild-type NPT by having a methionine at the position corresponding to amino acid 36 of SEQ ID NO: 1 and a glycine at the position corresponding to amino acid 216 of SEQ ID NO: 1. In certain embodiments, the NPT variants have reduced activity as assessed by techniques known to those of skill in the art or described herein.

[0180] In certain embodiments, provided herein is a non-naturally occurring NPT with neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO: 1 is a substitution with alanine. In some embodiments, provided herein is a non-naturally occurring NPT with neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues 36 and 182 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO: 1 is a substitution with aspartic acid. In certain embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with phenylalanine.In some embodiments, provided herein is a non-naturally occurring NPT with neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution of glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO: 1 is a substitution of asparagine. In certain embodiments, provided herein is a non-naturally occurring NPT with neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution of methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution of serine. In some embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a wild-type neomycin phosphotransferase with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine. In certain embodiments, the non-naturally occurring NPT has decreased activity as assessed by techniques known to those of skill in the art or described herein.

[0181] In some embodiments, wild-type NPT comprises an amino acid sequence at least 50%, at least 55%, or at least 60% identical to SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, wild-type NPT comprises an amino acid sequence at least 65%, at least 70%, or at least 75% identical to SEQ ID NO:1 or SEQ ID NO:44. In some embodiments, wild-type NPT comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, wild-type NPT comprises an amino acid sequence 50%-75%, 50%-80%, 50%-60%, 75%-95%, or 85%-95% identical to SEQ ID NO:1 or SEQ ID NO:44. In some embodiments, motif 1, motif 2, or motif 3 of the wild-type sequence is identical to motif 1, motif 2, or motif 3 of SEQ ID NO:1 or SEQ ID NO:44, respectively. In certain embodiments, motif 1, motif 2, and motif 3 of the wild-type sequence are identical to motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, the combination of motif 1, motif 2, and motif 3 of the wild-type sequence is identical to the combination of motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, motif 1, motif 2, or motif 3 of the wild-type sequence is at least 85%, at least 90%, or at least 95% identical to motif 1, motif 2, or motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In certain embodiments, motif 1, motif 2, and motif 3 of the wild-type sequence are at least 85%, at least 90%, or at least 95% identical to motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, the combination of motif 1, motif 2, and motif 3 of the wild-type sequence is at least 85%, at least 90%, or at least 95% identical to the combination of motif 1, motif 2, and motif 3 of SEQ ID NO: 1 or SEQ ID NO: 44, respectively.In some embodiments, the combination of motif 1, motif 2, and motif 3 of the wild-type sequence is at least 98% or at least 99% identical to the combination of motif 1, motif 2, and motif 3 of SEQ ID NO: 1 or SEQ ID NO: 44, respectively.

[0182] In certain embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine. In some embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid. In certain embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 218 of SEQ ID NO:1 is a substitution with phenylalanine.In some embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution of glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution of asparagine. In certain embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 218 of SEQ ID NO:1 is a substitution with serine. In some embodiments, provided herein is a non-naturally occurring NPT having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising the amino acid sequence of a neomycin phosphotransferase variant having wild-type neomycin phosphotransferase activity with amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine. In certain embodiments, the non-naturally occurring NPT variant has decreased activity as assessed by techniques known to those of skill in the art or described herein.

[0183] In certain embodiments, a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity comprises an amino acid sequence at least 60%, at least 65%, at least 70%, or at least 75% identical to SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity comprises an amino acid sequence at least 80%, at least 90%, or at least 98% identical to SEQ ID NO:1 or SEQ ID NO:44. In some embodiments, motif 1, motif 2, or motif 3 of a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity is identical to motif 1, motif 2, or motif 3 of SEQ ID NO:1 or SEQ ID NO:44, respectively. In certain embodiments, motif 1, motif 2, or motif 3 of a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity is identical to motif 1, motif 2, and motif 3 of SEQ ID NO:1 or SEQ ID NO:44, respectively. See, for example, Figures 6A-6B for the locations of motifs 1, 2, and 3 of aminoglycoside transferases. In some embodiments, the combination of motif 1, motif 2, and motif 3 of a neomycin phosphotransferase variant that has wild-type neomycin phosphotransferase activity is identical to the combination of motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, motif 1, motif 2, or motif 3 of a neomycin phosphotransferase variant that has wild-type neomycin phosphotransferase activity is at least 85%, at least 90%, or at least 95% identical to motif 1, motif 2, or motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44.In certain embodiments, motif 1, motif 2, and motif 3 of a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity are at least 85%, at least 90%, or at least 95% identical to motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, the combination of motif 1, motif 2, and motif 3 of a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity is at least 85%, at least 90%, or at least 95% identical to the combination of motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44. In some embodiments, the combination of motif 1, motif 2, and motif 3 of a neomycin phosphotransferase variant with wild-type neomycin phosphotransferase activity is at least 98% or at least 99% identical to the combination of motif 1, motif 2, and motif 3, respectively, of SEQ ID NO: 1 or SEQ ID NO: 44.

[0184] In certain embodiments, the NPT variant comprises the amino acid sequence of SEQ ID NO: 1 with one or two amino acid substitutions. In certain embodiments, the NPT variant is any one of the NPT variants listed in Table 1 provided herein. In another specific embodiment, the NPT variant is any one of the NPT variants listed in Table 2 provided herein.

[0185] In certain embodiments, NPT variants provided herein differ from SEQ ID NO:1 or SEQ ID NO:44 by having a methionine at amino acid position 36 of SEQ ID NO:1 or SEQ ID NO:44 and an alanine at amino acid position 210 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, NPT variants differ from SEQ ID NO:1 or SEQ ID NO:44 by having a methionine at amino acid position 36 of SEQ ID NO:1 or SEQ ID NO:44 and an aspartic acid at amino acid position 182 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, NPT variants differ from SEQ ID NO:1 or SEQ ID NO:44 by having a methionine at amino acid position 36 of SEQ ID NO:1 or SEQ ID NO:44 and a phenylalanine at amino acid position 218 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, NPT variants differ from SEQ ID NO:1 or SEQ ID NO:44 by having a glycine at amino acid position 216 of SEQ ID NO:1 or SEQ ID NO:44 and an asparagine at amino acid position 261 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, an NPT variant differs from SEQ ID NO:1 or SEQ ID NO:44 by having a methionine at amino acid position 36 of SEQ ID NO:1 or SEQ ID NO:44 and a serine at amino acid position 218 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, an NPT variant differs from SEQ ID NO:1 or SEQ ID NO:44 by having a methionine at amino acid position 36 of SEQ ID NO:1 or SEQ ID NO:44 and a glycine at amino acid position 216 of SEQ ID NO:1 or SEQ ID NO:44. In certain embodiments, an NPT variant provided herein is a double-point NPT variant of SEQ ID NO:1. For example, in some embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:38. In certain embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:39. In some embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:40. In certain embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:41. In some embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:42. In other embodiments, an NPT variant comprises the amino acid sequence of SEQ ID NO:43.

[0186] In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 12. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 13. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 14. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 15. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 16. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 17. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 18. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 19. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 21. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 22. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 23. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 24. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 25. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 26. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 27.In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 28. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 29. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 30. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 31. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 35.

[0187] In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 20. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 32. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 33. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 34. In certain embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 36. In some embodiments, NPT variants provided herein comprise an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 37.

[0188] In certain embodiments, bacterial cells transfected or transformed with nucleotide sequences encoding NPT variants provided herein exhibit reduced colony formation compared to bacterial cells transfected or transformed with a corresponding nucleotide sequence encoding wild-type NPT (e.g., SEQ ID NO: 1), as assessed by colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin. "Reduced colony formation" can be, for example, a 0.001% to 75% reduction in colonies compared to kanamycin-resistant colonies of bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT. In some embodiments, reduced colony formation is a 0.001% to 10% reduction compared to kanamycin-resistant colonies of bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT. In certain embodiments, reduced colony formation is a 0.01% to 6% reduction compared to kanamycin-resistant colonies of bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT.

[0189] In some embodiments, mammalian cells transfected or transformed with a nucleotide sequence encoding an NPT variant provided herein exhibit reduced colony formation compared to mammalian cells transfected or transformed with a nucleotide sequence encoding wild-type NPT (e.g., SEQ ID NO: 1), as assessed by a colony formation assay after two weeks of growth on tissue culture plates containing 500 μg / mL geneticin (G418). "Reduced colony formation" can be, for example, a 0.001% to 75% reduction in colonies compared to G418-resistant colonies of mammalian cells transfected with a nucleotide sequence encoding wild-type NPT. In some embodiments, reduced colony formation is a 0.001% to 10% reduction compared to G418-resistant colonies of mammalian cells transfected with a nucleotide sequence encoding wild-type NPT. In certain embodiments, reduced colony formation is a 0.01% to 6% reduction compared to G418-resistant colonies of mammalian cells transfected with a nucleotide sequence encoding wild-type NPT.

[0190] The NPT mutants or non-naturally occurring NPTs described herein confer resistance to certain antibiotics (e.g., neomycin, kanamycin, G418, or derivatives of any of the foregoing). In certain embodiments, expression of an NPT mutant or non-naturally occurring NPT described herein by a cell enables the cell to grow in the presence of a neomycin phosphotransferase substrate (e.g., neomycin, kanamycin, G418, or derivatives of any of the foregoing). In some embodiments, the mutant NPT or non-naturally occurring NPT comprises an amino acid sequence described in Section 8, below.

[0191] 7.2 Nucleic Acid Sequences In one aspect, provided herein are nucleic acids encoding the NPT variants described herein. In certain embodiments, provided herein are nucleic acid sequences comprising a nucleotide sequence encoding the NPT variants described herein. In another specific embodiment, provided herein are nucleic acid sequences comprising a nucleotide sequence encoding a non-naturally occurring NPT described herein. Due to the degeneracy of the code, any nucleotide sequence encoding an NPT variant or non-naturally occurring NPT is encompassed by the present disclosure. In certain embodiments, the nucleotide sequence encoding an NPT variant or non-naturally occurring NPT is codon-optimized (e.g., codon-optimized for expression in a particular subject or in cells derived from a particular subject). Techniques known in the art can be used to codon-optimize a nucleotide sequence encoding an NPT variant or non-naturally occurring NPT. The nucleic acid or nucleotide sequence may further comprise one or more regulatory elements (e.g., a promoter, an enhancer, etc.). In some embodiments, the nucleic acid or nucleotide sequence may further comprise one, two, or more, or all of the following: a promoter, an enhancer, an intron, and a poly-A sequence. In some embodiments, the nucleic acid or nucleotide sequence may further comprise a promoter and an origin of replication sequence.

[0192] In certain embodiments, a nucleic acid sequence or nucleotide sequence is isolated from the nucleic acid sequence in which it is found in nature. In certain embodiments, a nucleic acid sequence or nucleotide sequence is isolated from the organism in which it is found in nature. Furthermore, an "isolated" nucleic acid sequence, such as a cDNA molecule, may be substantially free of other cellular material or culture medium if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. For example, the language "substantially free" includes preparations of polynucleotides or nucleic acid molecules having less than about 15%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (particularly less than about 10%) of other materials, e.g., cellular material, culture medium, other nucleic acid molecules, chemical precursors, and / or other chemicals.

