A self-circularizing non-replicative phage vector (scnrpv) for gene therapy

Abstract

Provided herein are compositions and methods for phage vector for gene therapy. In particular, the present disclosure provides compositions and methods self-circularizing non-replicative phage vector (scNRPV) for targeting the microbiome for treating microbiota-associated diseases.

Description

Client Ref No. 24-0106Attny Docket No. UNC2.43224.601A Self-Circularizing Non-Replicative Phage Vector (scNRPV) for Gene TherapyRELATED APPLICATION INFORMATION

[0001] This application claims priority to U.S. Application No. 63 / 682,847, filed on August 14, 2024, the contents of which are herein incorporated by reference.FIELD OF THE INVENTION

[0002] Provided herein are compositions and methods for phage vectors for gene therapy. In particular, the present disclosure provides compositions and methods of self-circularizing non- replicative phage vectors (scNRPV) for targeting the microbiome for treating microbiota- associated diseases.GOVERNMENT SUPPORT

[0003] This invention was made with government support under Grant Number All 85808 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING STATEMENT

[0004] The contents of the electronic sequence listing titled UNC2_43224_601.xml (Size: 61,440 bytes; and Date of Creation: August 14, 2025) is herein incorporated by reference in its entirety.BACKGROUND

[0005] Human microbiomes are implicated in a multitude of diseases, yet a full mechanistic understanding of their causative relationships and, by extension, precision microbiome-targeted therapies has generally remained elusive- To thoroughly interrogate these relationships and to develop therapies capable of correcting aberrances, tools that can readily manipulate specific bacterial species within complex microbiota ecosystems in situ are needed. Similar to viral vectors that have dominated the human gene therapy landscape for the past 3 decades, bacteriophage (phage) vectors - bacterial viruses engineered to deliver transgenic DNA into target bacteria - possess many promising features for this task.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0006] Unfortunately, current phage vectors suffer from several important drawbacks that have limited their use for in situ microbiome engineering. Phage vectors can be either replicative or non-replicative. Replicative vectors, including many current phage-based vectors, involve substituting non-essential DNA in the phage genome with transgenic DNA, but otherwise retain the key genomic elements necessary for producing viable progeny in infected cells. While selfreplication aids in propagating transgene expression, this class of vector suffers from (i) a limited transgene packing capacity (imposed by the amount of non-essential DNA in the underlying phage genome), and (ii) risks of both target cell lysis and horizontal gene transfer, due to their replication-competent nature. These concerns are eliminated with the use of non-replicative phage vectors (NRPVs) packed exclusively with transgenic DNA, which offer the benefit of a far greater DNA packing capacity, but more importantly, do not pose a risk of horizontal gene transfer and can avoid significant target cell killing. For perspective, all vectors approved or in clinical development for human gene therapy are non-replicative, due to safety concerns from unmitigated vector replication. Unfortunately, the transduction efficiencies of current NRPVs are generally extremely poor.SUMMARY OF THE INVENTION

[0007] Provided herein are compositions and methods for phage vector for gene therapy. In particular, the present disclosure provides compositions and methods self-circularizing non- replicative phage vector (scNRPV) for targeting the microbiome for treating microbiota- associated diseases.

[0008] Provided herein is a bacterial production strain, wherein the production strain does not contain any serT (tRNASer^UGV) and serU (tRNASs^CGA^) tRNA (e.g., meaning there is no expression of serT and serU) and further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site.

[0009] In some embodiments, the production strain does not contain any TCG, TCA, or any TCG and TCA codons in any essential gene.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0010] The vector used in the bacterial production strain is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises at least 1, 2, 3, 4, or 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In still further embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector further comprises at least one origin of replication (ori). In further embodiments, the ori is located between the polynucleotide sequence encoding a first and second att site. In further embodiments, the ori is located outside of the polynucleotide sequence encoding a first and second att site. In some embodiments, the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0011] In some embodiments, the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, Propionibacterium acnes, a Corynebacterium, or any combination thereof. In further embodiments, the bacteria is E. coli. In other embodiments, the production strain is Syn61. In some embodiments, the recombinase is F1P, KD, X-INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0012] In some embodiments, the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCG codons in any essential gene. In further embodiments, there is at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0013] In some embodiments, the helper phage is derived from T7, Pl, X, OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, <pl 1, SEP1, vB_SepS_SEP9, PAD20,Client Ref No. 24-0106Attny Docket No. UNC2.43224.601M102AD, FNU1, Dp-1, Cp-1, HP1, MDA, phi-adh, Lv-1, Lu-1, vB Gva ABl, or any combinations thereof.

