bacterial host strain

Abstract

Disclosed are host bacterial strains, methods of making such host bacterial strains, and methods of using such host bacterial strains to improve plasmid production. In some embodiments, provided is an engineered bacterial host cell having a knock-out of SbcC, SbcD, or both, and a knock-out of one or all of recA, endA, and pgi. In some embodiments, provided are methods for replicating a vector in an engineered Escherichia coli host cell of the present disclosure.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 642,260, filed May 3, 2024, the entire contents of which are incorporated herein by reference.

[0003] References to sequence lists

[0004] The sequence list submitted via the USPTO Electronic Patent Filing System as an ST.26 XML file is incorporated herein by reference. The XML file for the sequence list is named 0802761_SequenceListing.xml, created on 2025-04-29, and is 161 KB (165,618 bytes) in size. Background Technology

[0005] The generation of viral vectors and plasmids containing structured DNA sequences, such as inverted and normal repeat sequences, is challenging and often leads to reduced yields and instability. Bacterial host cells used to propagate these vectors and plasmids have been engineered through genetic modification in an attempt to address these issues.

[0006] Although some engineered bacterial cells have incorporated mutation strategies to stabilize these types of DNA sequence structures, such as E. coli strains Stbl2, Stbl3, and Stbl4, they still suffer from reduced viability and productivity.

[0007] Therefore, there is a need for bacterial production strains (without ITR deletions or rearrangements) that can produce vectors containing palindromic and inverted repeat sequences in high yields, and that do not have problems with low stability or low viability. Summary of the Invention

[0008] This disclosure relates to host bacterial strains, methods for preparing such host bacterial strains, and methods for improving plasmid production using such host bacterial strains.

[0009] In some embodiments, an engineered bacterial host cell is provided having SbcC, SbcD or both knocked out, and one or all of recA, endA and pgi knocked out.

[0010] In some embodiments, methods for replicating vectors in engineered E. coli host cells of this disclosure are provided. Attached Figure Description

[0011] Figure 1A The pKD4 SbcCD targeted PCR fragment was depicted.

[0012] Figure 1B The SbcCD locus was described.

[0013] Figure 1C The integrated pKD4 PCR product of SbcCD knockout was depicted.

[0014] Figure 1D The scar sequence following FRT-mediated pKD4 kanR marker excision was depicted. Detailed Implementation

[0015] definition

[0016] As used herein, unless the context clearly indicates otherwise, the singular forms “a / an” and “the” may contain plural references.

[0017] The use of the term "or" in the claims and this disclosure is intended to mean "and / or" unless it is explicitly stated that it refers only to alternatives or that the alternatives are mutually exclusive.

[0018] When used with numerical values, the term “about” is intended to include + / - 10%. As an example rather than a limitation, if the number of amino acids identified is about 200, this would include 180 to 220 (plus or minus 10%).

[0019] As used herein, “engineered bacterial host cell” means a bacterial cell or cell line that has been modified by human intervention to induce genetic changes, including but not limited to (i) the deletion (e.g., knockout) of a naturally occurring gene or part of a gene; (ii) the insertion of genetic material into a genome that is not naturally present in the cell or cell line; or (iii) otherwise exposing the cell to agents (including but not limited to genetic material, peptides, proteins, or chemical entities) that reduce or promote the expression of naturally occurring genes.

[0020] As used herein, “gene knockout” means (i) the deletion of all or part of a target gene sufficient to silence or reduce the expression of the target gene to a level insufficient to maintain the normal function of the gene product; (ii) the deletion of all or part of the genetic element controlling the expression of the target gene, resulting in the target gene no longer being expressed or its expression level being insufficient to maintain its normal function; or (iii) reducing or silencing gene expression by exposing the target gene to an agent that acts directly on the gene or upstream signaling pathways. Non-limiting examples of methods for reducing or silencing gene expression or permanently deleting a gene or part thereof include small interfering RNA, microRNA, transcription activator-like effector nucleases (TALENs), the CRISPR / Cas9 system, transposons / transposases, and RNA-induced epigenetic silencing.

[0021]

[0022] As used herein, "mutations that reduce viability or yield" refers to mutations that reduce the viability or yield of a cell line relative to a cell line derived under the same culture conditions. It should be understood that such mutations can be engineered or naturally occurring.

[0023] As used herein, “endA” is a gene in *E. coli* that encodes a DNA-specific endonuclease. The endA gene encodes a protein having a sequence with 95% or higher sequence identity to the sequence in SEQ ID NO: 73 (MYRYLSIAAVVLSAAFSGPALAEGINSFSQA KAAAVKVHADAPGTFYCGCKINWQGKKGVVDLQSCGYQVRKNENRASRVEWEHVVPAWQFGHQRQCWQDGGRKNCAKDPVYRKMESDMHNLQPSVGEVNGDRGNFMYSQWNGGEGQYGQCAMKVDFKEKAAEPPARARGAIARTYFYMRDQYNLTLSRQQTQLFNAWNKMYPVTDWECERDERIAKVQGNHNPYVQRACQARKS).For example, the endA gene may have the sequence of SEQ ID NO: 74 (ATGTACCGTTATTTGTCTATTGCTGCGGTGG TACTGAGCGCAGCATTTTCCGGCCCGGCGTTGGCCGAAGGTATCAATAGTTTTTCTCAGGCGAAAGCCGCGGCGGTAAAAGTCCACGCTGACGCGCCCGGTACGTTTTATTGCGGATGTAAAATTAACTGGCAGGGCAAAAAAGGCGTTGTTGATCTGCAATCGTGCGGCTATCAGGTGCGCAAAAATGAAAACCGCGCCAGCCGCGTAGAGTGGGAACATGTCGTTCCCGCCTGGCAGTTCGGTCACCAGCGCCAGTGCTGGCAGGACGGTGGACGTAAAAACTGCGCTAAAGATCCGGTCTATCGCAAGATGGAAAGCGATATGCATAACCTGCAGCCGTCAGTCGGTGAGGTGAATGGCGATCGCGGCAACTTTATGTACAGCCAGTGGAATGGCGGTGAAGGCCAGTACGGTCAATGCGCCATGAAGGTCGATTTCAAAGAAAAAGCTGCCGAACCACCAGCGCGTGCACGCGGTGCCATTGCGCGCACCTACTTCTATATGCGCGACCAATACAACCTGACACTCTCTCGCCAGCAAACGCAGCTGTTCAACGCATGGAACAAGATGTATCCGGTTACCGACTGGGAGTGCGAGCGCGATGAACGCATCGCGAAGGTGCAGGGCAATCATAACCCGTATGTGCAACGCGCTTGCCAGGCGCGAAAGAGCTAA).

[0024]

[0025]

[0026] As used herein, “sbcB” is a gene in *E. coli* that encodes 3'-5' exonucleases. By way of example and not limitation, the sbcB gene may contain the sequence encoding a protein having the sequence SEQ ID NO: 81 (MMNDGKQQSTFLFHDYETFGTHPALDRPAQFA AIRTDSEFNVIGEPEVFYCKPADDYLPQPGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNFYDPYAWSWQHDNSRWDLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNAHDAMADVYATIAMAKLVKTRQPRLFDYLFTHRNKHKLMALIDVPQMKPLVHVSGMFGAWRGNTSWVAPLAWHPE). NRNAVIMVDLAGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKLVHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPSDNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLDYAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIV). For example, the sbcB gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 11.

[0027]

[0028] As used herein, “recD” is a gene present in *Escherichia coli* that encodes a subunit of the RecBCD enzyme complex (exonuclease V), which functions as a helicase-nuclease. By way of example and not limitation, the recD gene may contain a sequence encoding a protein having the sequence of SEQ ID NO: 83 (MKLQKQLLEAVEHKQLRPLDVQFALTVAGDEHPAVT). For example, the recD gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 13.

[0029] As used herein, “recJ” is a gene encoding an ssDNA-specific exonuclease found in E. coli. By way of example and not limitation, the recJ gene may contain a sequence encoding a protein having the sequence of SEQ ID NO: 84 (MKQQIQLRRREVDETADLPAELPPLLR). For example, the recJ gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 65.

[0030] As used herein, “uvrC” is a gene encoding a nuclease found in Escherichia coli and is a subunit of the UvrABC system. By way of example and not limitation, the uvrC gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 14.

[0031] As used herein, “mcrA” is a gene encoding a methylcytosine-specific nuclease found in Escherichia coli. By way of example and not limitation, the mcrA gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NO: 15.

[0032] As used herein, “mcrBC-hsd-mrr” is a gene encoding an endonuclease complex found in Escherichia coli. By way of example and not limitation, the mcrBC-hsd-mrr gene may contain a sequence having at least 90%, at least 95%, at least 99%, or 100% sequence identity with SEQ ID NOS: 16-21.

[0033] As used in this article, "fhuA" is a gene found in Escherichia coli that encodes an outer membrane protein involved in transport and also acts as a receptor.

[0034] As used in this article, "glnV" is the gene in *E. coli* that encodes the glutamine tRNA molecule and can also be referred to as the "supE" gene. In some cases, glnV carries the glnV44 (also known as supE44) mutation, which results in a succinate repressor allele of the glnV gene. GlnV44 encodes glutamine tRNA. gln CUA Glutamine is incorporated at the UAG codon.

[0035] As used in this article, "dcm" is the gene that encodes DNA cytosine methyltransferase, which exists in Escherichia coli.

[0036] As used herein, when referring to a specific cell, “derived from” means that the cell originates from a specific cell line or species in general. Alternatively, “derived from” may refer to the base cell line, species, or strain used to produce engineered bacterial host cells.

[0037] As used in this article, "DH5α" refers to an Escherichia coli strain / cell line with the following genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (r k -, m k +) gal- phoA supE44 λ-thi-1 gyrA96 relA1.

[0038] As used in this article, "DH1" refers to E. coli strains / cell lines with the following genotypes: F – λ –endA1recA1 relA1 gyrA96 thi-1 glnV44 hsdR17(r K – m K – ).

[0039] As used in this article, "JM107" refers to an Escherichia coli strain / cell line with the following genotype: endA1glnV44 thi-1 relA1 gyrA96 Δ(lac-proAB) [F' traD36 proAB + lacI q lacZΔM15]hsdR17(R K - m K + ) λ - .

[0040] As used in this article, "JM108" refers to an Escherichia coli strain / cell line with the following genotype: endA1 recA1gyrA96 thi-1 relA1 glnV44 Δ(lac-proAB) hsdR17 (r K - m K + ).

[0041] As used in this article, "JM109" refers to an Escherichia coli strain / cell line with the following genotypes: endA1glnV44 thi-1 relA1 gyrA96 recA1 mcrB + Δ(lac-proAB) e14- [F' traD36 proAB + lacI q lacZΔM15] hsdR17(r K - m K + ).