[0193] As used herein, the terms "nucleic acid" and "nucleotide" include deoxyribonucleotides, deoxyribonucleic acids, ribonucleotides, and ribonucleic acids, and polymeric forms thereof, including either single- or double-stranded forms. In certain embodiments, the terms "nucleic acid" and "nucleotide" include known analogs of natural nucleotides, such as peptide nucleic acids ("PNAs"), which have similar binding properties as a reference nucleic acid. In some embodiments, the terms "nucleic acid" and "nucleotide" refer to deoxyribonucleic acids (e.g., cDNA or DNA). In other embodiments, the terms "nucleic acid" and "nucleotide" refer to ribonucleic acids (e.g., mRNA or RNA).

[0194] In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 12. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 13. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 14. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 15. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 16. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 17. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 18. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 19. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 21. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 22. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 23. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 24. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 25. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 26. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 27. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 28. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 29. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 30. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 31. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 35.

[0195] In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 20. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 32. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 33. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 34. In certain embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 36. In some embodiments, provided herein is a nucleic acid sequence comprising the nucleotide sequence of SEQ ID NO: 37.

[0196] In certain embodiments, provided herein are nucleic acid sequences comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein and a second nucleotide sequence. The second nucleotide sequence may encode a protein or non-coding RNA of interest or may comprise a nucleotide sequence that disrupts an endogenous gene in a host cell. In some embodiments, provided herein are nucleic acid sequences comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein and a second nucleotide sequence encoding a protein or non-coding RNA of interest. In certain embodiments, the nucleic acid sequence may further comprise an additional nucleotide sequence (e.g., a transposon element). The nucleic acid sequence may further comprise one or more regulatory elements (e.g., a promoter, an enhancer, etc.), an origin of replication, and / or a poly-A sequence. In certain embodiments, the first and second nucleotide sequences are operably linked to the same promoter. In other embodiments, the first and second nucleotide sequences are operably linked to different promoters.

[0197] In certain embodiments, provided herein are nucleic acid sequences comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein, a second nucleotide sequence of a first fragment of a gene of interest, and a third nucleotide sequence of a second fragment of the gene of interest, wherein the second nucleotide sequence is adjacent to the first nucleotide sequence at the 5' end and the third nucleotide sequence is adjacent to the first nucleotide sequence at the 3' end, and the first and second fragments facilitate recombination and disruption of the gene of interest. In some embodiments, the nucleic acid sequence further comprises a loxP nucleotide sequence upstream of the second nucleotide sequence and a loxP nucleotide sequence downstream of the third nucleotide sequence. See, e.g., Guldener et al., Nucleic Acids Research 24(13):2519-2524 (1996) for methods for producing and using such nucleic acid sequences. The nucleic acid sequence may further comprise one or more regulatory elements (e.g., promoters, enhancers, etc.), poly-A sequences, etc.

[0198] In certain embodiments, provided herein are nucleic acid sequences comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein, a second nucleotide sequence encoding a protein of interest, a third nucleotide sequence comprising a first transposase sequence, and a fourth nucleotide sequence comprising a second transposase sequence, wherein the third nucleotide sequence is upstream of the first and second nucleotide sequences, and the fourth nucleotide sequence is downstream of the first and second nucleotide sequences. In some embodiments, the first transposase sequence is a Leap-In left transposase, and the second transposase is a Leap-In transposase. The nucleic acid sequence may further comprise one or more regulatory elements (e.g., promoters, enhancers, etc.), an origin of replication, and / or a poly-A sequence.

[0199] In particular embodiments, the nucleic acid sequences are those described in Section 8 below.

[0200] In certain embodiments, provided herein are nucleic acid sequences comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein and a transgene. The transgene may be the native gene sequence or may be modified, for example, to include codon optimization to suit expression in a particular host cell. The transgene may comprise a nucleotide sequence encoding a protein of interest or a non-coding RNA. In certain embodiments, the transgene is operably linked to one or more regulatory elements (e.g., promoters, enhancers, etc.).

[0201] The protein of interest can be, for example, a therapeutic protein or a detectable marker. In certain embodiments, the protein of interest is a hormone, growth factor, antibody, viral protein, enzyme, cytokine, or fragment thereof. In certain embodiments, the fragment is at least 8, at least 9, at least 10, at least 11, or at least 12 amino acids in length. In some embodiments, the protein of interest is an antigen (e.g., a viral, bacterial, fungal, or cancer antigen). In certain embodiments, the protein of interest is a viral protein, such as a capsid protein, an envelope protein, or a protein required for viral replication. The viral protein may be an adeno-associated virus (AAV), adenovirus, retrovirus, lentivirus, herpes simplex virus, vaccinia virus, or baculovirus protein. In some embodiments, the protein of interest is a peptide or polypeptide, which may be useful as a therapeutic assay or in a diagnostic assay.

[0202] The non-coding RNA can be, for example, an antisense RNA, a microRNA (miRNA), a short hairpin RNA (shRNA), a long non-coding RNA, a catalytic RNA (including, for example, a ribozyme), a ribosomal RNA, a tRNA, or a guide RNA for a CRISPR nuclease.

[0203] In some embodiments of the nucleic acid sequences provided herein, the nucleic acid sequence further comprises a nucleotide sequence encoding a selectable marker other than an NPT protein. A selectable marker, when introduced into a cell, confers a trait suitable for artificial selection. A selectable marker may, for example, confer resistance to an antibiotic or encode an enzyme required for the growth of eukaryotic cells under particular culture conditions. Selectable markers are well known in the art. In certain embodiments, the selectable marker is beta-lactamase, which confers ampicillin resistance. In some embodiments, the selectable marker is a fluorescent protein. In some embodiments, the term "selectable marker" is used interchangeably with "selectable marker."

[0204] Selectable markers that can be used include, but are not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:8-17 (1980)), and genes can be utilized in tk-, hgprt-, or aprt- cells, respectively. Antimetabolite resistance can also be used as a selection basis for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Cell 22:8-17 (1980)), which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:357 (1980)). al., Proc. Natl. Acad. Sci. USA 78:1527(1981)), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)), and md hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147(1984)).

[0205] 7.3 Vectors In another aspect, provided herein are vectors comprising nucleic acid sequences that include nucleotide sequences encoding an NPT variant or non-naturally occurring NPT described herein. In particular embodiments, provided herein are vectors comprising a nucleic acid sequence or nucleotide sequence described herein (e.g., Section 7.2 or Section 8). In some embodiments, provided herein are vectors comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein and a second nucleotide sequence encoding a protein or non-coding RNA of interest. In certain embodiments, provided herein are vectors comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein, a second nucleotide sequence of a first fragment of a gene of interest, and a third nucleotide sequence of the second fragment of the gene of interest, wherein the second nucleotide sequence is adjacent to the first nucleotide sequence at the 5' end and the third nucleotide sequence is adjacent to the first nucleotide sequence at the 3' end, and wherein the first and second fragments facilitate recombination and disruption of the gene of interest. In some embodiments, the vector further comprises a loxP nucleotide sequence upstream of the second nucleotide sequence and a loxP nucleotide sequence downstream of the third nucleotide sequence.

[0206] In certain embodiments, the vector is a vector described in Section 8, below.

[0207] In view of the present disclosure, any vector known to those of skill in the art can be used, such as a plasmid, cosmid, phage vector, or viral vector. In some embodiments, the vector is a recombinant expression vector such as a plasmid. The vector can include any elements for establishing the conventional functions of an expression vector, such as a promoter, a ribosome-binding element, a terminator, an enhancer, a selection marker, and an origin of replication. The promoter can be a constitutive, inducible, or reconfigurable promoter. Numerous expression vectors capable of delivering nucleic acids to cells are known in the art and can be used herein to produce proteins or non-coding RNAs within cells. Conventional cloning techniques or artificial gene synthesis can be used to generate recombinant expression vectors according to the embodiments provided herein. Such techniques are well known to those of skill in the art in view of the present disclosure.

[0208] In certain embodiments, the vector is a cloning vector containing a nucleic acid encoding an NPT variant. The cloning vector can be, for example, a plasmid, a phage, a virus, a cosmid, an episome, or a bacterial artificial chromosome. See also Section 7.4 for vectors, including expression vectors, encompassed herein.

[0209] 7.4 Methods for expressing NPT mutants In one aspect, provided herein are methods for producing an NPT variant or non-naturally occurring NPT as described herein, and optionally one or more additional proteins or non-coding RNAs.

[0210] In certain aspects, provided herein are cells (e.g., host cells) that express (e.g., recombinantly express) an NPT variant or non-naturally occurring NPT described herein, and optionally, one or more additional proteins or one or more non-coding RNAs, or both. In another aspect, provided herein are vectors (e.g., expression vectors) that comprise a nucleic acid sequence, the nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein, and optionally, one or more nucleotide sequences encoding one or more additional proteins or non-coding RNAs, or both, for recombinant expression in a host cell (e.g., a mammalian cell). Also provided herein are host cells that comprise a nucleic acid sequence comprising nucleotides encoding an NPT variant or non-naturally occurring NPT described herein, and optionally, one or more nucleotide sequences encoding one or more additional proteins or non-coding RNAs, or both. In certain embodiments, provided herein is a host cell comprising two vectors, a first vector comprising a nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT, and a second vector comprising a nucleic acid sequence comprising one or more nucleotide sequences encoding one or more additional proteins, or one or more non-coding RNAs, or both.

[0211] Examples of cells that can be used include those described in this section and in Sections 7.5 and 8 below. The cells can be primary cells or cell lines. In certain embodiments, the host cells are isolated from other cells. In other embodiments, the host cells are not found within the body of a subject. The term "subject" in the context of a cell or body refers to any organism (e.g., a bacterium or a mammal). The subject can be a human or a non-human mammal.

[0212] NPT variants or non-naturally occurring NPTs, and optionally one or more additional proteins or one or more non-coding RNAs, or both, can be produced by any method known in the art, such as, for example, chemical synthesis or recombinant expression techniques. The methods described herein utilize, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described in the references cited herein and are fully explained in the literature.For example, see Maniatis et al. (1982) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual Second Edition, Cold Spring Harbor Laboratory Press, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annual updates), Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press, Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press, Birren et al. (eds.) (1999) Genome Analysis: A Laboratory Manual Cold Spring Harbor Laboratory Press.

[0213] Proteins (e.g., NPT variants or non-native NPTs, and optionally, proteins of interest) can be prepared using a variety of techniques known in the art, including recombinant and phage display techniques, or a combination thereof. Examples of phage display methods include Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology. 57:191-280, International Application No. GB91 / 01134, International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO097 / 13844, and U.S. Pat. Nos. 5,698,426, 5,223,409, and 5,403,484. Nos. 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and 5,969,108.