[0014] In some embodiments, the transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini-protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0015] Provided herein is an engineered bacteria strain, wherein the engineered bacteria strain does not contain any serT and serU tRNAs and further wherein the production strain comprises a vector encoding a protein, wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the protein.

[0016] In some embodiments, the engineered bacteria strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0017] The vector used is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises at least 1, 2, 3, 4 or 5 TCA, TCG or TCA and TCG codons in the coding sequence of the protein. In some embodiments, the vector comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the protein. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the protein. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the protein. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the protein. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the protein. In still further embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the protein. In some embodiments, the vector further comprises at least one origin of replication (ori). In some embodiments, the vector further comprises a polynucleotide sequence encoding a packing signal (pac) or a conjugative transfer origin (oriT).

[0018] In some embodiments, the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perjringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium, or any combination thereof. In further embodiments, wherein the bacteria is E. coli. In some embodiments, the protein is the HIV-1 env gene, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes including endonucleases and exonucleases, toxin-antitoxin systems, eukaryotic RNase A, Bamase, MazF endoribonuclease, RelE, phage holins or endolysins, and / or ccdB toxin, and the human apoptosis modulator protein Bax.

[0019] Provided herein is a self-circularizing non-replicative phage vector (scNRPV) comprising: a non-replicative phage vector that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene; and a bacterial production strain wherein the production strain does not contain any serT and serU tRNA and further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA or TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site.

[0020] In some embodiments, the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0021] The vector used in the bacterial production strain is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises at least 1, 2, 3, 4, or 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 1TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In still further embodiments, the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector further comprises at least one origin of replication (ori). In further embodiments, theClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 ori is located between the polynucleotide sequence encoding a first and second att site. In further embodiments, the ori is located outside of the polynucleotide sequence encoding a first and second att site. In some embodiments, the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0022] In some embodiments, the bacteria is E. coll, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0023] In some embodiments, the bacteria is E. coli. In some embodiments, the production strain is Syn61.

[0024] In further embodiments, the recombinase is F1P, KD, X -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0025] In some embodiments, the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCG codons in any essential gene. In further embodiments, the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0026] In some embodiments, the helper phage is derived from T7, Pl, , OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, (pl 1, SEP1, vB_SepS_SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDA<D, phi-adh, Lv-1, Lu-1, vB_Gva_ABl, or combinations thereof.

[0027] In some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0028] Provided herein is a self-circularizing non-replicative phage vector (scNRPV) comprising: a vector that encodes a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG, or TCA and TCG codons in a coding sequence of the SSR and furtherClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and further wherein the scNRPV does not contain any TCG or TCA codons in any essential gene.

[0029] In further embodiments, the recombinase is F1P, KD, A, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0030] The vector used in the bacterial production strain is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises at least 1, 2, 3, 4, or 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 1TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In still further embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0031] In some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof. Provided herein is a composition comprising a scNRPV.

[0032] Provided herein is a method of producing a self-circularizing non-replicative phage comprising a nucleic acid or transgene of interest, the method comprising the steps of: infecting a bacterial production strain with a phage (non-replicative or replicative) that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene, wherein the production strain does not contain any serT or serU tRNA, and further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleicClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 acid or transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and still further wherein the vector is incorporated into progeny of the phage producing a selfcircularizing non-replicative phage vector; and recovering the self-circularizing non-replicative phage produced by the bacterial production strain.

[0033] In some embodiments, the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0034] In some embodiments, vector comprises at least 1, 2, 3, 4, or 5 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 1TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In still further embodiments, the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence. In some embodiments, the vector farther comprises at least one origin of replication (ori). In farther embodiments, the ori is located between the polynucleotide sequence encoding a fast and second att site. In farther embodiments, the ori is located outside of the polynucleotide sequence encoding a first and second att site. In some embodiments, the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0035] In some embodiments, the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium, or any combination thereof.

[0036] In some embodiments, the bacteria is E. coli. In some embodiments, the production strain is Syn61.