[0042] As used in this article, "MG1655" refers to an Escherichia coli strain / cell line with the following genotype: K-12 F – λ – ilvG –RFB-50 RPH-1. Exemplary genome sequences of *Escherichia coli* MG1655 include, but are not limited to, genome sequences with the following GenBank accession numbers: GCA_000005845.2, GCA_000269645.2, GCA_000273425.1, GCA_000482265.1, GCA_000517165.1, GCA_000801205.1. GCA_001308065.1, GCA_001544635.1, GCA_001566335.1, GCA_002843685.1, GCA_002966145.1, GCA_003627195.1, GCA_009767645.1, GCA_011750885.1, GCA_011750905.1, GCA_011750915.1, GCA_011750925.1, GCA_011750965.1, The genomes of GCA_011750975.1, GCA_011750995.1, GCA_011751015.1, GCA_013694185.1, GCA_015291845.1, GCA_018458765.1, GCA_020328175.1, GCA_025643415.1, GCA_025643435.1, GCA_025643455.1, GCA_025643475.1, and GCA_028618655.1, or ATCC® 700926 (also known as Migula strains, or Castellani and Chalmers strains).

[0043] As used in this article, "MG1655 ΔendA ΔrecA" refers to an Escherichia coli strain / cell line with the following genotype: K-12 F – λ – ilvG – rfb-50 rph-1 ΔendA ΔrecA.

[0044] As used in this article, "XL1Blue" refers to an E. coli strain / cell line with the following genotype: endA1gyrA96(nal R ) thi-1 recA1 relA1 lac glnV44 F'[ ::Tn10 proAB + lacI q Δ(lacZ)M15]hsdR17(r K - m K + ).

[0045] As used in this article, "GALG20" refers to genotype K-12 F. – λ – ilvG – The MG1655-derived Escherichia coli strain of rfb-50 rph-1 ΔendA ΔrecA Δpgi.

[0046] As used in this article, “REVIVER-pUC” refers to Escherichia coli strains / cell lines with the following genotypes: fhuA2Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1hsdR17 ΔSbcDC Δdcm.

[0047] As used in this article, "engineered mutation" should be understood as a mutation that is not naturally occurring, but rather a product of direct human intervention.

[0048] As used herein, the term "sequence identity" refers to the degree of identity between any given query sequence and a subject sequence. A subject sequence may, for example, have at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with a given query sequence. To determine the percentage of sequence identity, the query sequence (e.g., a nucleic acid sequence) is aligned with one or more subject sequences using any suitable sequence alignment program well known in the art, such as the computer program ClustalW (version 1.83, default parameters), which allows for alignment of nucleic acid sequences across their entire length (global alignment). (Chema et al., 2003 Nucleic Acids Res., 31:3497-500). In a preferred method, the sequence alignment program (e.g., ClustalW) calculates and aligns the best match between the query sequence and one or more subject sequences, allowing identity, similarity, and differences to be determined. Gaps of one or more nucleotides may be inserted into the query sequence, the subject sequence, or both to maximize the sequence alignment. For rapid alignment of nucleic acid sequences, appropriate default parameters can be selected for the specific alignment program. The output is the sequence alignment reflecting the relationship between sequences. To further determine the percentage of identity between the subject nucleic acid sequence and the query sequence, the sequences are aligned using the alignment program, the number of identical matches is divided by the length of the query sequence, and the result is multiplied by 100. Note that the percentage of identity can be rounded to the nearest decimal place. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0049] As used in this article, "HyperGRO" TM "Fermentation" refers to fed-batch fermentation, in which *E. coli* cells containing plasmids are grown at a reduced temperature for a portion of the fed-batch phase, during which the growth rate is limited. Subsequently, the temperature is increased, and growth continues at the increased temperature to accumulate plasmids. This temperature transition under limited growth rates improves plasmid yield and purity. HyperGRO TM As described in U.S. Patent No. 7,943,377, the entire contents of which are incorporated herein by reference.

[0050] As used in this article, “Rep protein” refers to a protein found in E. coli cells that is involved in the replication process.

[0051] As used in this article, the term "genome" refers to the nucleic acid sequence incorporated into the genome of a bacterial cell.

[0052] As used herein, “AAV vector” refers to an adeno-associated virus vector or an addendum viral vector. By way of example and not limitation, “AAV vector” includes self-complementary adeno-associated virus vectors (scAAV) and single-stranded adeno-associated virus vectors (ssAAV).

[0053] As used in this article, "amp" refers to ampicillin.

[0054] As used in this article, "ampR" refers to the ampicillin resistance gene.

[0055] As used in this article, the "bacterial region" refers to the area in a bacterial host where the desired vectors (such as plasmids) are propagated and selected.

[0056] As used in this article, "Cat" R "" refers to the chloramphenicol resistance gene.

[0057] As used herein, unless used in the context of a nucleotide or amino acid sequence, “ccc” or “CCC” means “covalently closed circular”.

[0058] As used in this article, "cI" refers to the λ suppressor.

[0059] As used in this article, "cITs857" refers to a λ repressor that further incorporates a temperature-sensitive C-to-T (Ala-to-Thr) mutation. cITs857 is a functional repressor at 28°C–30°C, but is largely inactivated at 37°C–42°C. It is also known as cI857 or cI857ts.

[0060] As used in this article, "cmv" or "CMV" refers to cytomegalovirus.

[0061] As used herein, "copy-cutting host strain" refers to an R6K origin-producing strain containing a phage φ80 attachment site chromosome integration copy of the arabinose-inducible CI857ts gene. Adding arabinose (e.g., to a final concentration of 0.2–0.4%) to the plate or culture medium induces pARA-mediated expression of the CI857ts repressor, which reduces copy number at 30°C through CI857ts-mediated downregulation of the pL promoter-expressing R6K Rep protein [i.e., further CI857ts-mediated more effective downregulation of the pL (OL1-G to T) promoter at 30°C]. Copy number induction is not impaired upon temperature transitions to 37°C–42°C because the CI857ts repressor is inactivated at these elevated temperatures. The copy-cutting host strain increases the temperature-upward copy number induction ratio of the R6K vector by reducing copy number at 30°C. This is advantageous for producing large, toxic, or easily dimerizing R6K origin vectors.

[0062] As used in this article, "dcm methylation" refers to methylation performed by Escherichia coli methyltransferase, which methylates the sequence CC(A / T)GG at the C5 position of the second cytosine.

[0063] As used herein, “eukaryotic region” refers to a plasmid region that encodes a eukaryotic sequence and / or a sequence required for the plasmid to function in a target organism. This includes plasmid vector regions required for the expression of one or more transgenes in a target organism, which include the RNA Pol II enhancer, promoter, transgene, and polyA sequence. It also includes plasmid vector regions required for the expression of one or more transgenes in a target organism using the RNA Pol I or RNA Pol III promoter, RNA Pol I or RNA Pol III promoter, or RNA. The eukaryotic region may optionally contain other functional sequences, such as eukaryotic transcription terminators, supercoil-induced DNA double-strand destabilization (SIDD) structures, S / MAR, boundary elements, etc. In lentiviral or retroviral vectors, the eukaryotic region contains flanking positive repeat sequences (LTRs); in AAV vectors, it contains flanking inverted terminal repeat sequences (IRTLs); and in transposon vectors, it contains flanking transposon IRTLs or IR / DR ends (e.g., Sleeping Beauty). In genome integration vectors, the eukaryotic region may encode homologous arms to guide targeted integration.

[0064] As used herein, “expression vector” refers to a vector used to express a gene of interest in a target organism, including but not limited to mRNA, protein antigen, protein therapeutic agent, shRNA, RNA or microRNA gene.

[0065] As used in this article, "gene of interest" refers to a gene that is to be expressed in a target organism.

[0066] As used herein, an "inverted repeat sequence" refers to a single-stranded sequence of nucleotides, downstream of which is its inverted complementary sequence. The nucleotide interpolation sequence between the initial sequence and the inverted complementary sequence can be of any length, including zero. When the interpolation length is zero, the complex sequence is a palindrome. It should be understood that inverted repeat sequences can appear in double-stranded DNA, and other inverted repeat sequences can appear within interpolation sequences.

[0067] As used in this article, "IR / DR" refers to an inverted repeat sequence that is repeated twice directly. For example, the Sleeping Beauty transposon IR / DR repeat sequence.

[0068] As used herein, “iterator” refers to a directly repeating DNA sequence at the origin of replication required for replication initiation. The R6K origin iterator repeat sequence is 22 bp and is described in WO 2019 / 183248 (see SEQ ID NO19-23 therein).

[0069] As used in this article, "ITR" refers to inverted terminal repeat sequence.

[0070] As used in this article, "lentiviral vector" refers to an integrated viral vector that can infect both dividing and non-dividing cells. It is also called a lentiviral transfer plasmid. The plasmid encodes expression units flanking the lentiviral LTR. The transfer plasmid, along with the lentiviral envelope and packaging plasmids required for virus particle preparation, is transfected into production cells.

[0071] As used in this article, "lentiviral envelope vector" refers to a plasmid that encodes an envelope glycoprotein.

[0072] As used in this article, "lentiviral packaging vector" refers to one or two plasmids that express the functions of the gag, pol, and Rev genes required for packaging lentiviral transfer vectors.

[0073] As used herein, "microcircle" refers to a covalently closed circular plasmid derivative in which the bacterial region has been removed from the parent plasmid through in vivo or in vitro site-specific recombination or in vitro restriction digestion / ligation. Microcircular vectors do not have the ability to replicate in bacterial cells.

[0074] As used in this article, “Nanoplasmid” TM "Vector" refers to a vector that combines an RNA selectivity marker with an R6K, ColE2, or ColE2-associated origin of replication, as described in WO2014077866 and WO 2014 / 035457, the full text of which is incorporated herein by reference.

[0075] As used herein, "NTC9385R" refers to the NTC9385R Nanoplasmid described in WO 2014 / 035457. TM The vector contains a spacer region encoding a NheI-trpA terminator-R6K origin RNA-OUT-KpnI bacterial region, which is linked to the eukaryotic region via flanking NheI and KpnI sites.

[0076] As used in this article, "PCR" refers to "polymerase chain reaction".

[0077] As used in this article, "pDNA" refers to plasmid DNA.

[0078] As used in this article, a “piggyback transposon” refers to a transposon system in which a PB transposon flanking an ITR is integrated into the genome via a simple cleavage and paste mechanism mediated by PB transposases. Transposon vectors typically contain a promoter-transgene-polyA expression cassette between the PB and ITRs, which is excised and integrated into the genome.

[0079] As used in this article, the "PL promoter" refers to the left-hand λ promoter. PL is a strong promoter that is repressed by the cI repressor, which binds to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for control of gene expression via thermal induction, as it is functional and suppresses gene expression at 30°C, but becomes inactive between 37°C and 42°C, thus allowing gene expression to occur.

[0080] As used herein, the “PL (OL1 G to T) promoter” refers to the λ promoter remaining after the OL1 G to T mutation. PL is a strong promoter that is repressed by the cI repressor, which binds to the OL1, OL2, and OL3 repressor binding sites. The temperature-sensitive cI857 repressor allows for control of gene expression via heat induction, as it is functional and represses gene expression at 30°C, but becomes inactive at 37°C–42°C, thus allowing gene expression to occur. As described in WO 2014 / 035457, reducing the binding of the cI repressor to OL1 through the OL1 G to T mutation results in increased promoter activity at 30°C and 37°C–42°C.