[0214] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce the NPT variant or non-naturally occurring NPT, and optionally the protein or non-coding RNA of interest can be purified or isolated. A vector (e.g., an expression vector) or nucleic acid or nucleotide sequence can be introduced into a cell (e.g., a host cell) by, for example, electroporation, transfection, infection, heat shock, microinjection, chromosomal transfer, or any technique known to those of skill in the art.

[0215] A variety of host-expression vector systems can be utilized to express the NPT mutant or non-native NPT, and optionally, the protein or non-coding RNA of interest. Such host-expression systems represent vehicles in which a coding sequence of interest may be produced and subsequently purified, but also cells that, when transformed or transfected with the appropriate nucleotide coding sequence, are capable of expressing the proteins described herein in situ. These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors, yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors, insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus), and plant cell systems (e.g., Chlamydomonas) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid). Examples of suitable expression vectors include, but are not limited to, green algae such as Nicotiana tabacum, Nicotiana reinhardtii, or tobacco plants, or mammalian cell lines (e.g., COS, CHO, BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa, and NIH 3T3 cells) harboring recombinant expression constructs comprising promoters derived from the genome of mammalian cells (e.g., metallothionein promoters) or promoters derived from mammalian viruses (e.g., adenovirus late promoters, vaccinia virus 7.5K promoters).

[0216] In bacterial systems, the number of expression vectors can be advantageously selected depending on the intended use of the non-coding RNA protein of interest to be expressed. In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. This virus grows in Spodoptera frugiperda cells. In mammalian host cells, many virus-based expression systems can be utilized. When using adenovirus as an expression vector, the protein of interest can be ligated to an adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) can result in a recombinant virus that is viable and capable of expressing the protein of interest in infected hosts (see, e.g., Logan & Shenk, 1984, Proc. Natl. Acad. Sci. USA, 81:355-359). Specific initiation signals may also be required for efficient translation of the inserted coding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., 1987, Methods in Enzymol. 153:51-544).

[0217] As used herein, the term "host cell" refers to any type of cell, e.g., a primary cell or a cell from a cell line. A host cell may be a primary cell such as a fibroblast, lymphocyte (e.g., B or T cell), epithelial cell, endothelial cell, neuron, astrocyte, hepatocyte, myocyte, chondrocyte, adipocyte, or stem cell (e.g., embryonic stem cell). Alternatively, a host cell may be an immortalized cell. In certain embodiments, the term "host cell" refers to a cell that has been transfected, infected, microinjected, or transformed with a nucleic acid or nucleotide sequence, or a cell that has been engineered to contain a nucleic acid or nucleotide sequence, and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid or nucleotide sequence due to mutations or environmental influences that may occur during the subsequent generation or integration of the nucleic acid or nucleotide sequence into the host cell genome.

[0218] Additionally, a host cell line can be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products can be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. By selecting the appropriate cell line or host system, the expressed foreign protein can be correctly modified and processed. To this end, eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, COS, MDCK, HEK293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20, and T47D, NS0 (a murine myeloma cell line), CRL7030, and HsS78Bst cells.

[0219] For long-term, high-yield production of recombinant proteins, stable expression is preferred. Rather than using expression vectors containing viral origins of replication, host cells can be transformed with a nucleic acid sequence (e.g., DNA) controlled by appropriate expression control elements (e.g., promoter, enhancer, sequence, transcription terminator, polyadenylation site, etc.) and a selectable marker (e.g., NPT mutant or non-native NPT). After introduction of the foreign DNA, engineered cells can be grown in enriched medium for a period of time (e.g., 1-2 days) and then switched to selective medium (e.g., medium containing an antibiotic such as neomycin, kanamycin, or G418 in the case of NPT mutant or non-native NPT). The selectable marker on the recombinant plasmid confers resistance to selection (e.g., neomycin, kanamycin, or G418 in the case of NPT mutant or non-native NPT) and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci, which can then be cloned and expanded into cell lines. This method can be advantageously used to engineer cell lines that express the protein.

[0220] In certain embodiments, provided herein are methods for generating host cells comprising a second nucleotide sequence, the method comprising: (a) introducing into a first host cell population a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT; and (ii) a second nucleotide sequence (e.g., a second nucleotide sequence encoding a second protein or non-coding RNA); (b) growing the first host cell population in the presence of kanamycin, neomycin, or G418, or a derivative thereof, to generate colonies; and (c) selecting colonies of cells that grow in the presence of kanamycin, neomycin, or G418. In some embodiments, provided herein are methods for generating host cells comprising a second nucleotide sequence, the method comprising: (a) growing a first host cell population in the presence of kanamycin, neomycin, or G418, or a derivative thereof, to generate colonies, wherein a first nucleic acid sequence is introduced into the first host cell population, the first nucleic acid sequence comprising (i) a first nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT, and (ii) a second nucleotide sequence (e.g., a second nucleotide sequence encoding a second protein or non-coding RNA); and (b) selecting cell colonies that grow in the presence of kanamycin, neomycin, or G418. In certain embodiments, the first host cell population produces fewer and / or smaller colonies than a second host cell population transfected or transformed with a second nucleic acid sequence and grown in the presence of kanamycin, neomycin, or G418, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence.In certain embodiments, the first host cell population produces 50-100, 100-1,000, 1,000-5,000, 5,000-10,000, 1,000-10,000, 10,000-15,000, 5,000-15,000, 15,000-25,000, or 10,000-25,000 times fewer colonies than a second host cell population transfected or transformed with a second nucleic acid sequence and grown in the presence of kanamycin, neomycin, or G418, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a second nucleotide sequence. In certain embodiments, the first host cell population comprises a higher copy number of a first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with the second nucleic acid sequence, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence. In certain embodiments, the first host cell population comprises 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher copies of a first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with the second nucleic acid sequence, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a second nucleotide sequence. In some embodiments, the first host cell population comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold higher copy number of a first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with the second nucleic acid sequence, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence.In certain embodiments, the first host cell population achieves a higher level of expression of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with the second nucleic acid sequence, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence. In certain embodiments, the first host cell population achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 5-1000 fold higher level of expression of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with the second nucleic acid sequence, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence. In some embodiments, the first host cell population achieves at least 5-fold, at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher expression levels of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with the second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a second nucleotide sequence. Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure the abundance of the second nucleotide sequence relative to a single-copy endogenous gene in the genome of the host cell). Expression of the second nucleotide sequence can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassays (e.g., Western blot or immunocytochemistry). Additionally, for some proteins encoded by the second nucleotide sequence, protein activity (e.g., enzymatic activity) can be assessed.

[0221] In certain embodiments, provided herein are methods for generating host cells comprising a transgene, the method comprising: (a) introducing into a first host cell population a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding an NPT variant or a non-native NPT; and (ii) a transgene; (b) growing the first host cell population in the presence of kanamycin, neomycin, or G418, or a derivative thereof, to generate colonies; and (c) selecting colonies of cells that grow in the presence of kanamycin, neomycin, or G418. In some embodiments, provided herein are methods for generating host cells comprising a transgene, the method comprising: (a) growing a first host cell population in the presence of kanamycin, neomycin, or G418, or a derivative thereof, to generate colonies, wherein a first nucleic acid sequence is introduced into the first host cell population, the first nucleic acid sequence comprising (i) a first nucleotide sequence encoding an NPT mutant or non-native NPT and (ii) a transgene, and (b) selecting cell colonies that grow in the presence of kanamycin, neomycin, or G418. In certain embodiments, the first host cell population produces fewer and / or smaller colonies than a second host cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a transgene, and grown in the presence of kanamycin, neomycin, or G418. In certain embodiments, the first host cell population produces 10 to 100, 100 to 1,000, 1,000 to 5,000, 5,000 to 10,000, 1,000 to 10,000, 10,000 to 15,000, 5,000 to 15,000, 15,000 to 25,000, or 10,000 to 25,000 times fewer colonies than a second host cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a transgene and a third nucleotide sequence encoding a wild-type NPT protein, and grown in the presence of kanamycin, neomycin, or G418.In certain embodiments, the first host cell population comprises a higher copy number of a first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In certain embodiments, the first host cell population comprises 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher copies of a first nucleic acid sequence compared to the copy number of a second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In some embodiments, the first host cell population comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold higher copy numbers of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In certain embodiments, the first host cell population achieves a higher level of expression of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a second nucleic acid sequence, wherein the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene.In certain embodiments, the first host cell population achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1,000 fold, or 5-1000 fold higher level of expression of the second nucleotide sequence compared to the level of expression of the second nucleotide sequence achieved by a second cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In some embodiments, the first host cell population achieves a level of transgene expression that is at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher than the level of transgene expression achieved by a second cell population transfected or transformed with a second nucleic acid sequence, the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and the transgene. Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure transgene abundance relative to a single-copy endogenous gene in the genome of the host cell). Transgene expression can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassays (e.g., Western blot or immunocytochemistry). Additionally, for some proteins encoded by the transgene, protein activity (e.g., enzymatic activity) can be assessed.

[0222] In certain embodiments, provided herein are methods for generating host cells comprising a second nucleotide sequence, the method comprising: (a) introducing into a first host cell population: (1) a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT; and (2) a second nucleic acid sequence comprising a second nucleotide sequence (e.g., a second nucleotide sequence encoding a second protein or non-coding RNA); (b) growing the first host cell population in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) to generate colonies; and (c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof). In some embodiments, provided herein are methods for generating host cells comprising a second nucleotide sequence, the method comprising: (a) growing a first host cell population in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) to generate colonies, wherein a first nucleic acid sequence and a second nucleic acid sequence are introduced into the first host cell population, wherein the first nucleic acid sequence comprises a first nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT, and the second nucleic acid sequence comprises a second nucleotide sequence (e.g., a second nucleotide sequence encoding a second protein or non-coding RNA); and (b) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof).In certain embodiments, the first host cell population produces fewer and / or smaller colonies than a second host cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence and grown in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof), wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. In certain embodiments, the first host cell population produces 50-100, 100-1,000, 1,000-5,000, 5,000-10,000, 1,000-10,000, 10,000-15,000, 5,000-15,000, 15,000-25,000, 10,000-25,000, or 10,000-25,000 times fewer colonies than a second host cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence and grown in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof), wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. In certain embodiments, the first host cell population comprises a higher copy number of the first nucleic acid sequence and / or the second nucleic acid sequence compared to the copy number of the third nucleic acid sequence and / or the fourth nucleic acid sequence achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence.In certain embodiments, the first host cell population comprises 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher copies of the first nucleic acid sequence and / or the second nucleic acid sequence compared to the copy number of the third nucleic acid sequence and / or the fourth nucleic acid sequence achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. In some embodiments, the first host cell population comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher copy numbers of the first and / or second nucleic acid sequences compared to the copy number of the third and / or fourth nucleic acid sequences achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. In certain embodiments, the first host cell population achieves a higher level of expression of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence.In certain embodiments, the first host cell population achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1,000 fold, or 5-1000 fold higher level of expression of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. In some embodiments, the first host cell population achieves at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher expression levels of the second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the second nucleotide sequence. Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure the abundance of the second nucleotide sequence relative to a single-copy endogenous gene in the genome of the host cell). Expression of the second nucleotide sequence can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassays (e.g., Western blot or immunocytochemistry). Additionally, for some proteins encoded by the second nucleotide sequence, the activity of the protein (eg, enzymatic activity) can be assessed.