[0081] As used in this article, "plasmid" refers to an extrachromosomal DNA molecule that is separate from chromosomal DNA and can replicate independently of chromosomal DNA.

[0082] As used in this article, "plasmid copy number" refers to the number of plasmid copies per cell. An increase in plasmid copy number indicates an increase in plasmid yield.

[0083] As used in this article, "Pol" refers to polymerase.

[0084] As used in this article, "Pol I" refers to Escherichia coli DNA polymerase I.

[0085] As used in this article, "Pol III" refers to Escherichia coli DNA polymerase III.

[0086] As used herein, “Pol III-dependent origin of replication” refers to an origin of replication that does not require Pol I, such as the rep protein-dependent R6Kγ origin of replication. Many other Pol III-dependent origins of replication are known in the art, many of which are summarized in del Solar et al., ibid., 1998 (which are incorporated herein by reference).

[0087] As used herein, “polyA” refers to a polyadenylation signal or site. Polyadenylation is the addition of a poly(A) tail to an RNA molecule. Polyadenylation signals contain a sequence motif recognized by the RNA cleavage complex. Most human polyadenylation signals contain the AAUAAA motif and conserved sequences at their 5' and 3'. Commonly used polyA signals are derived from rabbit β-globin, bovine growth hormone, and early or late SV40 polyA signals.

[0088] As used herein, "polyA repeat sequence" refers to a continuous sequence of adenine nucleotides that are repetitive sequences. Similarly, "polyG repeat sequence" refers to a continuous sequence of guanine nucleotides that are repetitive sequences, "polyC repeat sequence" refers to a continuous sequence of cytosine nucleotides that are repetitive sequences, and "polyT repeat sequence" refers to a continuous sequence of thymine nucleotides that are repetitive sequences. The "mRNA vector" contains a polyA repeat sequence.

[0089] As used in this article, "pUC origin" refers to the replication origin derived from pBR322, which has a G-to-A transition that increases copy number at elevated temperatures and lacks the negative ROP regulator.

[0090] As used in this article, "pUC-free" refers to a plasmid that does not contain a pUC origin.

[0091] As used in this article, "pUC plasmid" refers to a plasmid containing a pUC origin.

[0092] As used herein, “R6K plasmid” refers to a plasmid having an R6K or R6K-derived origin of replication, such as NTC9385R, NTC9685R, NTC9385R2-O1, NTC9385R2-O2, NTC9385R2a-O1, NTC9385R2a-O2, NTC9385R2b-O1, NTC9385R2b-O2, NTC9385Ra-O1, NTC9385Ra-O2, NTC9385RaF, and NTC9385RbF vectors, as well as modified and alternative vectors containing an R6K origin of replication as described in WO 2014 / 035457 and WO2019 / 183248. Alternative R6K vectors known in the art include, but are not limited to, pCOR vectors (Gencell), pCpG-free vectors (Invivogen), and CpG-free Oxford University vectors (including pGM169).

[0093] As used herein, “R6K origin of replication” refers to a region specifically recognized by the R6K Rep protein to initiate DNA replication, including but not limited to R6K γ origin of replication sequences (SEQ ID NO: 43-44, 46, and 60, respectively). It also includes the CpG-free form as described in Drocourt et al., U.S. Patent 7,244,609, which is incorporated herein by reference (SEQ ID NO: 63).

[0094] As used herein, “R6K origin of replication-RNA-OUT bacterial origin” contains R6K origin of replication and RNA-OUT selectivity markers for reproduction (SEQ ID NO: 50-59, respectively).

[0095] As used herein, a “Rep protein-dependent plasmid” is a plasmid in which replication depends on the trans-provided replication (Rep) protein. Examples include the R6K origin of replication, the ColE2-P9 origin of replication, and the ColE2-associated origin of replication plasmids, in which the Rep protein is expressed from the host strain genome. Many other Rep protein-dependent plasmids are known in the art, many of which are summarized in del Solar et al., ibid., 1998, Microbiol. Mol. Biol. Rev. 62:44-464 (which is incorporated herein by reference).

[0096] As used in this article, "retroviral vector" refers to an integrated viral vector that can infect dividing cells. It is also called a transfer plasmid. The plasmid encodes expression units flanking retroviral LTRs. The transfer plasmid, along with the envelope and packaging plasmids required for viral particle preparation, is transfected into production cells.

[0097] As used in this article, "retroviral envelope vector" refers to a plasmid that encodes an envelope glycoprotein.

[0098] As used in this article, "retroviral packaging vector" refers to the plasmid that encodes the retroviral gag and pol genes required for packaging retroviral transfer vectors.

[0099] As used in this article, “RNA-IN” refers to RNA-IN encoded by Insertion Sequence 10 (IS10), a complementary and antisense RNA to a portion of RNA, RNA-OUT. When RNA-IN is cloned into the untranslated leader sequence of mRNA, the annealing of RNA-IN with RNA-OUT reduces the translation of the gene encoded downstream of RNA-IN.

[0100] As used herein, “selective markers of RNA-IN regulation” refers to genome-expressed, RNA-IN-regulated selective markers. In the presence of a plasmid-carried RNA-OUT antisense repressor RNA (SEQ ID NO: 48), the expression of proteins encoded downstream of RNA-IN (e.g., having the sequence gccaaaaatcaataatcagacaacaagatg (SEQ ID NO: 68)) is suppressed. Selective markers of RNA-IN regulation are configured such that RNA-IN regulates 1) proteins that are lethal or toxic to the cells themselves or by generating toxic substances (e.g., SacB), or 2) repressor proteins that are lethal or toxic to the cells by inhibiting the transcription of genes essential for the growth of the bacterial cells (e.g., the murA essential gene regulated by the RNA-IN tetR repressor gene). For example, a genome-expressing RNA-IN-SacB cell line for RNA-OUT plasmid selection / propagation is described in WO 2008 / 153733. Alternative selective markers described in the art may replace SacB.

[0101] As used herein, “RNA-OUT” refers to RNA-OUT encoded by insert sequence 10 (IS10), an antisense RNA that hybridizes with a transposon gene expressed downstream of RNA-IN and reduces its translation. The sequences of RNA-OUT RNA (SEQ ID NO:48) and RNA-IN-SacB cell lines expressing complementary RNA-IN SacB genomes can be modified to incorporate alternative functional RNA-IN / RNA-OUT binding pairs, such as those described in Mutalik et al., 2012 Nat Chem Biol 8:447, including but not limited to RNA-OUT A08 / RNA-IN S49 pairs, RNA-OUT A08 / RNA-IN S08 pairs, and CpG-free modifications of RNA-OUT A08 that change the CG in the RNA-OUT 5' TTCGC sequence to a non-CpG sequence. CpG-free RNA-OUT can be prepared by using various alternative substitutions that remove two CpG motifs (mutating each CpG to CpA, CpC, CpT, ApG, GpG, or TpG).

[0102] As used herein, “RNA-OUT selective marker” refers to an RNA-OUT selective marker DNA fragment containing E. coli transcription promoter and terminator sequences flanking the RNA-OUT RNA. RNA-OUT selective markers utilizing RNA-OUT promoter and terminator sequences flanking DraIII and KpnI restriction enzyme sites, and a custom RNA-IN-SacB cell line for genome expression propagation using RNA-OUT plasmids, are described in WO 2008 / 153733 and are incorporated herein by reference. The RNA-OUT promoter and terminator sequences flanking the RNA-OUT RNA can be replaced with heterologous promoter and terminator sequences. For example, the RNA-OUT promoter can be replaced with CpG-free promoters known in the art (e.g., the I-EC2K promoter or the P5 / 6 5 / 6 or P5 / 6 6 / 6 promoter described in WO2008 / 153733 and incorporated herein by reference). The 2 CpG RNA-OUT selectivity marker, with two CpG motifs removed from the RNA-OUT promoter, is given as SEQ ID NO: 49. The RNA-IN-SacB cell line for RNA-OUT plasmid propagation described in WO 2008 / 153733, or any cell line with RNA-IN-SacB described in WO 2008 / 153733, can be used to select for sucrose resistance in vectors incorporating the CpG-free RNA-OUT selectivity marker. Alternatively, the RNA-IN sequence in these cell lines can be modified to incorporate a 1 bp variation to perfectly match the CpG-free RNA-OUT region complementary to RNA-IN.

[0103] As used herein, “RNA selection marker” refers to an expressed nontranslated RNA carried by a plasmid that regulates the expression of target genes on chromosomes to provide selection. This could be a nonsense repressor tRNA carried by a plasmid that regulates nonsense repressor-selective chromosomal targets, as described in Crouzet J and Soubrier F, U.S. Patent 6,977,174, 2005, which is incorporated herein by reference. This could also be an antisense repressor RNA carried by a plasmid, as cited in the non-restrictive list herein, including RNA-OUT (WO 2008 / 153733) for targets that repress RNA-IN regulation, RNAI encoded by the pMB1 plasmid origin for targets that repress RNAII regulation (Grabherr R, Pfaffenzeller I. 2006 US Patent Publication US20060063232; Cranenburgh RM. 2009; US Patent 7,611,883), RNAI encoded by the pMU720 origin for the IncB plasmid for targets that repress RNAII regulation (Wilson IW, Siemering KR, Praszkier J, Pittard AJ. 1997. J Bacteriol 179:742-53), Sok at the ParB locus for the R1 plasmid for targets that repress Hok regulation, and FlmB at the Flm locus for the F plasmid for targets that repress flmA regulation (Morsey). (MA, 1999 US Patent US5922583). RNA selectivity markers can be other naturally occurring antisense repressor RNAs known in the art, such as those described in Wagner EGH, Altuvia S, Romby P. 2002. Adv Genet 46:361-98 and Franch T and Gerdes K. 2000. CurrentOpin Microbiol 3:159-64. RNA selectivity markers can also be engineered repressor RNAs, such as synthetic small RNAs expressed from SgrS, MicC, or MicF scaffolds, as described in Na D, Yoo SM, Chung H, Park H, Park JH, Lee SY. 2013. Nat Biotechnol 31:170-4. RNA selectivity markers can also be engineered repressor RNAs that, as part of a selectivity marker, repress target RNAs fused to a target gene to be regulated (such as SacB as described in US 2015 / 0275221).

[0104] As used in this article, "SacB" refers to the structural gene encoding fructan sucrase in Bacillus subtilis. SacB expression is toxic in Gram-negative bacteria in the presence of sucrose.

[0105] As used in this article, “selective biomarker” or “selective biomarker” refers to a selective biomarker, such as a kanamycin resistance gene or an RNA selective biomarker.

[0106] As used in this article, "shRNA" refers to short hairpin RNA.

[0107] As used in this article, "Sleeping Beauty transposon" refers to a transposon system in which SB transposons flanking IR / DR transposons are integrated into the genome via a simple cleavage and paste mechanism mediated by SB transposase. Transposon vectors typically contain a promoter-transgene-polyA expression cassette between IR / DR transposases, which is excised and integrated into the genome.

[0108] As used herein, a "spacer region" refers to the region connecting the 5' and 3' ends of a eukaryotic region sequence. The 5' and 3' ends of the eukaryotic region are typically separated by bacterial origins of replication and bacterial selectivity markers in plasmid vectors (bacterial regions), thus many spacer regions consist of bacterial regions. In the Pol III-dependent origin of replication vector of this invention, this spacer region is preferably less than 1000 bp.

[0109] As used herein, “structured DNA sequence” refers to a DNA sequence capable of forming replication-repressive secondary structures (Mirkin and Mirkin, 2007. Microbiology and Molecular Biology Reviews 71:13-35). This includes, but is not limited to, inverted repeats, palindromic repeats, orthogonal repeats, IR / DR repeats, homopolymeric repeats, or repeats containing eukaryotic promoter enhancers or eukaryotic origins of replication.

[0110] As used in this article, "SV40 origin" refers to the simian virus 40 genomic DNA containing the origin of replication.

[0111] As used in this article, "SV40 enhancer" refers to simian virus 40 genomic DNA containing 72 bp and optionally 21 bp enhancer repeat sequences.

[0112] As used in this article, "TE buffer" refers to a solution containing approximately 10 mM Tris pH 8 and 1 mM EDTA.

[0113] As used in this article, "TetR" refers to the tetracycline resistance gene.

[0114] As used herein, “transfection” refers to methods for delivering nucleic acids into cells [e.g., poly(lactide-co-glycolic acid) (PLGA), ISCOM, liposomes, vesicles, virions, block copolymers, Prönkel block copolymers, chitosan and other biodegradable polymers, microparticles, microspheres, calcium phosphate nanoparticles, nanoparticles, nanocapsules, nanospheres, poloxamine nanospheres, electroporation, nuclear transfection, piezoelectric permeation, acoustic permeation, iontophoresis, ultrasound, SQZ high-speed cell deformation-mediated membrane rupture, corona plasma, plasma-enhanced delivery, tissue-tolerant plasma, laser microperforation, shock wave energy, magnetic field, non-contact magnetic permeation, gene gun, microneedles, microdermabrasion, hydrodynamic delivery, high-pressure tail vein injection, etc.], as known in the art and incorporated herein by reference. Transfection of DNA into *E. coli* (commonly referred to as transformation) is typically performed using chemically competent or electrocompetent *E. coli* cells, using standard methods known in the art and incorporated herein by reference.

[0115] As used in this article, "transgenic" refers to a gene of interest cloned into a vector for expression in a target organism.

[0116] As used in this article, "transposase vector" refers to a vector that encodes a transposase.

[0117] As used in this article, "transposon vector" refers to a vector that encodes a transposon, which is a substrate for transposase-mediated gene integration.

[0118] As used in this article, "ts" means temperature sensitive.

[0119] As used in this article, "UTR" refers to the untranslated region of mRNA (5' or 3' of the coding region).

[0120] As used in this article, "vector" refers to a gene delivery medium, including viral (e.g., alphavirus, poxvirus, lentivirus, retrovirus, adenovirus, adenovirus-associated virus, etc.) and non-viral (e.g., plasmid, MIDGE, transcriptionally active PCR fragment, microloop, bacteriophage, nanoplasmid) substances. TM (etc.) carriers. These are well known in the art and are included herein by reference.

[0121] As used in this article, "vector backbone" refers to the eukaryotic and bacterial regions of the vector, without transgene or target antigen coding regions.

[0122] Engineered bacterial host cells

[0123] This disclosure relates to engineered bacterial host strains with improved stability and replication of transfected plasmids, including but not limited to those containing structured DNA sequences such as ITR and AAV vectors. The engineered bacterial host strains comprise at least one gene knockout selected from the group consisting of SbcC and SbcD, and at least one gene knockout selected from the group consisting of endA, recA, and pgi. The engineered bacterial host strains may also comprise gene knockouts of SbcC, SbcD, endA, recA, and pgi.

[0124] In some embodiments, the engineered bacterial host cells do not contain engineered mutations that reduce viability or yield in any of sbcB, recB, recD, and recJ (and optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof). In some embodiments, the engineered bacterial host cell contains the genes sbcB, recB, recD, and recJ, and optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof, wherein the genes sbcB, recB, recD, and recJ, and optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof (if present), encode a functional gene product.

[0125] In some embodiments, the engineered bacterial host cell is derived from an *E. coli* cell line, and in some embodiments, it is genetically homologous to the cell line / strain from which it is derived. In some embodiments, the *E. coli* cell line is selected from the group consisting of: DH5α, DH1, JM107, JM108, JM109, XL1Blue, and MG1655.

[0126] In some embodiments, the engineered bacterial host cell contains a gene knockout of one or both of the SbcC and SbcD genes and is otherwise homogeneous with the strain from which it is derived, the strain being selected from the group consisting of MG1655, MG1655 ΔendA ΔrecA, and MG1655 ΔendA ΔrecA Δpgi (GalG20). Without conflicting with any of the foregoing embodiments, the engineered *E. coli* host cell may also not contain engineered mutations that reduce viability or yield in any or all of sbcB, recB, recD, and recJ (and at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof). Therefore, in some embodiments, the engineered *E. coli* host cell also does not contain any engineered mutations that reduce viability or yield, engineered mutations, or any mutations in uvrC. In other embodiments, the engineered *E. coli* host cells do not contain any engineered mutations that reduce viability or yield, engineered mutations, or any mutations in *mcrA*. In still other embodiments, the engineered *E. coli* host cells do not contain any engineered mutations that reduce viability or yield, engineered mutations, or any mutations in *mcrBC-hsd-mrr*. In yet another embodiment, the engineered *E. coli* host cells do not contain any engineered mutations that reduce viability or yield, engineered mutations, or any mutations in *mcrA* and *mrBC-hsd-mrr*.

[0127] To the extent that it does not conflict with any of the foregoing embodiments, the engineered *E. coli* host cells may also exclude mutations that reduce viability or yield in any or all of sbcB, recB, recD, and recJ (and at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof). Therefore, in some embodiments, the engineered *E. coli* host cells also exclude any mutations that reduce viability or yield, engineered mutations, or any mutations in uvrC. In other embodiments, the engineered *E. coli* host cells also exclude any mutations that reduce viability or yield, engineered mutations, or any mutations in mcrA. In still other embodiments, the engineered *E. coli* host cells also exclude any mutations that reduce viability or yield, engineered mutations, or any mutations in mcrBC-hsd-mrr. In yet still other embodiments, the engineered *E. coli* host cells also exclude any mutations that reduce viability or yield, engineered mutations, or any mutations in mcrA and mrBC-hsd-mrr.

[0128] Therefore, without conflicting with any of the foregoing embodiments, the engineered bacterial host cell comprises the genes sbcB, recB, recD, and recJ, and optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof, wherein the sbcB, recB, recD, and recJ genes, and optionally, at least one of uvrC, mcrA, mcrBC-hsd-mrr, and combinations thereof (if present), encodes a functional gene product. Thus, in some embodiments, the engineered *E. coli* host cell comprises the sbcB gene, wherein the sbcB gene encodes a functional gene product. In some embodiments, the engineered *E. coli* host cell comprises the recB gene, wherein the recB gene encodes a functional gene product. In some embodiments, the engineered *E. coli* host cell comprises the recD gene, wherein the recD gene encodes a functional gene product. In some embodiments, the engineered *E. coli* host cell comprises the recJ gene, wherein the recJ gene encodes a functional gene product. Additionally, in some embodiments, the engineered *E. coli* host cell further comprises the uvrC gene, wherein the uvrC gene encodes a functional gene product. In other embodiments, the engineered *E. coli* host cell further comprises the mcrBC-hsd-mrr gene operon, wherein the mcrBC-hsd-mrr gene operon encodes a functional gene product. In still other embodiments, the engineered *E. coli* host cell further comprises both the mcrA gene and the mrBC-hsd-mrr gene operon, wherein the mcrA gene encodes a functional gene product and the mrBC-hsd-mrr gene operon encodes a functional gene product.

[0129] In any of the foregoing embodiments, gene knockout of the engineered bacterial host cell may be SbcC knockout. Alternatively, in some embodiments, gene knockout of the engineered bacterial host cell may be SbcD knockout. In any of the foregoing embodiments, gene knockout of the engineered bacterial host cell may be both SbcC and SbcD knockout.

[0130] In any of the foregoing embodiments, the engineered bacterial host cell may also contain a genomic antibiotic resistance marker. By way of example, and not limitation, the genomic antibiotic resistance marker may be kanR, which contains a sequence having at least 90%, at least 95%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 23 (kanR, 795 bp). By way of further example, and not limitation, the genomic antibiotic resistance marker may be kanR, which contains a sequence encoding a protein having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 36 (kanR). By way of even further example, the genomic antibiotic resistance marker may be a chloramphenicol resistance marker, a gentamicin resistance marker, a kanamycin resistance marker, a spectinomycin and streptomycin resistance marker, a trimethoprim resistance marker, or a tetracycline resistance marker. Alternatively, in any of the foregoing embodiments, the bacterial host cell may not contain a genomic antibiotic resistance marker.

[0131] In any of the foregoing embodiments, the engineered bacterial host cell may further comprise a Rep protein suitable for culturing Rep protein-dependent plasmids. By way of example and not limitation, the engineered bacterial host cell may comprise a genomic nucleic acid sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 26 (P42L-P106I-F107S-P113S, 918 bp), SEQ ID NO: 27 (P42L-Δ106-107-P113S, 912 bp), SEQ ID NO: 28 (P42L-P106L-F107S, 918 bp), and SEQ ID NO: 29 (P42L-P113S, 918 bp). As a further example and not a limitation, engineered bacterial host cells may contain a genomic nucleic acid sequence encoding a Rep protein that has at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with an amino acid sequence selected from the group consisting of: SEQ ID NO: 39 (P42L-P106I-F107S-P113S), SEQ ID NO: 40 (P42L-Δ106-107-P113S), SEQ ID NO: 42 (P42L-P106L-F107S), SEQ ID NO: 41 (P42L-P113S), SEQ ID NO: 34 (ColE2 wild type), SEQ ID NO: 35 (ColE2 mutant G194D). It should be understood that the nucleic acid sequence encoding the Rep protein in any of the foregoing embodiments can be controlled by the PL promoter, and if a λ repressor, such as cITs857, is present in the genome, such a PL promoter can achieve temperature-sensitive expression of the Rep protein. By way of example and not limitation, the PL promoter may have a sequence having at least 95%, at least 98%, at least 99%, or 100% sequence identity with ttgacataaa taccactggc ggtgatact (PL promoter (-35 to -10); SEQ ID NO: 68), ttgacataaa taccactggc gtgatact (PL promoter OL1-G (-35 to -10); SEQ ID NO: 69), or ttgacataaa taccactggc gttgatact (PL promoter OL1-G to T (-35 to -10); SEQ ID NO: 70).It should be further understood that, in the case where the Rep protein is an R6K Rep protein such as SEQ ID NO: 39-42, the vector transfected into engineered bacterial host cells may contain an R6K origin of replication, and alternatively, in the case where the Rep protein is a ColE2 Rep protein, the vector transfected into engineered bacterial host cells may contain a ColE2 origin of replication.