[0223] In certain embodiments, provided herein are methods for generating host cells comprising a transgene, the method comprising: (a) introducing into a first host cell population: (1) a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or a non-native NPT; and (2) a second nucleic acid sequence comprising a transgene (e.g., a transgene encoding a second protein or non-coding RNA); (b) growing the first host cell population in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) to generate colonies; and (c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof). In some embodiments, provided herein are methods for generating host cells comprising a transgene, the method comprising: (a) growing a first host cell population in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) to generate colonies, wherein a first nucleic acid sequence and a second nucleic acid sequence are introduced into the first host cell population, wherein the first nucleic acid sequence comprises a first nucleotide sequence encoding an NPT mutant or a non-naturally occurring NPT, and the second nucleic acid sequence comprises a transgene (e.g., a transgene encoding a second protein or non-coding RNA); and (b) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof). In certain embodiments, the first host cell population produces fewer colonies than a second host cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence and grown in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof), wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein, and the fourth nucleic acid sequence comprises a transgene.In certain embodiments, the first host cell population produces 100-1,000, 1,000-5,000, 5,000-10,000, 1,000-10,000, 10,000-15,000, 5,000-15,000, 15,000-25,000, 10,000-25,000 times fewer colonies than a second host cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence and grown in the presence of a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof), wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises a transgene. In certain embodiments, the first host cell population comprises a higher copy number of the first nucleic acid sequence and / or the second nucleic acid sequence compared to the copy number of the third nucleic acid sequence and / or the fourth nucleic acid sequence achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises a transgene. In certain embodiments, the first host cell population comprises 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher copies of the first nucleic acid sequence and / or the second nucleic acid sequence compared to the copy number of the third nucleic acid sequence and / or the fourth nucleic acid sequence achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein, and the fourth nucleic acid sequence comprises a transgene.In some embodiments, the first host cell population comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher copy numbers of the first and / or second nucleic acid sequences compared to the copy numbers of the third and / or fourth nucleic acid sequences achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein, and the fourth nucleic acid sequence comprises a transgene. In certain embodiments, the first host cell population achieves a higher level of transgene expression compared to the expression level of the transgene achieved by a second host cell population transfected or transformed with the third and fourth nucleic acid sequences, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein, and the fourth nucleic acid sequence comprises a transgene. In certain embodiments, the first host cell population achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1,000 fold, or 5-1000 fold higher level of transgene expression compared to the level of transgene expression achieved by a second cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the transgene. In some embodiments, the first host cell population achieves at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher levels of transgene expression compared to the level of transgene expression achieved by a second cell population transfected or transformed with a third nucleic acid sequence and a fourth nucleic acid sequence, wherein the third nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and the fourth nucleic acid sequence comprises the transgene.Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure the abundance of the transgene relative to a single-copy endogenous gene in the genome of the host cell). Expression of the transgene can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassays (e.g., Western blot or immunocytochemistry). Additionally, for some proteins encoded by the transgene, protein activity (e.g., enzymatic activity) can be assessed.

[0224] In certain embodiments, the NPT variant or non-native NPT is one described in Section 7.1 or Section 8. In some embodiments, the transgene is one described in Section 7.2.

[0225] Methods generally known in the art in the field of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli, et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol. 150:1, which are incorporated herein by reference in their entireties.

[0226] Host cells can be co-transfected with two or more expression vectors described herein. The two vectors can contain the same selectable marker (e.g., an NPT mutant or a non-naturally occurring NPT) that allows equal expression of the target protein or non-coding RNA. Host cells can be co-transfected with different amounts of two or more expression vectors. For example, host cells can be transfected with any one of the following ratios of the first and second expression vectors: 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, or 1:50.

[0227] Alternatively, a single vector can be used that encodes and expresses the NPT mutants or non-naturally occurring NPTs described herein and a protein or non-coding RNA of interest. Expression vectors can be monocistronic or multicistronic. Multicistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10, or more genes / nucleotide sequences, or in the range of 2-5, 5-10, or 10-20. For example, a bicistronic nucleic acid construct can include, in the following order: a promoter, a first gene (e.g., an NPT mutant or non-naturally occurring NPT), and a second gene (e.g., a protein or non-coding RNA of interest). In such expression vectors, transcription of both genes can be driven by a promoter, but translation of mRNA from the first gene can be via a cap-dependent scanning mechanism, and translation of mRNA from the second gene can be via a cap-independent mechanism, e.g., an IRES.

[0228] Once a protein of interest described herein is produced by recombinant expression, it can be purified by any method known in the art for purifying proteins, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly Protein A chromatography followed by affinity chromatography for a specific antigen, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard technique for purifying proteins. Additionally, the protein of interest can be fused to a heterologous polypeptide sequence known in the art (e.g., a Flag tag or a His tag) to facilitate purification.

[0229] In certain embodiments, a protein described herein (e.g., an NPT variant or non-native NPT, or protein of interest) is isolated or purified. Generally, an isolated protein is one that is substantially free of other proteins other than the isolated protein. For example, in certain embodiments, preparations of the proteins described herein are substantially free of cellular material and / or chemical precursors. The term "substantially free of cellular material" includes preparations of the proteins described herein in which the protein is separated from cellular components of the cells from which it is isolated or recombinantly produced. Thus, a protein described herein that is substantially free of cellular material includes preparations of the protein having less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5%, or 0.1% (by dry weight) of heterologous protein (also referred to herein as "contaminating protein") and / or variants of the protein, e.g., different post-translationally modified forms of the protein or other different versions of the protein. When the protein is recombinantly produced, it will also generally be substantially free of culture medium. That is, the culture medium represents less than about 20%, 10%, 2%, 1%, 0.5%, or 0.1% of the volume of the protein preparation. When a protein is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., separated from chemical precursors or other chemicals involved in the synthesis of the protein. Thus, such preparations of protein have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or compounds other than the protein of interest. In certain embodiments, the proteins described herein are isolated or purified.

[0230] 7.5 cells In another aspect, provided herein are host cells. In certain embodiments, the host cell comprises a vector comprising a nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT. In some embodiments, the host cell comprises a nucleic acid sequence or a nucleotide sequence described herein (e.g., Section 7.2 or Section 8). In certain embodiments, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:20. In another particular embodiment, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:32. In another particular embodiment, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:33. In another particular embodiment, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:34. In another particular embodiment, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:36. In another particular embodiment, the host cell comprises a nucleic acid sequence comprising SEQ ID NO:37.

[0231] In some embodiments, the host cell comprises an NPT mutant or non-native NPT described herein (e.g., Section 7.1 or Section 8). In certain embodiments, the host cell expresses an NPT mutant or non-native NPT described herein (e.g., Section 7.1 or Section 8).

[0232] Any of the host cells described herein (e.g., Section 7.4 or Section 8) or any host cell known to one of skill in the art in view of the present disclosure can be used for recombinant expression of the NPT variants or non-native NPTs described herein (e.g., Section 7.1 or Section 8). For example, such host cells can be cultured to co-express the NPT variant or non-native NPT and a transgene when nucleic acid sequences encoding the NPT variant or non-native NPT and the transgene are introduced into the cells. See, e.g., Sections 7.4 and 8 for exemplary host cells.

[0233] In certain embodiments, the cell (e.g., a host cell) is an in vitro or ex vivo cell. In certain embodiments, the host cell is isolated from cells that have not been transfected or transformed with a nucleotide sequence encoding an NPT variant or non-native NPT. The host cell can be any type of cell described herein or known in the art.

[0234] In some embodiments, the host cell is a bacterial or eukaryotic cell. In certain embodiments, the host cell is a yeast, insect, mammalian, or plant cell. In embodiments in which the host cell is a bacterial cell, the cell is an E. coli cell. Exemplary E. coli cells can be, for example, but are not limited to, E. coli TG1 or BL21 cells.

[0235] In some embodiments, the host cell is a mammalian cell. In certain embodiments, the host cell is derived from a human cell line. Suitable mammalian cells include, for example, CHO and HEK239 cells, and variants thereof (e.g., CHO-DG44 or CHO-K1 cells).

[0236] In certain embodiments, the host cells are immortalized cell lines, hi some embodiments, the host cells are HEK293, CHO, PER.C6, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

[0237] In certain embodiments, the host cell is a primary cell, such as, but not limited to, a fibroblast or a blood cell (e.g., a B cell or a T cell). In some embodiments, the host cell is an embryonic stem cell.

[0238] In some embodiments, the host cell is an insect cell. In certain embodiments, the host cell is a plant cell.

[0239] Cultured immortalized cells can be transfected with nucleic acids encoding NPT mutants or non-native NPTs for short-term (transient) or long-term (stable) expression, depending on whether the introduced nucleic acid integrates into the host cell genome. Transient DNA expression typically lasts for 24-72 hours, while stable DNA expression potentially allows for permanent overexpression of the protein.

[0240] According to certain embodiments, the recombinant expression vector is introduced into the host cell by conventional methods, such as chemical transfection, heat shock, or electroporation, so as to allow efficient expression of the recombinant nucleic acid sequence.

[0241] In certain embodiments, a nucleic acid or nucleotide sequence encoding an NPT variant or non-naturally occurring NPT described herein is stably integrated into the genome of a cell (e.g., a host cell). The nucleic acid or nucleotide sequence can be randomly integrated into the genome of a cell (e.g., a host cell). Alternatively, the nucleic acid or nucleotide sequence can be integrated into a specific location in the genome of a cell (e.g., a host cell). Multiple copies of a nucleic acid or nucleotide sequence can be integrated into the genome of a cell (e.g., a host cell). For example, a host cell can contain 5, 10, 15, 20, 25, or more copies of a nucleic acid or nucleotide sequence integrated into its genome. In some embodiments, the transgene is one described herein (e.g., Section 7.2).

[0242] In some embodiments, the host cell is a mammalian cell and the nucleic acid or nucleotide sequence encoding the NPT variant or non-native NPT, and optionally a transgene, is introduced into the cell by transfection, transduction, infection, microinjection or chromosomal transfer.

[0243] In some embodiments, the second nucleotide sequence encodes a protein of interest or a non-coding RNA described herein (eg, Section 7.2).