[0132]

[0133]

[0134] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆SbcDC::kanR.

[0135] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi ∆SbcDC.

[0136] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi Δdcm ΔSbcDC::kanR

[0137] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi Δdcm ΔSbcDC

[0138] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S(P3-), SpecR StrepR; ∆SbcDC::kanR.

[0139] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S(P3-), SpecR StrepR; ∆SbcDC.

[0140] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC::kanR.

[0141] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC.

[0142] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC::kanR ∆dcm.

[0143] In some implementations, engineered bacterial host cells with the following genotypes are provided: F– λ– ilvG–rfb-50 rph-1 ΔendA ΔrecA Δpgi; ∆SbcDC ∆dcm.

[0144] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)dcm-; ΔSbcDC.

[0145] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)dcm-; ∆SbcDC::kanR.

[0146] In some implementations, engineered bacterial host cells with the following genotype are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; ΔSbcDC.

[0147] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC::kanR.

[0148] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attφ80::pARA-CI857tsPc-RNA-IN-SacB, tetR; ∆SbcDC.

[0149] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attφ80::pARA-CI857tsPc-RNA-IN-SacB, tetR; ∆SbcDC::kanR.

[0150] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts, tetR;∆SbcDC.

[0151] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN-SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts, tetR;∆SbcDC::kanR.

[0152] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC.

[0153] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20)attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857ts Pc-RNA-IN- SacB, tetR; ∆SbcDC::kanR.

[0154] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20) dcm- attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL(OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857tsPc-RNA-IN- SacB, tetR; ∆SbcDC.

[0155] In some implementations, engineered bacterial host cells with the following genotypes are provided: MG1655 ΔendA ΔrecA Δpgi (GalG20) dcm- attλ:: Pc-RNA-IN- SacB, catR; attHK022::pL(OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attφ80::pARA-CI857tsPc-RNA-IN- SacB, tetR; ∆SbcDC::kanR.

[0156] It should be understood that any of the above-described embodiments may, to the extent applicable, be incorporated into DH5α, DH1, JM107, JM108, JM109, and XL1Blue strains containing gene knockouts of endA, recA, pgi, SbcD, and SbcC. It should also be understood that any of the above-described embodiments may, to the extent applicable, be incorporated into REVIVER-pUC strains containing gene knockouts of endA, recA, and pgi.

[0157] In any of the foregoing embodiments, the engineered bacterial host cell may also contain a vector. By way of example and not limitation, the vector may be a non-viral transposon vector, such as a transposase vector, Sleeping Beauty transposon vector, Sleeping Beauty transposase vector, PiggyBac transposon vector, PiggyBac transposase vector, expression vector, etc.; a non-viral gene editing vector, such as a homology-directed repair (HDR) / CRISPR-Cas9 vector; or a viral vector, such as an AAV vector, AAV repcap vector, AAV helper vector, Ad helper vector, lentiviral vector, lentiviral envelope vector, lentiviral packaging vector, retroviral vector, retroviral envelope vector, retroviral packaging vector, mRNA vector, etc.

[0158] In any of the foregoing embodiments, where the bacterial host cell also comprises a vector, the vector may comprise a nucleic acid sequence having a palindromic sequence. A palindromic sequence can be understood as a nucleic acid sequence in a double-stranded DNA molecule, wherein a sequence read in one direction on one strand matches a sequence read in the opposite direction on the complementary strand, such that there is a complementary portion along one strand, wherein there are no intercalation sequences between the complementary portions. By way of example and not limitation, the complementary sequences of the palindromic sequence may each comprise about 10 to about 200 base pairs, about 15 to about 200 base pairs, about 20 to about 200 base pairs, about 25 to about 200 base pairs, about 30 to about 200 base pairs, about 40 to about 200 base pairs, about 50 to about 200 base pairs, about 75 to about 200 base pairs, or about 100 to about 200 base pairs. Approximately 15 to 200 base pairs, approximately 10 to 150 base pairs, approximately 15 to 150 base pairs, approximately 20 to 150 base pairs, approximately 25 to 150 base pairs, approximately 30 to 150 base pairs, approximately 30 to 150 base pairs, approximately 40 to 150 base pairs, approximately 50 to 150 base pairs, approximately 100 to 150 base pairs, approximately 10 to 140 About 15 to about 140 base pairs, about 20 to about 140 base pairs, about 25 to about 140 base pairs, about 30 to about 140 base pairs, about 30 to about 140 base pairs, about 40 to about 140 base pairs, about 50 to about 140 base pairs, about 100 to about 140 base pairs, about 10 to about 100 base pairs, about 15 to about 100 base pairs, about 20 to About 100 base pairs, about 25 to about 100 base pairs, about 30 to about 100 base pairs, about 40 to about 100 base pairs, about 50 to about 100 base pairs, or about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 base pairs.

[0159] In any of the foregoing embodiments, where the bacterial host cell also includes a vector, the vector may contain a nucleic acid sequence having at least one positive repeat sequence. By way of example, and not limitation, the at least one positive repeat sequence may contain about 40 to 150 nucleotides, about 60 to about 120 nucleotides, or about 90 nucleotides. By way of further example, and not limitation, the at least one positive repeat sequence may be a simple repeat sequence, comprising a short sequence of DNA consisting of multiple repetitions of a single base, such as a polyA repeat sequence, a polyT repeat sequence, a polyC repeat sequence, or a polyG repeat sequence, wherein the simple repeat sequence comprises about 40 to 150 consecutive repeat sequences of the same base, about 60 to 120 consecutive repeat sequences of the same base, or about 90 consecutive repeat sequences of the same base. By way of further example, and not limitation, the polyA repeat sequence may contain 40 to 150 consecutive adenine nucleotides, 60 to 120 consecutive adenine nucleotides, or about 90 adenine nucleotides.

[0160] In any of the foregoing embodiments, where the bacterial host cell also contains a vector, the vector may contain inverted repeat sequences, forward repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences. As a further example, the vector may contain sequences selected from the group consisting of: polyA repeat sequences, SV40 origin of replication, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITR, CMV enhancers, and SV40 enhancers. By way of example, and not limitation, an AAV vector may contain an AAV ITR. In some embodiments, where the bacterial host cell also contains a vector, the vector may contain a nucleic acid sequence having at least one inverted repeat sequence, which may also be an inverted terminal repeat sequence, such as, by way of example, and not limitation, an AAV ITR. Therefore, in any of the foregoing embodiments, the vector may contain an AAV ITR. It should be understood that an inverted repeat sequence is a single-stranded sequence of nucleotides, downstream of which is its inverted complementary sequence. It should be further understood that the single-stranded sequence may be part of a double-stranded vector. The nucleotide interpolation sequence between the initial sequence and the inverted complementary sequence can be of any length, including zero. When the interpolation length is zero, the complex sequence is a palindrome. When the interpolation length is greater than zero, the complex sequence is an inverted repeat. In any of the foregoing embodiments, the interpolation sequence can be from 1 to approximately 2000 base pairs. By way of example and not limitation, inverted repeat sequences (which may also be inverted terminal repeat sequences) may comprise about 1 to about 2000 base pairs, about 5 to about 2000 base pairs, about 10 to about 2000 base pairs, about 25 to about 2000 base pairs, about 50 to about 2000 base pairs, about 100 to about 2000 base pairs, about 250 to about 2000 base pairs, about 500 to about 2000 base pairs, about 750 to about 2000 base pairs, about 1000 to about 2000 base pairs, about 1250 to about 2000 base pairs, about 1500 to about 2000 base pairs, about 1750 to about 2000 base pairs, and so on. Intercalated sequences of 0 base pairs, about 1 to about 100 base pairs, about 1 to about 50 base pairs, about 1 to about 25 base pairs, about 1 to about 20 base pairs, about 1 to about 10 base pairs, about 1 to about 5 base pairs, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 base pairs.By way of example and not limitation, the complementary portions of the inverted repeat sequence may each contain approximately 10 to approximately 200 base pairs, approximately 15 to approximately 200 base pairs, approximately 20 to approximately 200 base pairs, approximately 25 to approximately 200 base pairs, approximately 30 to approximately 200 base pairs, approximately 40 to approximately 200 base pairs, approximately 50 to approximately 200 base pairs, approximately 75 to approximately 200 base pairs, approximately 100 to approximately 200 base pairs, etc. base pairs, about 15 to about 200 base pairs, about 10 to about 150 base pairs, about 15 to about 150 base pairs, about 20 to about 150 base pairs, about 25 to about 150 base pairs, about 30 to about 150 base pairs, about 30 to about 150 base pairs, about 40 to about 150 base pairs, about 50 to about 150 base pairs, about 100 to about 150 base pairs, about 10 to about 14 0 base pairs, about 15 to about 140 base pairs, about 20 to about 140 base pairs, about 25 to about 140 base pairs, about 30 to about 140 base pairs, about 30 to about 140 base pairs, about 40 to about 140 base pairs, about 50 to about 140 base pairs, about 100 to about 140 base pairs, about 10 to about 100 base pairs, about 15 to about 100 base pairs, about 20 to Approximately 100 base pairs, approximately 25 to approximately 100 base pairs, approximately 30 to approximately 100 base pairs, approximately 40 to approximately 100 base pairs, approximately 50 to approximately 100 base pairs, or approximately 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 base pairs. By way of example and not limitation, the at least one inverted repeat sequence may comprise an AAV ITR repeat sequence comprising ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcgcagagagggagtggccaactccatcactaggggttcct (5' AAV ITR; SEQ ID NO: 73) and aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcgcagagagggagtggccaa (3' AAV ITR; SEQ ID NO: 73). 74) A sequence having at least 95%, at least 95%, at least 98%, at least 99%, or 100% sequence identity.

[0161] Alternatively, in any of the foregoing embodiments, where the bacterial host cell also contains a vector, the vector cannot contain a nucleic acid sequence having a palindromic sequence, a forward repeat sequence, or an inverted repeat sequence.

[0162] In any of the foregoing embodiments, the vector may be an AAV vector. In some embodiments, where the vector is an AAV vector, the AAV vector contains an AAV ITR. In other embodiments, the vector may be a lentiviral vector, a lentiviral envelope vector, or a lentiviral packaging vector. In other embodiments, the vector may be a retroviral vector, a retroviral envelope vector, or a retroviral packaging vector. In other embodiments, the vector may be a transposase vector or a transposon vector. In a further embodiment, the vector may be an mRNA vector. By way of example and not limitation, the mRNA vector may contain a polyA repeat sequence as described in this disclosure.

[0163] In any of the foregoing embodiments, the vector may be a plasmid. In any of the foregoing embodiments, the vector may be a Rep protein-dependent plasmid.

[0164] In any of the foregoing embodiments, the vector may also contain an RNA-selectivity marker. By way of example and not limitation, the RNA-selectivity marker may be RNA-OUT. As a further example and not a limitation, RNA-OUT may have at least 95%, at least 98%, at least 99%, or 100% sequence identity with sequences selected from the group consisting of SEQ ID NO: 47 (gtagaattgg taaagagagt cgtgtaaaat atcgagttcg cacatcttgttgtctgatta ttgatttttg gcgaaaccat ttgatcatat gacaagatgt gtatctacct taacttaatgattttgataa aaatcatta) and SEQ ID NO: 49 (gtagaattgg taaagagagt tgtgtaaaatattgagttcg cacatcttgt tgtctgatta ttgatttttg gcgaaaccat ttgatcatat gacaagatgtgtatctacct taacttaatg attttgataa aaatcatta). In some implementations, the engineered bacterial host cell may contain a corresponding RNA-IN sequence to allow regulation of downstream markers via RNA-OUT, and the RNA-OUT sequence corresponds to RNA-IN.

[0165] In any of the foregoing embodiments, the vector may also contain RNA-OUT antisense repressor RNA. By way of example and not limitation, the RNA-OUT antisense repressor RNA may have a sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 48.

[0166] In any of the foregoing embodiments, the vector may also contain a bacterial origin of replication. By way of example and not limitation, the bacterial origin of replication may be selected from the group consisting of R6K, pUC, and ColE2. As a further example, and not a limitation, the bacterial origin of replication can be an R6Kγ origin of replication having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 43 (ggcttgttgt ccacaaccgt taaaccttaaaagctttaaa agccttatat attctttttt ttcttataaa acttaaaacc ttagaggcta tttaagttgctgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaac atgagagctt agtacgttagccatgagagc ttagtacgtt agccatgagg gtttagttcg ttaaacatga gagcttagta cgttaaacatgagagcttag tacgtactat caacaggttg aactgctgat c), SEQ ID NO: 44 (ggcttgttgtccacaaccat taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaa acttaaaaccttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttag tacgtgaaacatgagagctt agtacattag ccatgagagc ttagtacatt agccatgagg gtttagttca ttaaacatgagagcttagta cattaaacat gagagcttag tacatactat caacaggttg aactgctgat c),SEQ IDNO: 45 (aaaccttaaa acctttaaaa gccttatata ttcttttttt tcttataaaa cttaaaaccttagaggctat ttaagttgct gatttatatt aattttattg ttcaaacatg agagcttagt acatgaaacatgagagctta gtacattagc catgagagct tagtacatta gccatgaggg tttagttcat taaacatgagagcttagtac attaaacatg agagcttagt acatactatc aacaggttga actgctgatc),SEQ ID NO: 46 (tgtcagccgt taagtgttcc tgtgtcactg aaaattgctt tgagaggctc taagggcttctcagtgcgtt acatccctgg cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag ccttatatattctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg atttatatta attttattgttcaaacatga gagcttagta cgtgaaacat gagagcttag tacgttagcc atgagagctt agtacgttagccatgagggt ttagttcgtt aaacatgaga gcttagtacg ttaaacatga gagcttagta cgtgaaacatgagagcttag tacgtactat caacaggttg aactgctgat cttcagatc) and SEQ ID NO: 60 (ggcttgttgt ccacaaccgt taaaccttaa aagctttaaa agccttatat attctttttt ttcttataaaacttaaaacc ttagaggcta tttaagttgc tgatttatat taattttatt gttcaaacat gagagcttagtacgtgaaac atgagagctt agtacgttag ccatgagagc ttagtacgtt agccatgagg gtttagttcgttaaacatga gagcttagta cgttaaacat gagagcttag tacgttaaac atgagagctt agtacgtactatcaacaggt tgaactgctg atc), SEQ ID NO: 30 (ColE2 origin (+7), 45 bp), SEQ ID NO: 31 (ColE2 origin (+7, CpG-free), 45 bp), SEQ ID NO: 32 (ColE2 origin (Min), 38 bp), SEQ ID NO: 33 (ColE2 origin (+16), 60 bp) and SEQ ID NO: 22 (pUC, 784 bp).

[0167] In any of the foregoing embodiments, the engineered bacterial host cell may further comprise a pUC-free eukaryotic microcircle expression vector, which may comprise: (i) a eukaryotic region sequence encoding a gene of interest and having 5' and 3' ends; and (ii) a spacer region of less than 1000, preferably less than 500 base pairs, connecting the 5' and 3' ends of the eukaryotic region sequence, and comprising an R6K bacterial origin of replication and RNA-OUT selectivity marker. By way of example and not limitation, the R6K bacterial origin of replication and RNA-OUT selectivity marker may have sequences as described in this disclosure and as known in the art. Alternatively, in any of the foregoing embodiments, the engineered bacterial cell may further comprise a covalently closed circular plasmid having: a backbone comprising a Pol III-dependent R6K origin of replication and RNA-OUT selectivity marker, wherein the backbone is less than 1000 bp, preferably less than 500 bp; and an insert comprising a structured DNA sequence. By way of example, and not limitation, a structured DNA sequence may contain sequences selected from the group consisting of: inverted repeat sequences, forward repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, and eukaryotic promoter enhancer sequences. As further examples, a structured DNA sequence may contain sequences selected from the group consisting of: polyA repeat sequences, SV40 origin of replication, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, AAV ITR, CMV enhancer, and SV40 enhancer. By way of example, and not limitation, the insert may be a transposase vector, an AAV vector, or a lentiviral vector. By way of example, and not limitation, a Pol III-dependent R6K origin of replication may have a sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 60. By way of example, and not limitation, an RNA-OUT selectivity marker may be an RNA-IN regulatory RNA-OUT functional variant having at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 47 or SEQ ID NO: 49. As a further example, an RNA-OUT selectivity marker may be an RNA-OUT antisense repressor RNA. By way of example, and not limitation, an RNA-OUT antisense repressor RNA may have a sequence having at least 90%, at least 95%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 48.

[0168] As disclosed herein, methods for gene knockout or knockdown are well known in the art, including, by way of example and not limitation, the methods disclosed in the embodiments herein (recombinant engineering), as well as P1 phage transduction, large-scale genome transfer, and CRISPR / Cas9. It should be understood that gene knockout can result in the elimination of protein expression or the expression of nonfunctional proteins. Therefore, the SbcCD complex may or may not be present in the bacterial host strains of this disclosure; however, if present, it is nonfunctional in the case of knockout, or has reduced activity as a nuclease in the case of knockdown. It should be understood that embodiments of this disclosure may include knockout or knockdown of SbcC, SbcD, or both.

[0169] Unbound by theory, it is anticipated that knocking out SbcC or SbcD alone would be sufficient to achieve the desired effects of this invention, as both proteins are essential subunits of the SbcCD nuclease (Connelly JC and Leach DR, Genes Cells 1:285, 1996). The bacterial sbcC and sbcD genes encode nucleases involved in palindromic sequence lethality and genetic recombination (Connelly JC and Leach DR, Genes Cells 1:285, 1996).

[0170] Methods for producing vectors in engineered bacterial host cells

[0171] The desired vector can be produced in engineered bacterial host cells using a fermentation protocol suitable for transfection in appropriate engineered bacterial host cells according to this disclosure. In one embodiment, the vector can be produced via fed-batch fermentation (such as HyperGRO). TM (Fermentation) is used to produce this. Typically, this method involves growing engineered bacterial host cells at a reduced temperature during the first part of a fed-batch stage, which may be under growth-limiting conditions, followed by raising the temperature to a higher temperature during the second part of the fed-batch stage. For example, the reduced temperature could be approximately 28-30°C. o C, and the higher temperature can be about 37-42°C. o C. As an example, the first part can be approximately 12 hours, and the second part can be approximately 8 hours. It should be understood that, in the case of fed-batch fermentation with an upward temperature shift, engineered bacterial host cells can possess a λ repressor and a Rep protein, which in turn... L Under the control of the promoter, the PL promoter can be adjusted by the λ suppressor, which can be temperature-sensitive.

[0172] In any of the foregoing embodiments, the plasmid yield after incubating transfected host cells under conditions sufficient for vector replication can be higher than that of cell lines that have derived engineered bacterial host cells and have been treated under the same conditions. In any of the foregoing embodiments, the plasmid yield after incubating transfected host cells under conditions sufficient for vector replication can be higher than that of SURE2, SURE, Stbl2, Stbl3, or Stbl4 cells treated under the same conditions.

[0173] It should be understood that in any of the foregoing embodiments, the engineered *E. coli* host cells may contain knockdown of SbcC, SbcD, or both, or knockdown of recA, endA, and pgi, rather than knockout. Knockdown may result in reduced expression and / or decreased activity of the SbcCD complex. The reduction may be at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more.

[0174] The bacterial host strains and methods of this disclosure will now be described with reference to the following non-limiting embodiments.

[0175] Example

[0176] In the following embodiments, for shake flask production, the proprietary Plasmid+ is used. ® Shaking medium. Seed cultures are started from glycerol stock solution or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates are incubated at 30°C–32°C; cells are resuspended in medium and used to prepare 500 mL Plasmid+. ® The shake flask provides approximately 2.5 OD600 inoculum containing either 50 μg / mL antibiotic (for ampR or kanR selection plasmids) or 0.5% sucrose (for RNA-OUT selection plasmids). Shake the flask until saturation at the indicated growth temperature.

[0177] In the following embodiments, as described in U.S. Patent No. 7,943,377, a proprietary fed-batch culture medium (NTC3019, HyperGRO) was used in a New Brunswick BioFlo 110 bioreactor. TM HyperGRO (culture medium) TMFermentation. Seed cultures were started from glycerol stock solutions or colonies and streaked onto LB agar plates containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 6% sucrose (for RNA-OUT selection plasmids). The plates were incubated at 30°C–32°C; the cells were resuspended in the medium and used to provide approximately 0.1% inoculum for fermentation, containing 50 μg / mL antibiotics (for ampR or kanR selection plasmids) or 0.5% sucrose (for RNA-OUT plasmids). HyperGRO TM The temperature change is shown in the figure.

[0178] In the following examples, culture samples were collected at key points and at regular intervals throughout all fermentation periods. The biomass (OD600) and plasmid yield of the samples were analyzed immediately. When plasmid yield was determined, analysis was performed by quantifying plasmids obtained from the Qiagen Spin Miniprep kit formulation, as described in U.S. Patent No. 7,943,377. Briefly, cells were lysed in an alkaline environment, clarified, and the plasmid column was purified and eluted prior to quantification. Plasmid quality was determined by agarose gel electrophoresis (AGE) analysis, performed on a 0.8–1% Tris / acetate / EDTA (TAE) gel as described in U.S. Patent No. 7,943,377.

[0179] Example 1: Preparation of SbcCD knockout strain

[0180] As described in Datsenko and Wanner, PNAS USA 97:6640-6645 (2000), SbcCD knockout strains were generated using Red Gam recombinant cloning. PCR amplification of the pKD4 plasmid (Datsenko and Wanner, 2000) was performed using the following primers to introduce the SbcC and SbcD targeting homologous arms.