[0244] In certain embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence comprises a higher copy number of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein of interest or a non-coding RNA) encoding an NPT variant or non-naturally occurring NPT, and the second nucleic acid sequence comprises a third nucleotide sequence and the second nucleotide sequence encoding a wild-type NPT protein. In some embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher copy numbers of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein of interest or a non-coding RNA) encoding an NPT variant or non-naturally occurring NPT, and the second nucleic acid sequence comprises a third nucleotide sequence and a second nucleotide sequence encoding a wild-type NPT protein.In some embodiments, a first population of host cells transformed or transfected with a first nucleic acid sequence comprises 2-20 fold, 2-100 fold, 2-500 fold, 2-1000 fold, 50-100 fold, 50-500 fold, 50-1000 fold, or 500-1000 fold higher copies of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second population of host cells transfected or transformed with a second nucleic acid sequence, wherein the first population of cells comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein of interest or a non-coding RNA), and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a second nucleotide sequence. In certain embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence achieves a higher level of expression of a second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein of interest or a non-coding RNA) encoding an NPT variant or non-naturally occurring NPT, and the second nucleic acid sequence comprises a third nucleotide sequence and the second nucleotide sequence encoding a wild-type NPT protein.In certain embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 5-1000 fold higher level of expression of a second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-naturally occurring NPT and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein of interest or a non-coding RNA), and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a second nucleotide sequence. In some embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence achieves at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher levels of expression of a second nucleotide sequence compared to the expression level of the second nucleotide sequence achieved by a second cell population transfected or transformed with a second nucleic acid sequence, the first cell population comprising a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a second nucleotide sequence (e.g., a second nucleotide sequence encoding a protein or non-coding RNA of interest), the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and the second nucleotide sequence. Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure the abundance of the second nucleotide sequence relative to a single-copy endogenous gene in the genome of the host cell). Expression of the second nucleotide sequence can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassay (e.g., Western blot or immunocytochemistry).Additionally, for some proteins encoded by the second nucleotide sequence, the activity of the protein (eg, enzymatic activity) can be assessed.

[0245] In certain embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence comprises a higher copy number of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a transgene, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In some embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence comprises at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, or at least 20-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 500-fold, or at least 1000-fold higher copy numbers of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence and a transgene encoding an NPT variant or non-native NPT, and the second nucleic acid sequence comprises a third nucleotide sequence and a transgene encoding a wild-type NPT protein. In some embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence comprises 2-20 fold, 2-100 fold, 2-500 fold, 2-1000 fold, 50-100 fold, 50-500 fold, 50-1000 fold, or 500-1000 fold higher copies of the first nucleic acid sequence compared to the copy number of the second nucleic acid sequence achieved by a second host cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a transgene, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene.In certain embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence achieves a higher level of transgene expression compared to the level of transgene expression achieved by a second cell population transfected or transformed with a second nucleic acid sequence, the first cell population comprising a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a transgene, and the second nucleic acid sequence comprising a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In certain embodiments, a first population of host cells transformed or transfected with a first nucleic acid sequence achieves a 5-25 fold, 10-25 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 5-1000 fold higher level of transgene expression compared to the level of transgene expression achieved by a second population of cells transfected or transformed with a second nucleic acid sequence, wherein the first population of cells comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a transgene, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene. In some embodiments, a first host cell population transformed or transfected with a first nucleic acid sequence achieves at least 10-fold, at least 25-fold, at least 50-fold, at least 100-fold, at least 200-fold, at least 250-fold, at least 500-fold, or at least 1,000-fold higher levels of transgene expression compared to the level of transgene expression achieved by a second cell population transfected or transformed with a second nucleic acid sequence, wherein the first cell population comprises a first nucleic acid sequence comprising a first nucleotide sequence encoding an NPT variant or non-native NPT and a transgene, and the second nucleic acid sequence comprises a third nucleotide sequence encoding a wild-type NPT protein and a transgene. Copy number can be determined using any technique known in the art (e.g., copy number can be measured using digital droplet PCR to measure the abundance of the transgene relative to a single-copy endogenous gene in the genome of the host cell).Transgene expression can be assessed at the RNA level by quantitative reverse transcription PCR (qPCR) or at the protein level by immunoassays (e.g., Western blot or immunocytochemistry). Additionally, for some proteins encoded by the transgene, protein activity (e.g., enzymatic activity) can be assessed.

[0246] In some embodiments, the transgene is one described herein (e.g., Section 7.2). In some embodiments, the NPT variant or non-native NPT is one described herein (e.g., Section 7.1 or Section 8).

[0247] In certain embodiments, the host cell is a virus-producing cell line containing a nucleic acid or nucleotide sequence encoding an NPT variant or non-native NPT described herein. The virus-producing cell line may express capsid proteins or other surface proteins (e.g., envelope proteins), proteins required for replication, or both. Suitable virus-producing cell lines may be for AAV, adenovirus, retrovirus, lentivirus, herpes simplex virus, vaccinia virus, or baculovirus. The virus-producing cell line may be used, for example, to generate viruses for gene therapy or vaccination purposes.

[0248] In certain embodiments, provided herein is a virus-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT comprises: (1) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) an amino acid substitution at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) an amino acid substitution at amino acid residues corresponding to SEQ ID NO:1 (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine;or (6) a first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; (ii) a second nucleic acid sequence encoding one or more viral proteins, wherein the one or more viral proteins are capsid proteins, or envelope proteins, viral proteins required for replication, or both.

[0249] In some embodiments, the virus-producing cell line comprises the NPT mutant nucleic acid sequence of any one of SEQ ID NOs: 20, 32, 33, 34, 36, or 37.

[0250] In certain embodiments of the virus-producing cell lines provided herein, the encoded one or more viral proteins can be, for example, an AAV capsid protein, an AAV rep protein, an adenoviral E1 region protein required for adenoviral replication, a retroviral envelope protein, a retroviral gag protein, or a retroviral reverse transcriptase, or a combination thereof. For example, the one or more viral proteins can be a retroviral envelope protein, a gag protein, and a reverse transcriptase.

[0251]

[0023] In another embodiment, provided herein is an antigen-producing cell line comprising one or more nucleic acid sequences, wherein the one or more nucleic acid sequences are selected from the group consisting of: (i) a non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising: (1) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is with alanine; (2) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, where the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is with aspartic acid; (3) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1; (4) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO: 1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO: 1 is a substitution with asparagine; (5) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (6) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO: 1,An antigen-producing cell line is provided, comprising: (i) a first nucleic acid sequence encoding a non-naturally occurring NPT, the first nucleic acid sequence comprising the amino acid sequence of a wild-type neomycin phosphotransferase having amino acid substitutions at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1, where the amino acid substitution is a substitution of methionine, and at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1, where the amino acid substitution is a substitution of glycine; and (ii) a second nucleic acid sequence encoding one or more antigens.

[0252] In some embodiments, the antigen-producing cell line comprises the NPT mutant nucleic acid sequence of any one of SEQ ID NOs: 20, 32, 33, 34, 36, and 37.

[0253] In certain embodiments, the antigen-producing cell line comprises a nucleic acid sequence encoding a viral, bacterial, or fungal antigen, hi other embodiments, the antigen-producing cell line comprises a nucleic acid sequence encoding a cancer antigen.

[0254] In certain embodiments, provided herein are in vitro or ex vivo cells that express a non-naturally occurring NPT, wherein the non-naturally occurring NPT is attenuated compared to wild-type neomycin phosphotransferase, and the non-naturally occurring NPT comprises the amino acid sequence of any one of SEQ ID NOs: 38, 39, 40, 41, 42, and 43.

[0255] In certain embodiments, where the host cell is a bacterial cell transfected or transformed with a nucleotide sequence encoding an NPT variant or non-native NPT provided herein, the bacterial cell exhibits reduced colony formation as assessed by a colony formation assay after 48 hours of growth on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin, compared to bacterial cells transfected or transformed with a nucleotide sequence encoding wild-type NPT.

[0256] In certain embodiments, the host cell is a mammalian cell transfected with a nucleotide sequence encoding an NPT variant or a non-native NPT provided herein, as compared to mammalian cells transfected with a nucleotide sequence encoding wild-type NPT, exhibiting reduced colony formation as assessed by a colony formation assay after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418).

[0257] In certain embodiments, the host cell comprises a first nucleic acid sequence encoding an NPT variant or non-naturally occurring NPT and a second nucleic acid sequence encoding a second protein or non-coding RNA.

[0258] In some embodiments, the second protein or non-coding RNA is described herein (e.g., Section 7.2). In some embodiments, the host cell or host cell population is produced by a method described herein (e.g., Section 7.4 or Section 8).

[0259] 7.6 How to use In certain embodiments, the NPT variants or non-naturally occurring NPTs described herein, or the nucleotide sequences encoding the NPT variants or non-naturally occurring NPTs described herein, are used in any way that one of skill in the art would use wild-type NPT. In certain embodiments, the NPT variants or non-naturally occurring NPTs described herein, or the nucleotide sequences encoding the NPT variants or non-naturally occurring NPTs described herein, are used in any way that one of skill in the art would use a selectable marker. In certain embodiments, the NPT variants or non-naturally occurring NPTs described herein, or the nucleotide sequences encoding the NPT variants or non-naturally occurring NPTs described herein, are used as described herein.

[0260] In certain embodiments, a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) is used to select for host cells (e.g., mammalian host cells) transformed or transfected with a nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or non-native NPT described herein and an exogenous sequence, such that the host cell has the exogenous sequence stably integrated into its chromosome. Transfection, transduction, infection, microinjection, or chromosome transfer can be used to introduce the nucleic acid sequence into the host cell. This methodology can be used to express a protein of interest or to disrupt a gene by insertional mutagenesis (e.g., by inserting DNA by homologous recombination or transposon insertion).

[0261] In certain embodiments, host cells harboring large numbers of stable episomes (replicating, non-integrating plasmids, such as those containing the EBNA1 OriP sequence and expressing EBNA1 and an NPT mutant or non-native NPT described herein) can be selected for using, for example, neomycin, kanamycin, or G418. In certain embodiments, the high copy number is 5-10 fold, 5-15 fold, 2-5 fold, 2-10 fold, 2-15 fold, 10-20 fold, 10-50 fold, 10-100 fold, 50-100 fold, 50-200 fold, 50-500 fold, 100-500 fold, 100-1000 fold, 500-1000 fold, or 2-1000 fold higher than that achieved when a nucleotide sequence encoding wild-type NPT is used in place of the NPT mutant or non-native NPT.

[0262] In certain embodiments, short-term culture of host cells (e.g., mammalian cells) with a neomycin phosphotransferase substrate (e.g., kanamycin, neomycin, or G418, or a derivative thereof) can be used to enrich for cells that have received constructs expressing NPT mutants or non-native NPTs described herein, as well as other co-transfected nucleic acid sequences (e.g., DNA or RNA) encoding proteins or non-coding RNAs that do not incorporate the NPT mutant or non-native NPT-expressing constructs. For example, some cells are difficult to transfect, and enriching for cells that have received and expressed the NPT gene can also enrich for cells that have received a co-transfected Crispr construct, thus reducing the need for screening to identify cells with a desired modification (e.g., a gene knockout).

[0263] In certain embodiments, host cells engineered to express an NPT variant or a non-native NPT as described herein may be used to select for host cells that have undergone gene amplification using, for example, neomycin, kanamycin, G418, or derivatives thereof. For example, inhibitors of DHFR can be used in this manner to "amplify" a chromosomal region containing a transgene integrated into a host cell (e.g., a mammalian cell such as a CHO cell).

[0264] In certain embodiments, nucleotide sequences encoding NPT variants or non-native NPTs described herein may be used as selection genes when generating cell lines by chromosome transfer, such as the generation of human-hamster hybrids or the transfer of chromosomes between cells by cell fusion.