[0181] SEQ ID NO 1 (SbccR-pKD4):

[0182] CCCTCTGTATTCATTATCCTGCTGAATAGTTATTTCACTGCAAACGTACTCATATGAATATCCTCCTTAG

[0183] SEQ ID NO 2 (SbcdF-pKD4):

[0184] TCTGTTTGGGTATAATCGCGCCCATGCTTTTTCGCCAGGGAACCGTTATGTGTAGGCTGGAGCTGCTTCG

[0185] Purification of 1.6 kb PCR product (SEQ ID NO: 5) Figure 1A The cells were digested with DpnI (to eliminate the template plasmid). The host strain containing the SbcCD gene to be knocked out was transformed with the pKD46-RecA recombinant engineered plasmid (WO 2008 / 153731, the full text of which is incorporated herein by reference). Transformants were selected for ampicillin resistance. Electrocompetent cells of the transformed cell line were prepared by growing them in LB medium containing 50 μg / mL ampicillin, with an OD600 of approximately 0.05. Arabinose was added to 0.2% to induce expression of the recombinant engineered gene. Cells were grown to mid-logarithmic phase and then resuspended in 10% glycerol at 1 / 200 of their original volume by centrifugation. 5 μL of purified PCR product digested with DpnI was electroporated into 25 μL of electrocompetent cells, followed by the addition of 1 mL of SOC medium. Cells were grown at 30°C for 2 hours, seeded on LB agar plates containing 20 μg kanamycin, and grown overnight at 37°C. ΔSbcDC::kanR was screened for single kanR colonies using the SbcDF and SbcCR primers described below.

[0186] SEQ ID NO 3 (SbcDF primer): cgtctcgccatgatttgccctg

[0187] SEQ ID NO 4 (SbcCR primer): cgttatgcgccagctccgtgag

[0188] Host: The product of SbcDF and SbcCR primers = 4.8 kb ( Figure 1B (SEQ ID NO: 6)

[0189] The host ΔSbcDC::kanR:SbcDF and SbcCR primer products = 1.9 kb ( Figure 1C (SEQ ID NO: 7)

[0190] The temperature-sensitive pKD46-recApa plasmid was removed from the cell line by growth at 37°C–42°C. The ampicillin sensitivity of individual kanR colonies was also verified.

[0191] For host strains with antibiotic resistance plasmids (e.g., pUC origin of replication; antibiotic selection; R6K origin of replication; antibiotic selection), the kanR chromosomal marker was removed from ∆SbcDC::kanR using FRT recombination, as described (Datsenko and Wanner, ibid., 2000). In short, the ∆SbcDC::kanR cell line was transformed with the pCP20 FRT plasmid (Datsenko and Wanner, ibid., 2000), and the transformants were grown at 30°C and selected for ampicillin resistance. Single colonies were streaked onto LB agar plates (without ampicillin) to obtain single colonies and grown at 43°C to remove temperature-sensitive pCP20 plasmids. Single colonies on 43°C LB plates were streaked onto LB amp and LB kan plates to verify the loss of the ampR pCP20 plasmid and kanR excision, respectively. ΔSbcDC was screened by PCR using SbcDF and SbcCR primers for single amp and kan-sensitive colonies. Figure 1D The PCR products of SbcDF and SbcCR primers are 0.53 kb in size. Figure 1D As shown (SEQ ID NO: 8).

[0192] The gene can be replaced by deletion of the pKD4 gene-specific PCR product using Red Gam recombination, and the pKD4 kanR chromosomal marker can be removed using pCP20-mediated FRT excision as described above. Examples of genes deleted in this manner include dcm and fhuA.

[0193] The pUC replication origin of MG1655 ΔendA ΔrecA Δpgi (GalG20)

[0194] The starting strain had the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi, and was designated GalG20. After SbcCD knockout and kanR excision, the knockout strain (GalG20 ∆SbcDC) had the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecA Δpgi∆SbcDC. This strain can be used to generate plasmids with pUC bacterial origins of replication.

[0195] pUC replication origin

[0196] Another strain was generated from GalG20 ∆SbcDC. After knocking out dcm and excising kanR, the knockout strain (GalG20 ∆SbcDC ∆dcm) had the following genotype: F– λ– ilvG– rfb-50 rph-1 ΔendA ΔrecAΔpgi ∆SbcDC ∆dcm (GALG20-REVIVER-pUC). This strain can be used to generate plasmids with pUC bacterial origins of replication.

[0197] Other ∆SbcDC strains constructed in this manner for generating pUC replication origins include DH5α ∆SbcDC (starting strain DH5α; ∆SbcDC strain genotype F-φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1gyrA96 relA1 ∆SbcDC) and E. cloni 5-α ∆SbcDC Δdcm (starting strain E. cloni 5-α; ∆SbcDC strain genotype F-fhuA2 Δ(argF-lacZ)U169 phoA glnV44 Φ80 Δ(lacZ)M15 gyrA96recA1 relA1 endA1 thi-1) disclosed in WO 2021 / 183827. hsdR17 ∆SbcDC Δdcm).

[0198] R6K copy start point

[0199] Another strain was generated from GalG20 ∆SbcDC ∆dcm by integrating a heat-inducible R6K rep protein cassette (attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR) into the host genome, as described in WO 2014 / 035457, to generate a new strain, GalG20 R6K Rep ∆SbcDC ∆dcm, with the genotype: GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecRStrepR; ∆SbcDC ∆dcm. This strain can be used to generate plasmids with R6K bacterial origin of replication.

[0200] R6K replication origin with RNA-OUT selection

[0201] Another strain was generated from GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S(P3-), SpecR StrepR; ∆SbcDC ∆dcm by integrating a constitutively expressed RNA-IN sacB box (Pc-RNA-IN-SacB, catR) into the host genome, as described in WO 2014 / 035457, to generate a new strain with the following genotype: GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-),SpecR StrepR; attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC ∆dcm. Another strain was generated from GalG20attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attλ::Pc-RNA-IN-SacB, catR; ∆SbcDC ∆dcm by integrating an arabinose-inducible CI857ts λ repressor containing a second copy of the RNA-IN-SacB expression cassette (::pARA-CI857ts, tetR) into the host genome, as described in WO 2014 / 035457, to generate a new strain with the following genotype: GalG20 attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; attλ:: Pc-RNA-IN-SacB,catR; attφ80::pARA-CI857ts, tetR; ∆SbcDC ∆dcm(GALG20-REVIVER-R6K). This strain can be used to generate Nanoplasmids with R6K bacterial origin of replication and RNA-OUT antibiotic-free selection markers. TMVectors. Other ∆SbcDC strains constructed in this manner for generating R6K origin of replication / RNA-OUT vectors include DH5α ∆SbcDC (REVIVER-NP) disclosed in WO 2021 / 183827 (starting strain DH5α ∆SbcDC; final REVIVER-NP strain genotype F-φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+)gal- phoA supE44 λ- thi-1 gyrA96 relA1 ∆SbcDC; attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; 2) attλ:: Pc-RNA-IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR) and E. cloni 5-α ∆SbcDC Δdcm (REVIVER-R6K) (starting strain E. cloni 5-α; final REVIVER-R6K strain genotype F-ΔfhuA Δ(argF-lacZ)U169 phoA ΔPhoEglnV44 Φ80 Δ(lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17; attHK022::pL(OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecR StrepR; 2) attλ:: Pc-RNA-IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR).

[0202] pUC replication origin with RNA-OUT selection

[0203] Additional strains were generated from GalG20 ∆SbcDC ∆dcm by integrating a constitutively expressed RNA-INsacB box (Pc-RNA-IN-SacB, catR) into the host genome, as described in WO 2014 / 035457, to produce new strains with the following genotype: GalG20 attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC ∆dcm. Additional strains were generated from GalG20 ∆SbcDC by integrating a constitutively expressed RNA-INsacB box (Pc-RNA-IN-SacB, catR) into the host genome, as described in WO 2014 / 035457, to produce new strains with the following genotype: GalG20 attλ:: Pc-RNA-IN-SacB, catR; ∆SbcDC. These strains can be used to generate vectors with pUC bacterial origin of replication and RNA-OUT antibiotic-free selection markers.

[0204] Example 2: Performance of SbcCD knockout strain with AAV ITR vector: ITR stability

[0205] The application of the GALG20 ∆SbcDC host strain in improving the stability of AAV ITR-containing vectors was evaluated using the following: 5.5 kb AAV2 R6K origin-RNA-OUT marker Nanoplasmid in the GALG20 ∆SbcDC ∆dcm R6K replication origin with RNA-OUT host selection. TM The vector; and the 12 kb pUC origin-kanR AAV vector in GALG20 ∆SbcDC ∆dcm.

[0206] To evaluate the ability of the GALG20 ∆SbcDC host strain to stabilize AAV ITR, the AAV ITR vector was transformed into the GALG20 ∆SbcDC host strain. Intact ITRs of single colonies were screened by SmaI and AhdI digestion, and correct clones were then submitted to the CCIB DNA Core Laboratory (Cambridge MA) at Massachusetts General Hospital (MGH) for complete plasmid sequencing via next-generation sequencing. The 5.5 kb AAV2 R6K origin-of-replication marker Nanoplasmid was found in the GALG20 ∆SbcDC ∆dcm R6K origin of replication, which exhibits RNA-OUT host selection. TMThe vector, after restriction digestion, showed that 10 / 10 selected colonies had intact ITRs, and after next-generation sequencing, 9 / 9 selected colonies had intact ITRs. For the 12 kb pUC origin-kanR AAV vector in the GALG20 ∆SbcDC ∆dcm pUC origin vector host, after restriction digestion, 6 / 6 selected colonies had intact ITRs, and after next-generation sequencing, 6 / 6 selected colonies had intact ITRs. This demonstrates ITR stability during transformation in the pUC and R6K origin-kanR AAV vector host strains of GALG20 ∆SbcDC.

[0207] Example 3: Performance of SbcCD knockout strain with AAV ITR vector: HyperGRO TM Fermentation

[0208] Use the following items in HyperGRO TM The application of the GALG20 ∆SbcDC host strain in improving the stability and yield of AAVITR-containing vectors was evaluated during fermentation: compared with DH5α ∆SbcDC and E. cloni 5-α ∆SbcDC ∆dcm R6K replication origin with RNA-OUT selection, the 5.5 kb AAV2 R6K origin-RNA-OUT marker anoplasmid was significantly improved in the GALG20 ∆SbcDC ∆dcm R6K replication origin with RNA-OUT selection. TM The vector (GALG20-REVIVER-R6K); and the 12 kb pUC initiation-kanR AAV vector (GALG20-REVIVER-pUC) in GALG20 ∆SbcDC ∆dcm compared with DH5α, DH5α ∆SbcDC, E. cloni 5-α ∆SbcDC ∆dcm and Stbl3. The results are summarized in Tables 1 and 2.

[0209] Table 1: pAAV Nanoplasmid TM Vector (5.5 kb) (R6K origin, RNA-OUT selector) HyperGRO TM Fermentation evaluation

[0210]

[0211] a Incubate at 30°C for 12 hours, then increase to 37°C and continue for 24-36 hours until OD decreases or cleavage occurs. Maintain at 25°C. The culture medium contains 0.2% arabinose.