[0265] In certain embodiments, embryonic stem cells are engineered to contain a nucleotide sequence encoding an NPT mutant or non-native NPT described herein, the npt gene is introduced into a chromosome during homologous recombination in the embryonic stem cells (creating a heterozygous insertion), and higher concentrations of G418 can be used to select for rare cells that have inherited two knockout chromosomes by non-disjunction events. This allows for some analysis of the knockout phenotype by characterization of the cells in vitro or in vivo, without the need to first introduce the cells into mice and breed the mice to generate homozygotes.

[0266] In certain embodiments, highly active gene promoters in host cells can be identified by genome-wide screening using transposons engineered with promoterless NPT mutant nucleotide genes or non-native NPT genes placed downstream of a splice acceptor. Transposons inserted into genes with highly active promoters that activate NPT expression can be selected using appropriate levels of neomycin phosphotransferase substrate (e.g., neomycin, kanamycin, or G418, or their derivatives). The identity of the associated gene and promoter can then be identified by characterizing the transposon insertion site in surviving cells.

[0267] In certain embodiments, host cells (e.g., bacteria) transformed with a first nucleotide sequence encoding an NPT variant or non-native NPT and one or more covalently linked additional nucleotide sequences can be selected by culturing the cells with an appropriate neomycin phosphotransferase substrate (e.g., neomycin, kanamycin, or G418, or derivatives thereof). The nucleotide sequence encoding the NPT gene can be present in a cloning vector, virus, or genomic insert in the host cell.

[0268] In certain embodiments, a plasmid containing a nucleotide sequence encoding an NPT mutant or non-native NPT that is expressed only in bacteria may be used to generate a gene therapy product, including, for example, a lentivirus or AAV. The highly attenuated nature of the NPT mutant or non-native NPT makes any aberrant packaging and delivery of the nucleotide sequence encoding the NPT mutant or non-native NPT into a patient very safe, since the gene activity is very low.

[0269] In certain embodiments, concatemers of DNA can be made, for example, by ligating a linear fragment containing a nucleotide sequence encoding a gene of interest and an NPT mutant or non-native NPT to a fragment containing a bacterial origin of replication, transforming host cells, and selecting for surviving cells with multiple copies of the genes ligated together using, for example, neomycin, kanamycin, or G418, or derivatives thereof, which can be used to generate head-to-tail arrays of genes that can be delivered to mammalian host cells and result in higher frequency of multicopy insertions into the host chromosome.

[0270] In certain embodiments, nucleotide sequences encoding NPT variants or non-naturally occurring NPTs may be used wherever G418 and other NPT substrates are toxic to cells (e.g., yeast, bacteria, insect cells, animal cells, plants, and any pathogens of these organisms).

[0271] In some embodiments, nucleotide sequences encoding NPT variants or non-naturally occurring NPTs are used as described in Section 8.

[0272] 7.7 Kit In another aspect, kits are provided herein. In one embodiment, the kits provided herein comprise, in a container, a nucleic acid sequence comprising a nucleotide sequence encoding an NPT mutant or non-naturally occurring NPT. In another embodiment, the kits provided herein comprise, in a container, a nucleic acid sequence or a vector (e.g., an expression vector) comprising a nucleotide sequence encoding an NPT mutant or non-naturally occurring NPT. In another embodiment, the kits comprise, in a container, a cDNA or genomic library or individual clone comprising a nucleic acid sequence or a nucleotide sequence encoding an NPT mutant or non-naturally occurring NPT. In some embodiments, the NPT mutant nucleic acid sequence is one described in Section 7.2 or Section 8. In certain specific embodiments, the NPT mutant nucleic acid sequence is selected from the group consisting of SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:37. In some embodiments, the kits further comprise, in a container, neomycin, kanamycin, or G418, or a derivative of any of the foregoing. In certain embodiments, the kit comprises, in a container, a cell (e.g., a host cell) into which a nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or non-naturally occurring NPT, or a vector (e.g., an expression vector) comprising the nucleic acid sequence or nucleotide sequence encoding an NPT variant or non-naturally occurring NPT, can be introduced. In some embodiments, the kit further comprises, in a container, a cell (e.g., a host cell) into which a nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or non-naturally occurring NPT, or a vector (e.g., an expression vector) comprising the nucleic acid sequence or nucleotide sequence encoding an NPT variant or non-naturally occurring NPT, can be introduced.

[0273] In certain embodiments, provided herein are kits comprising, in a container, a vector comprising a nucleic acid sequence, the nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT. The vector can be a plasmid, a phage, a virus, a cosmid, or a bacterial artificial chromosome. In some embodiments, provided herein are kits comprising, in a container, a genomic sequence, a cDNA sequence, a genomic library, or an individual clone comprising a nucleic acid sequence, the nucleic acid sequence comprising a nucleotide sequence encoding an NPT variant or a non-naturally occurring NPT. In some embodiments, the kit further comprises, in a container, neomycin, kanamycin, or G418, or a derivative of any of the foregoing.

[0274] In some embodiments, the kits include, in a container, synthetic DNA fragments or fragments not propagated in living cells that encode fragments of an NPT mutant or non-native NPT described herein. The two or more complementary fragments of the NPT mutant or non-native NPT can be separate parts in a vector, and the NPT mutant gene or non-native NPT is reconstituted from the separate parts when introduced into a host cell.

[0275] In some embodiments, provided herein is a kit comprising a host cell described herein in a container. [Example]

[0276] 8.1 Example 1: Identification of NPT Mutants with Reduced Activity This example describes how NPT mutants were generated and screened for reduced phosphotransferase activity.

[0277] Construction of plasmid expression vectors Plasmid vector P313 was constructed (Figure 1, SEQ ID NO:2). It encodes an mCherry fluorescent protein expression cassette containing the human elongation factor alpha promoter and first intron (SEQ ID NO:3), the mCherry coding region (SEQ ID NO:4), and the SV40 polyadenylation signal (SEQ ID NO:5). P313 encodes the neomycin phosphotransferase (NPT) protein derived from transposon Tn5 (aminoglycoside phosphotransferase 3'-IIa) (SEQ ID NO:1; nucleotide sequence comprising SEQ ID NO:6) driven by the mouse phosphoglycerate kinase promoter (SEQ ID NO:7) for expression in mammalian cells and by the E. coli laczya promoter (SEQ ID NO:8) for expression in bacteria. NPT transcription is terminated in mammalian cells by the herpes simplex virus thymidine kinase polyadenylation signal (SEQ ID NO:9). This plasmid also encodes an ampicillin resistance gene (SEQ ID NO:10) and the pUC57 plasmid origin of replication (SEQ ID NO:11).

[0278] Plasmids containing mutations in the NPT gene were generated by replacing a portion of the NPT open reading frame with a DNA fragment generated by gene synthesis (Integrated DNA Technologies, Coralville, IA). Plasmid P313 was digested with an appropriate pair of unique restriction endonucleases (including BspE1, Tth111I, RsrII, and AvrII) to generate the recipient vector. The cloning mixture contained 5 μl of 2x HiFi cloning mix, 50 ng of synthetic DNA, and 509 ng of digested vector. The mixture was incubated at 50°C for 15 minutes and cooled to 4°C. 2 μl was transformed into Top10 competent cells (Invitrogen) or Stellar competent cells (Clontech), plated on LB-carbenicillin plates, and incubated at 37°C. A single colony was inoculated into a 5 ml LB-carbenicillin culture and grown overnight at 37°C in a shaking incubator. DNA was purified using a Qiagen spin miniprep kit (Qiagen). Plasmid sequences were confirmed by DNA sequencing (GENEWIZ, Plainfield, NJ). NPT activity was screened on plates containing kanamycin at concentrations of 25 μg / mL (KAN25), 50 μg / mL (KAN50), 75 μg / mL (KAN75), and 100 μg / mL (KAN100), as described below.

[0279] Screening for NPT mutants in bacteria Overnight cultures grown in LB-carbenicillin were serially diluted in PBS and plated onto LB-carbenicillin, LB-KAN25, and LB-KAN100 plates and incubated for 24 hours. Colonies were counted, incubated for an additional 24 hours at 37°C, and recounted. Plasmids that showed significantly reduced colony numbers but were absent on KAN100 plates compared to KAN25 and carbenicillin plates were replated onto carbenicillin, KAN25, KAN50, KAN75, and KAN100 plates, incubated, and counted as above. The colony counts obtained after 48 hours of incubation are shown in Table 1.

[0280] [Table 1]

[0281] result Two of the single-site point mutants resulted in a complete loss of activity in this assay (G205E and D208G). Only two of the remaining eight mutants showed reduced activity in this assay. Mutant R211G also grew more slowly on KAN100 plates, even though the total number of colonies was similar to growth at lower KAN concentrations. D261N was unable to grow on KAN100 plates and produced approximately half the number of colonies on the other KAN plates. Four of the mutants that showed full activity in our assay (G210A, Y218S, Y218F, and V36M) have previously been reported to confer reduced resistance to kanamycin (Blázquez (1991) Mol. Microbiol. 5:1511-1518; Kocabiyik (1992) Biochem. Biophys. Res. Commun. 185:925-931; Kocabiyik (1992) FEMS Microbiol Lett 93:199-202). These NPT mutants may be expressed at higher levels than in previous studies through the use of high-copy plasmids and / or stronger bacterial promoters.

[0282] Double mutants containing D261N were constructed to identify those with even lower activity. Four D261N double mutants were completely defective, and one (E182D;D261N) was severely defective (2 percent of colonies on KAN25 versus carbenicillin plates, and no growth at other kanamycin concentrations). Only two clones produced colonies on KAN25 plates, but the number of colonies was similar to that on carbenicillin plates (i.e., clone N (D216G;D261N) and clone O (D227G;D261N)). One mutation appeared to partially complement the D261N mutation, allowing growth on KAN100 plates, albeit with reduced efficiency compared to growth on carbenicillin plates (clone K (H188L;D261N)).

[0283] Four clones (S, T, U, and V) had full activity independently, but combined two mutations previously reported to have reduced activity (Blázquez (1991) Mol. Microbiol. 5:1511-1518; Kocabiyik (1992) Biochem. Biophys. Res. Commun. 185:925-931; Kocabiyik (1992) FEMS Microbiol Lett 93:199-202). Two additional clones combined V36M with mutations not tested above. The mutation H188S reportedly reduces resistance to kanamycin (Blázquez (1991) Mol. Microbiol. 5:1511-1518), while the mutation E182D reportedly reduces resistance to G418 but not kanamycin (Yenofsky (1990) Proc. Natl. Acad. Sci. USA 87:3435-3439). Clones containing the V36M;H188S mutation were completely defective. Only three clones retained the ability to grow on KAN25 plates, while the remaining two clones only showed growth defects on KAN100 plates (1 and 0 colonies for clones U (V36M; Y218F) and W (V36M and E182D), respectively). These results surprisingly demonstrate that combining certain mutations that individually have little or no effect on NPT activity can generate double-mutant NPTs with activity suitable for many applications.

[0284] 8.2 Example 2: Mutant NPT Proteins as Selections Markers in HEK293 cells To demonstrate that the attenuated NPT gene cassette-containing plasmids can still confer resistance to G418 in human cells, we transfected some of the constructed plasmids into HEK293 cells and subjected them to colony formation assays. DNA was purified from 200 ml LB cultures containing carbenicillin using Qiagen's HiSpeed ​​maxiprep kit according to the manufacturer's instructions.

[0285] For transfection, 2E7 HEK293 cells were plated into eight T-75 flasks in 40 ml of growth medium (DMEM + 10% FBS + 1x PenStrep) and incubated at 37°C. The transfection was assembled in a 15 ml Corning tube and contained 22 μg of DNA, 3 ml of OptiMEM, and 66 μl of Fugene-6 transfection reagent at 37°C. The transfection mixture was vortexed briefly and incubated for 15 minutes in a CO2 incubator at 37°C. Growth medium (2 ml) was added, and the entire mixture was added to the previously plated flask of HEK293 cells. The flasks were incubated at 37°C. After 48 hours, all flasks contained cells with bright red fluorescence. The flasks were washed with 10 ml of PBS and 1 ml of TryPLE and incubated at 37°C for 5 minutes. Cells were washed from the flask with 10 ml of growth medium and subsequently replated into a T150 flask in 25 ml of medium and incubated at 37°C for 48 hours. Cells were then harvested from the growth surface as before, and cell density was determined using a duplicate reading using a Countess cell counter. Serial dilutions were plated onto duplicate 150 mm plates with Nuclon Delta Surface in 50 ml of selective growth medium (DMEM + 10% FBS + 1x PenStrep + 500 μg / ml geneticin). Plates were incubated for 18 days, and plates containing transfections of plasmids P313, C, and S were stained and photographed. Plates from other transfections were incubated for an additional 13 days before staining and photographing. For staining, the medium was gently removed by pipetting. Cells were covered with 10 ml of staining solution (0.4% methylene blue in 50% methanol) and incubated at room temperature for 10 minutes. The staining solution was removed by pipetting, the cells were washed with 5 ml of 100% methanol and allowed to air dry, and the plates were photographed using a Bio-Rad Imaging station.

[0286] result The results from the colony formation assay are shown in Table 2. Four of the mutant constructs generated G418-resistant colonies at frequencies ranging from 5.5% to 0.004% of the frequency measured for construct P313, which carries the wild-type NPT gene.

[0287] In this assay, colony formation frequency is an indirect measure of NPT protein activity. Cells that express more mutant NPT compared to other cells in a population of transfected cells, whether due to more multicopy integration of the expression cassette and / or a more favorable genomic location of cassette integration, are able to survive and form colonies when grown in the presence of G418. The results of this example demonstrate that the use of NPT mutants with reduced activity as selectable markers can be used to reduce the time and effort required to screen multiple colonies for stably integrated, high-transgene-expressing cells.

[0288] Three of the mutant constructs with the most attenuated phenotypes in bacteria did not generate G418-resistant colonies from plated 1E7 cells. It is possible that these mutant proteins are completely inactive in mammalian cells, but that cells expressing sufficiently high levels may survive selection. Such markers may be useful in combination with methods that are more efficient at generating high-copy-number integrations, such as retroviral infection or transposition.

[0289] [Table 2]

[0290] 8.3 Example 3: Transgene introduction with transposase This example demonstrates the integration of mCherry and NPT expression cassettes into human cells using transposase activity. The NPT mutants described herein are used in this example.

[0291] Three different constructs with the structures shown in Figure 2 were generated. These constructs differed from each other in that they contained nucleic acid sequences encoding either wild-type neomycin phosphotransferase, mutant 1 (P725) neomycin phosphotransferase (V36M; G210A), or mutant 2 (P726) neomycin phosphotransferase (E182D; D261N). The constructs were electroporated into human VPC cells (HEK293 variant) with or without Leap-In transposase RNA (ATUM Design, Newark, CA). Cells were plated onto 150 mm plates and cultured under neomycin selection for 2 weeks. Cells were then stained and measured for colony formation. Eight to 12 colonies were selected from different unstained plates, and mCherry copy numbers were measured relative to the endogenous glutamine synthetase gene using droplet digital PCR (ddPCR).

[0292] result Results from the colony formation assay are shown in Figure 3, which demonstrate that the NPT mutant dramatically reduced the efficiency of colony formation not through transposition but through random integration of the expression construct. Figure 4 shows a photograph of a stable pool of cells generated using the transposase; the color produced by mCherry expression is clearly evident under normal white light illumination when compared to non-transformed cells that lack color.

[0293] Results from measuring mCherry copy number in selected clones are shown in Figure 5. The results demonstrate that NPT mutant-containing cells have consistently higher average copy numbers of the linked mCherry transgene compared to those with wild-type NPT. Most of the clones generated by random integration of the construct with the wild-type NPT gene were little, if any, fluorescent, whereas most of the clones derived by random integration of the two mutant NPT genes were fluorescent. This can be interpreted as meaning that for survival during G418 selection, the mutant NPT gene must be expressed at a higher level than the wild-type NPT gene, whether by increased copy number or integration into a preferred genomic location, which results in increased expression of the mCherry transgene.

[0294] Enzymatic integration of the transgene into the host chromosome by transposition is much more efficient than random integration, resulting in a higher average copy number than with the wild-type NPT gene. The mutant NPT gene also increases copy number compared to the use of the wild-type NPT gene, which provides an advantage when gene delivery or transposition is inefficient, such as with large constructs.

[0295] 9. Implementation form The present invention provides the following non-limiting embodiments.

[0296] In one set of embodiments, the following is provided: A1. A non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT is: (a) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (b) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid; (c) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO:1 is a substitution with phenylalanine; (d) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine; (e) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at amino acid residue 218 of SEQ ID NO: 1 is a substitution with serine; or (f) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; A non-naturally occurring neomycin phosphotransferase (NPT) comprising the amino acid sequence of a wild-type neomycin phosphotransferase having the following structure: A2. A non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, wherein the non-naturally occurring NPT is: (a) amino acid substitutions at positions 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at position 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at position 210 of SEQ ID NO:1 is a substitution with alanine; (b) amino acid substitutions at positions 36 and 182 of SEQ ID NO: 1, wherein the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 182 of SEQ ID NO: 1 is a substitution with aspartic acid; (c) amino acid substitutions at positions 36 and 218 of SEQ ID NO: 1, wherein the amino acid substitution at position 36 of SEQ ID NO: 1 is a substitution with methionine and the amino acid substitution at position 218 of SEQ ID NO: 1 is a substituti...

Claims

1. A non-naturally occurring neomycin phosphotransferase (NPT) having neomycin phosphotransferase activity, the non-naturally occurring NPT comprising: (a) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 210 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 210 of SEQ ID NO:1 is a substitution with alanine; (b) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 182 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 182 of SEQ ID NO:1 is a substitution with aspartic acid; (c) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO:1 is a substitution with phenylalanine; (d) amino acid substitutions at amino acid residues corresponding to amino acid residues 216 and 261 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 261 of SEQ ID NO:1 is a substitution with asparagine; (e) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 218 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 218 of SEQ ID NO:1 is a substitution with serine; or (f) amino acid substitutions at amino acid residues corresponding to amino acid residues 36 and 216 of SEQ ID NO:1, wherein the amino acid substitution at the amino acid residue corresponding to amino acid residue 36 of SEQ ID NO:1 is a substitution with methionine and the amino acid substitution at the amino acid residue corresponding to amino acid residue 216 of SEQ ID NO:1 is a substitution with glycine; and optionally, said amino acid sequence of the wild-type neomycin phosphotransferase is SEQ ID NO:

1.

2. The non-naturally occurring NPT of claim 1, wherein the non-naturally occurring NPT has attenuated neomycin phosphotransferase activity as a selectable marker compared to wild-type NPT.

3. Bacterial cells transfected or transformed with a nucleotide sequence encoding the non-native NPT exhibit reduced colony formation as assessed by a colony formation assay after growth for 48 hours on plates containing 25 μg / mL, 75 μg / mL, or 100 μg / mL kanamycin, compared to bacterial cells transfected or transformed with a nucleotide sequence encoding a wild-type NPT, and optionally the bacterial cells are E. coli; or 2. The non-naturally occurring NPT of claim 1, wherein mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding said non-naturally occurring NPT exhibit reduced colony formation as assessed by a colony formation assay after two weeks of growth on tissue culture plates in medium containing 500 μg / mL geneticin (G418) compared to mammalian cells transfected with the same expression vector but comprising a nucleotide sequence encoding wild-type NPT, and optionally said mammalian cells are HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, or mouse C127 cells.

4. The non-naturally occurring NPT of claim 1, wherein geneticin (G418) resistant colonies of mammalian cells transfected with an expression vector comprising a nucleotide sequence encoding the non-naturally occurring NPT are generated at a frequency ranging from 0.001% to 75% compared to G418 resistant colonies of mammalian cells transfected with the same expression vector except that it comprises a nucleotide sequence encoding the wild-type NPT.

5. The non-naturally occurring NPT of claim 1, wherein the non-naturally occurring NPT comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 38 (V36M, G210A), SEQ ID NO: 39 (V36M, E182D), SEQ ID NO: 40 (V36M, Y218F), SEQ ID NO: 41 (D216G, D261N), SEQ ID NO: 42 (V36M, Y218S), and SEQ ID NO: 43 (V36M, D216G).

6. A first nucleotide sequence encoding the non-naturally occurring NPT of claim 1, or 10. A method for the preparation of a nucleic acid sequence comprising: a first nucleotide sequence encoding a non-naturally occurring NPT according to claim 1; and a second nucleotide sequence encoding a second protein or a non-coding RNA, optionally wherein the second protein is a therapeutic protein; Optionally, the first nucleotide sequence comprises the nucleotide sequence of SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, or SEQ ID NO:

37.

7. A vector comprising the nucleic acid sequence of claim 6.

8. 6. An in vitro or ex vivo host cell comprising a non-naturally occurring NPT according to any one of claims 1 to 5, optionally wherein the host cell is a bacterium, a yeast cell, a mammalian cell, a plant cell, and optionally wherein the mammalian cell is a human cell.

9. 8. An in vitro or ex vivo host cell comprising the nucleic acid sequence of claim 6 or the vector of claim 7, optionally wherein said nucleic acid sequence is stably integrated into the genome of said host cell, optionally wherein said host cell is a bacterium, a yeast cell, a mammalian cell, a plant cell, and optionally wherein said mammalian cell is a human cell.

10. 1. A method for selecting cells having a high copy number of a transgene, a high expression level of a transgene, or both, from a population of host cells into which said transgene has been introduced, comprising the steps of: (a) injecting into a population of host cells (i) a first nucleotide sequence encoding the non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; (ii) introducing a nucleic acid sequence comprising a second nucleotide sequence comprising the transgene; (b) selecting from the population of host cells into which the nucleic acid sequence has been introduced those cells which grow in the presence of a neomycin phosphotransferase substrate; Optionally, the nucleic acid sequence is stably integrated into the genome of the selected cell, optionally the host cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, optionally the mammalian cell is a human cell, and optionally the neomycin phosphotransferase substrate is neomycin, kanamycin, or geneticin (G418).