[0212] bAll cell lines are R6K origins of replication with RNA-OUT selection derivatives, containing the following genome-integrated copies: 1) attHK022::pL (OL1-G to T) P42L-P106I-F107S P113S (P3-), SpecRStrepR; 2) attλ:: Pc-RNA-IN-SacB, catR; and 3) attφ80::pARA-CI857ts, tetR.

[0213] c The DH5α ∆SbcDC strain was disclosed in WO 2021 / 183827 "Bacterial Host Strains".

[0214] d E. cloni 5-alpha ΔSbcDC Δdcm Genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal-phoA supE44 λ- thi-1 gyrA96relA1 fhuA2 glnV44 ΔSbcDC Δdcm

[0215] Table 2: pAAV vector (12 kb pUC origin - kanR) HyperGRO TM Fermentation evaluation

[0216]

[0217] a Transformed from 30°C to 42°C at 55 OD600 for 9 hours, and maintained at 25°C.

[0218] b Heat to 37°C, 24-36 hours

[0219] c Transform at 30°C to 37°C at 55°C OD600 until OD decreases or cleavage occurs, and maintain at 25°C.

[0220] d 30°C, transition to 37°C after 30 hours, until OD decreases or pyrolysis occurs, maintain at 25°C.

[0221] eE. cloni 5-alpha ΔSbcDC Δdcm Genotype: F- φ80lacZΔM15 Δ(lacZYA-argF) U169 recA1 endA1 hsdR17 (rk-, mk+) gal- phoA supE44 λ- thi-1 gyrA96relA1 fhuA2 glnV44 ΔSbcDC Δdcm

[0222] f The DH5α ∆SbcDC strain was disclosed in WO 2021 / 183827.

[0223] Compared to the Stbl3, DH5α, DH5α ∆SbcDC, or E. cloni 5-α ∆SbcDC ∆dcm hosts with AAV ITR vectors, the GALG20 ∆SbcDC ∆dcm host strains GALG20-REVIVER-R6K and GALG20-REVIVER-pUC showed improved plasmid production and / or plasmid quality. The GALG20 ∆SbcDC ∆dcm host also stabilized AAV ITRs during cell line transformation. The improved yield of GALG20 ∆SbcDC ∆dcm was unexpected, as it has been reported that fed-batch HyperGRO... TM The plasmid productivity during fermentation was similar to that of GALG20 and DH5α (Goncalves et al., (2014) Journal of Biotechnology 186:119-127).

[0224] The aforementioned embodiments can be repeated using MG1655 derivatives with additional gene knockouts (e.g., relA, ackA, topA, pta, poxB, fruR, pykA, pykF, purR, stress-inducible DNA polymerase, transposon or transposase gene deletion, rac prophage deletion, etc.) or through promoter upregulation mutations or ectopic gene copies of polA, ligA, gyrAB, trxA, grx1, zwf, rpiA, prsA, priA, priB, dnaT, and priC).

[0225] All references cited in this article (including publications, patent applications and patents) are hereby incorporated by reference to the extent that each reference is individually and specifically indicated by reference and is presented in the whole of this article.

[0226] Unless otherwise stated, the terms “comprising,” “having,” “including,” and “containing” should be interpreted as open-ended terms (i.e., meaning “including but not limited to”). Unless otherwise specified herein, descriptions of value ranges herein are intended only as a shorthand method of individually referring to each individual value falling within the range, and each individual value is incorporated into this specification as if individually described herein. All methods described herein may be performed in any suitable order unless otherwise stated herein or otherwise clearly contradicted by the context. Unless otherwise stated, the use of any and all instances or exemplary language (e.g., “such”) provided herein is intended only to better illustrate the invention and does not constitute a limitation on the scope of the invention. No language in the specification should be construed as indicating that any element not protected by the claims is essential for the practice of the invention.

[0227] This document describes preferred embodiments of the invention, including the best modes known to the inventors for carrying out the invention. Variations of those preferred embodiments will become apparent to those skilled in the art upon reading the foregoing description. The inventors intend that those skilled in the art will adopt these variations where appropriate, and the inventors intend that the invention be practiced in ways different from those specifically described herein. Therefore, the invention includes all modifications and equivalents to the subject matter set forth in the appended claims, where permitted by applicable law. Furthermore, the invention covers any combination of the foregoing elements in all possible variations, unless otherwise stated herein or otherwise clearly contradicted by the context.

Claims

1. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises at least one gene selected from SbcC and SbcD and at least one gene selected from the group consisting of endA, recA and pgi, with gene knockout.

2. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises SbcC and SbcD and at least one gene selected from endA, recA and pgi, with gene knockout.

3. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises gene knockouts of SbcC, SbcD, endA, and recA.

4. An engineered bacterial host cell, wherein the engineered bacterial host cell comprises gene knockouts of SbcC, SbcD, endA, recA, and pgi.

5. The engineered bacterial host cell according to any one of claims 1 to 4, further comprising the sbcB gene, recB gene, recD gene and recJ gene, wherein there is no engineered mutation in any of the sbcB, recB, recD and recJ genes that reduces viability or yield.

6. The engineered bacterial host cell according to any one of claims 1 to 5, further comprising at least one gene selected from the group consisting of uvrC, mcrA and mcrBC-hsd-mrr, wherein the engineered bacterial host cell does not contain any engineered mutations that reduce viability or yield in at least one gene selected from uvrC, mcrA and mcrBC-hsd-mrr.

7. The engineered bacterial host cell according to any one of claims 1 to 6, further comprising a gene selected from fhuA and glnV.

8. The engineered bacterial host cell according to any one of claims 1 to 6, further comprising the fhuA gene and the glnV gene.

9. The engineered bacterial host cell according to any one of claims 1 to 8, further comprising a gene knockout of the dcm gene.

10. The engineered bacterial host cell according to any one of claims 1 to 9, wherein the engineered bacterial host cell does not contain the supE44 gene.

11. The engineered bacterial host cell according to any one of claims 1 to 10, wherein the engineered bacterial host cell is derived from an Escherichia coli (E. coli) cell line.

12. The engineered bacterial host cell of claim 11, wherein the Escherichia coli cell line is selected from the group consisting of: DH5α, DH1, JM107, JM108, JM109, XL1Blue and MG1655.

13. The engineered bacterial host cell of claim 11, wherein the Escherichia coli cell line is MG1655.

14. An engineered bacterial host cell, wherein the engineered bacterial host cell is derived from an Escherichia coli cell line, wherein the Escherichia coli cell line is selected from the group consisting of GalG20, MG1655 and MG1655 ΔendA ΔrecA, and wherein the engineered bacterial host cell comprises a gene knockout of at least one gene selected from the group consisting of SbcC and SbcD.

15. An engineered bacterial host cell, wherein the engineered bacterial host cell is derived from an Escherichia coli cell line, wherein the Escherichia coli cell line is selected from the group consisting of GalG20, MG1655 and MG1655 ΔendA ΔrecA, and wherein the engineered bacterial host cell comprises gene knockout of the SbcC gene and the SbcD gene.

16. The engineered host cell of any one of claims 14 to 15, wherein the engineered bacterial host cell comprises the sbcB gene, recB gene, recD gene and recJ gene, and wherein there is no engineered mutation in any of the sbcB, recB, recD and recJ genes that reduces viability or yield.

17. The engineered bacterial host cell according to any one of claims 1 to 16, wherein the engineered bacterial host cell further comprises a genomic nucleic acid sequence encoding a Rep protein, wherein the Rep protein comprises an amino acid sequence having at least 90% sequence identity with a sequence selected from the group consisting of: SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 34 and SEQ ID NO:

35.

18. The engineered bacterial host cell according to any one of claims 1 to 17, further comprising a genomic nucleic acid sequence encoding a temperature-sensitive λ repressor.

19. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive λ repressor is cITs857.

20. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive λ repressor comprises an amino acid sequence having at least 90% sequence identity with SEQ ID NO:

37.

21. The engineered bacterial host cell of claim 18, wherein the temperature-sensitive λ repressor is a chromosomal integrated copy of the phage φ80 attachment site of the arabinose-inducible CITs857 gene.

22. The engineered bacterial host cell of any one of claims 5 and 16, wherein the sbcB gene comprises a sequence having at least 90% sequence identity with SEQ ID NO: 11, wherein the recB gene comprises a sequence having at least 90% sequence identity with SEQ ID NO: 12, wherein the recD gene comprises a sequence having at least 90% sequence identity with SEQ ID NO: 13, and wherein the recJ gene comprises a sequence having at least 90% sequence identity with SEQ ID NO:

65.

23. The engineered bacterial host cell of any one of claims 1 to 22, further comprising a vector, wherein the vector comprises a nucleic acid sequence having an inverted repeat sequence, an orthogonal repeat sequence, or a palindromic sequence.

24. The engineered bacterial host cell according to any one of claims 1 to 22, further comprising a vector, wherein the vector is an AAV vector, a lentiviral vector, a retroviral vector, or an mRNA vector.

25. The engineered bacterial host cell according to any one of claims 1 to 24, further comprising a plasmid vector.

26. The engineered bacterial host cell of claim 25, wherein the plasmid vector is a pUC-free eukaryotic microcircle expression vector, the pUC-free eukaryotic microcircle expression vector comprising: (i) a eukaryotic region sequence encoding a gene of interest and having 5' and 3' ends; and (ii) a spacer region of less than 1000 base pairs in length, the spacer region connecting the 5' and 3' ends of the eukaryotic region sequence and comprising the R6K bacterial origin of replication and RNA selectivity marker.

27. The engineered bacterial host cell of claim 26, wherein the gene of interest comprises a structured DNA sequence selected from inverted repeat sequences, forward repeat sequences, homopolymeric repeat sequences, eukaryotic origin of replication, polyA repeat sequences, SV40 origin of replication, viral LTR, lentiviral LTR, retroviral LTR, transposon IR / DR repeat sequences, Sleeping Beauty transposon IR / DR repeat sequences, and AAV ITR.

28. A method for improving carrier generation, comprising: Provide engineered bacterial host cells as described in any one of claims 23 to 27; And incubate the engineered bacterial host cell under conditions sufficient to replicate the vector.

29. The method of claim 28, wherein the step of incubating the engineered bacterial host cells under conditions sufficient to replicate the vector is performed by fed-batch fermentation, wherein the fed-batch fermentation includes incubating the engineered bacterial host cells at approximately 25°C during a first portion of the fed-batch phase. o C to approximately 32 o The food is grown at a first temperature of C, and then the temperature is raised to approximately 37°C during the second part of the batch feeding phase. o C to approximately 42 o The second temperature of C.

30. The engineered bacterial host cell of claim 25, wherein the engineered bacterial host cell provides an improved yield of the plasmid vector compared to a reference engineered bacterial host cell that does not contain at least one of endA, recA, and pgi gene knockout, and does not lose the stability and integrity of the plasmid vector compared to the reference engineered bacterial host cell.

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