11. (a) the selected cells contain between 2 and 1000 times more copies of the transgene compared to the copy number of the transgene in a second set of cells following selection of a second host cell population grown in the presence of a neomycin phosphotransferase substrate, the second host cell population having been transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene; (b) the selected cells achieve a 10-1000 fold higher expression level of the transgene compared to the expression level of the transgene by a second set of cells following selection of a second host cell population grown in the presence of a neomycin phosphotransferase substrate, the second host cell population being transfected or transformed with a nucleic acid sequence comprising a nucleotide sequence encoding a wild-type NPT protein and the transgene; or The method of claim 10, wherein (c) both (a) and (b).

12. the selected cells have a high copy number of the transgene; the selected cells have a high expression level of the transgene; the selected cells have integrated between 5 and 100 copies of the transgene into their genomic DNA; or The method of claim 10 or 11, wherein the selected cells have integrated 1 to 5 copies of the transgene into their genomic DNA.

13. 12. The method of claim 10 or 11, wherein the transgene comprises a viral gene or a growth factor gene, or wherein the transgene encodes a protein or a non-coding RNA, optionally wherein the non-coding RNA is selected from the group consisting of antisense RNA, microRNA (miRNA), short hairpin RNA (shRNA), long non-coding RNA, catalytic RNA, ribosomal RNA, transfer RNA (tRNA), and guide RNA for Crispr nuclease, or optionally wherein the protein is a therapeutic protein or antigen.

14. A method for using a plasmid or a transposon comprising a nucleic acid sequence encoding the non-naturally occurring NPT according to claim 1 as a selectable marker, comprising: (a) introducing into a host cell the plasmid or transposon comprising the nucleic acid sequence encoding the non-naturally occurring NPT; (b) growing the cells in the presence of a neomycin phosphotransferase substrate; Optionally, the host cell is a bacterial cell, a yeast cell, a mammalian cell, a plant cell, optionally wherein the mammalian cell is a human cell, and optionally wherein the neomycin phosphotransferase substrate is neomycin, kanamycin, or geneticin (G418).

15. 15. The method of claim 14, wherein the plasmid or transposon further comprises a second nucleotide sequence encoding a protein or a non-coding RNA, optionally wherein the protein is a viral protein or a therapeutic protein, or optionally wherein the non-coding RNA is selected from the group consisting of short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, guide RNA for Crispr nuclease, catalytic RNA, ribosomal RNA, and transfer RNA (tRNA).

16. 1. A method of making a host cell comprising a second nucleotide sequence, comprising: (a) introducing or co-introducing into a host cell population a first nucleic acid sequence comprising: (i) a first nucleotide sequence encoding a non-naturally occurring NPT of claim 1; and (ii) a second nucleotide sequence comprising a transgene encoding a second protein or non-coding RNA, optionally wherein the second protein is a therapeutic protein or antigen, or optionally wherein the non-coding RNA is selected from the group consisting of short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, guide RNA for Crispr nuclease, catalytic RNA, ribosomal RNA, and transfer RNA (tRNA); (b) growing the host cell population in the presence of a neomycin phosphotransferase substrate to generate colonies; (c) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate; The method, wherein the host cell is a mammalian cell, optionally wherein the mammalian cell is selected from the group consisting of HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, and mouse C127 cells, and optionally wherein the neomycin phosphotransferase substrate is selected from the group consisting of neomycin, kanamycin, and geneticin (G418).

17. 1. A method of making a host cell comprising a second nucleotide sequence, comprising: (a) growing a host cell population comprising: (i) a first nucleic acid sequence comprising a first nucleotide sequence encoding a non-naturally occurring NPT of claim 1; and (ii) a second protein or non-coding RNA, optionally wherein the second protein is a therapeutic protein or antigen, or optionally wherein the non-coding region comprises a transgene encoding the second protein or non-coding RNA selected from the group consisting of short hairpin RNA (shRNA), microRNA (miRNA), antisense RNA, guide RNA for Crispr nuclease, catalytic RNA, ribosomal RNA, and transfer RNA (tRNA), in the presence of a neomycin phosphotransferase substrate to generate colonies; (b) selecting cell colonies that grow in the presence of the neomycin phosphotransferase substrate; The method, wherein the host cell is a mammalian cell, optionally wherein the mammalian cell is selected from the group consisting of HEK293 cells, CHO cells, PER.C6 cells, mouse NS0 cells, fibrosarcoma HT-1080 cells, mouse Sp2 / 0 cells, BHK cells, and mouse C127 cells, and optionally wherein the neomycin phosphotransferase substrate is selected from the group consisting of neomycin, kanamycin, and geneticin (G418).

18. 18. The method of claim 16 or 17, further comprising culturing the selected cell colonies.

19. 17. A host cell produced by the method of claim 16.

20. 1. A method for producing a stable cell line expressing a therapeutic protein or enzyme, comprising: (a) introducing into a population of host cells one or more nucleic acid sequences, said two or more nucleic acid sequences comprising: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; and (ii) a second nucleic acid sequence encoding said therapeutic protein or enzyme; (b) selecting from the cell population of step (a) cells that proliferate in the presence of geneticin (G418); (c) culturing the selected cells to produce stable cell lines expressing the therapeutic protein or enzyme; Optionally, the method, wherein said therapeutic protein is an antibody or an antibody fragment.

21. 21. A stable cell line produced by the method of claim 20.

22. 1. A method for producing a virus-producing cell line, comprising: (a) introducing into a population of host cells one or more nucleic acid sequences, the two or more nucleic acid sequences comprising: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; and (ii) a second nucleic acid sequence encoding one or more viral proteins, the one or more viral proteins being selected from the group consisting of a capsid protein, an envelope protein, a viral protein required for replication, and any combination thereof; (b) selecting from said cell population cells that grow in the presence of a neomycin phosphotransferase substrate; (c) expanding the selected cells to produce a virus-producing cell line; The one or more viral proteins comprise an AAV capsid protein, an AAV capsid protein and an AAV rep protein, an envelope protein, an adenoviral E1 region protein required for adenoviral replication, a retroviral envelope protein, a retroviral gag protein, a retroviral reverse transcriptase, or a retroviral envelope protein, a gag protein, and a reverse transcriptase.

23. Two or more nucleic acid sequences, the two or more nucleic acid sequences comprising: (a) a first nucleic acid sequence encoding the non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; (b) a second nucleic acid sequence encoding one or more viral proteins, wherein the one or more viral proteins are selected from the group consisting of capsid proteins, envelope proteins, viral proteins required for replication, and any combination thereof; A virus-producing cell line, wherein the one or more viral proteins comprise an AAV capsid protein, an AAV capsid protein and an AAV rep protein, an envelope protein, an adenoviral E1 region protein required for adenoviral replication, a retroviral envelope protein, a retroviral gag protein, a retroviral reverse transcriptase, or a retroviral envelope protein, a gag protein, and a reverse transcriptase.

24. 1. A method for producing a cell line expressing an antigen, comprising: (a) introducing into a host cell population one or more nucleic acid sequences, the two or more nucleic acid sequences comprising: (i) a first nucleic acid sequence encoding a non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; and (ii) a second nucleic acid sequence encoding an antigen, optionally wherein the antigen is a viral antigen, a bacterial antigen, a fungal antigen, or a cancer antigen; (b) selecting from the cell population of step (a) cells that proliferate in the presence of geneticin (G418); (c) culturing the selected cells to produce a cell line expressing the antigen; the cell line is a mammalian cell line, optionally wherein the mammalian cell line is a human cell line, or The method, wherein the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

25. Two or more nucleic acid sequences, the two or more nucleic acid sequences comprising: (a) a first nucleic acid sequence encoding the non-naturally occurring neomycin phosphotransferase (NPT) of claim 1; (b) a second nucleic acid sequence encoding one or more antigens, optionally wherein the one or more antigens are viral, bacterial, fungal, or cancer antigens; the cell line is a mammalian cell line, optionally wherein the mammalian cell line is a human cell line, or The antigen-producing cell line, wherein the cell line is a CHO, PER.C6, mouse NS0, HEK293, fibrosarcoma HT-1080, mouse Sp2 / 0, BHK, or mouse C127 cell line.

26. A selectable marker means for conferring resistance to kanamycin when introduced into a bacterial cell and for conferring resistance to geneticin (G418) when introduced into a mammalian cell, optionally wherein said selectable marker means comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, and SEQ ID NO:

37.

27. 1. A method for producing a production cell line, comprising: (a) transforming a bacterial or mammalian cell with an expression vector to produce a transformed cell, said expression vector comprising a nucleic acid sequence encoding one or more viral proteins, and a means for growing said transformed cell in the presence of kanamycin if said transformed cell is a bacterial cell, and a means for growing said transformed cell in the presence of geneticin (G418) if said transformed cell is a mammalian cell; (b) culturing the transformed cells in the presence of kanamycin or G418 to obtain a producer cell line, wherein the producer cell line expresses one or more viral proteins selected from the group consisting of AAV, adenovirus, retrovirus, lentivirus, herpes simplex virus, vaccinia virus, and baculovirus; Optionally, the nucleotide sequence is a nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase comprising an amino acid sequence selected from the group of SEQ ID NOs: 38, 39, 40, 41, 42, and 43.

28. 1. A method for selecting cells having stable chromosomal integration of an exogenous nucleic acid sequence, comprising: (a) transforming a population of eukaryotic cells with an exogenous nucleic acid sequence that includes a means for growth in the presence of geneticin (G418); (b) culturing the population of transformed cells in the presence of G418 to generate colonies of transformed cells capable of growing in the presence of G418; (c) selecting cells from the colonies generated in step (b) to obtain cells having stable chromosomal integration of said exogenous nucleic acid, optionally wherein said exogenous nucleic acid sequence further comprises a transgene expressed in said selected cells, or optionally wherein said exogenous nucleic acid sequence disrupts expression of a gene endogenous to said selected cells; Optionally, the nucleotide sequence is a nucleotide sequence encoding a non-naturally occurring neomycin phosphotransferase comprising an amino acid sequence selected from the group of SEQ ID NOs: 38, 39, 40, 41, 42, and 43.

29. 1. A method for selecting mammalian cells carrying stable episomes, comprising: (a) transforming a population of mammalian cells with a plasmid containing a means for growth in the presence of geneticin (G418); (b) culturing the population of transformed cells in the presence of G418 to generate colonies of transformed cells capable of growing in the presence of G418; (c) selecting cells from the colonies generated in step (b) to obtain cells having stable episomes containing the plasmid, optionally wherein the plasmid further comprises an EBNA1 OriP nucleic acid sequence, and wherein the selected cells express EBNA1.

30. 1. A method for selecting mammalian cells that transiently express a transgene, comprising: (a) introducing into a population of mammalian cells a nucleic acid encoding a transgene and a means for growth in the presence of geneticin (G418); (b) culturing the mammalian cell population in the presence of G418 for 48 to 72 hours; (c) selecting mammalian cells from said cultured population of mammalian cells growing in the presence of G418, wherein said selected mammalian cells transiently express said transgene, optionally wherein said transgene comprises a nucleic acid sequence encoding a Crispr endonuclease or a Crispr guide RNA.

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