[0037] In further embodiments, the recombinase is F1P, KD, A, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0038] In some embodiments, the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCG codons in any essential gene. In further embodiments, the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0039] In some embodiments, the helper phage is derived from T7, Pl, , OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, <pl 1, SEP1, vB SepS SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDAO, phi-adh, Lv-1, Lu-1, vB_Gva_ABl, or combinations thereof.

[0040] In some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0041] In other embodiments, the method further comprises purifying the self-circularizing non- replicative phage vector produced. Provided herein is a self-circularizing non-replicative phage vector.

[0042] Provided herein is a method of modifying bacteria. In some aspects, the bacteria to be modified are located in or on a subject, such as a human or non-human animal. In other aspects, the bacteria to be modified are located in an environment, such as in or on a plant or in soil, water, or air.BRIEF DESCRIPTION OF THE DRAWINGS

[0043] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0044] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0045] Figure 1 shows that Phage vector transduction is limited by DNA linearity. Figure 1 A shows a schematic of a phagemid packed into T7 NRPVs (attP / attB, Bxbl recombinaseClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 attachment sites). Figure IB shows phagemid-packed vectors (as determined by ddPCR) plotted against the number of cells transduced by the vectors when co-incubated with target E. coli. Dashed line = line of identity, n = 3. Figure 1C shows double-stranded DNA bacteriophages (phages), and viral vectors derived from them, deliver DNA to the cytosol of target bacteria as a linear molecule. In this form, transduced DNA is prone to degradation and cannot replicate. Recombination can convert transduced DNA from linear to circular, protecting it from degradation and allowing it to replicate. Figure ID shows the number of E. coli cells stably transduced (i.e. that both received and maintained vector DNA) by T7 vectors packed with the phagemid DNA described in Figure 1 A (wt, wild-type E. coli DH5a; HR, DH5a expressing RED proteins; SSR, DH5a expressing the Bxbl site specific recombinase (SSR). One-way ANOVA with Tukey’s post hoc test, n = 3, ***p<0.0005, DF = 6. Figure IE shows the percent of total target cells transduced by T7 NRPVs. One-way ANOVA with Tukey’s post hoc test, n = 3, ****p<0.00005, DF = 6. Figure IF shows the amount of transduced target cells as a percentage of the number of phagemid-packed NRPVs dosed. One-way ANOVA with Tukey’s post hoc test, n = 3, ***p<0.0005, DF = 6. (G) Fluorescence intensity of target cell populations (under no selective pressure) transduced by T7 NRPVs as a function of time, n=3.

[0046] Figure 2 shows the production of self-circularizing phage vectors requires stringent recombinase repression. Figure 2A shows “self-circularizing” non-replicative phage vectors (scNRPVs), packed with transgenic DNA that encodes a site-specific recombinase (SSR) transcriptional unit and the attachment sites (attP / attB) that the SSR acts on in cis, deliver this DNA cargo to target cells, where it undergoes recombination-mediated self-circularization. Figure 2B shows that to produce T7 scNRPVs, a strain of E. coli — containing the transgenic vector cargo cassette encoding in a plasmid and a helper plasmid, encoding the T7 tail fiber gene (gpl7) — is infected by a gpl7 knockout T7 variant. The recombination reaction must be prevented in this strain. Figure 2C shows schematics of a NanoLuc transcriptional units with elements used to repress to repress expression compared to tRNA deficiency-based repression. Tet, tetR-repressible operator site; cin, cinR-repressible operator site; lac, laci-repressible operator site; yiU, a terminator-forming theophylline-inducible riboswitch; RBS, ribosome binding site;HHAz, a theophylline-inducible hammerhead ribozyme; aTc, anhydrotetracycline (inhibits tetR repression of tetO); HSL, JV-(3-Hydroxytetradecanoyl)-DL-homoserine lactone (inhibits cinR repression of cinO); IPTG, Isopropyl B-D-l -thiogalactopyranoside (inhibits laclClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 repression of lacO). Figure 2D shows luminescence levels of E. coli strains containing variants of the plasmid in (C), n = 2. Figure 2E shows the percent of phagemid molecules purified from the total production strain population that underwent recombination (att, attachment site presence in the phagemid), n = 3. Figure 2F shows the dynamic range of luminescence for each construct in Figure 2D, n =2.

[0047] Figure 3 shows recoded T7 phage infect a tRNA-deficient vector production E. coli strain. tRNA-codon decoding maps for T7 in Figure 3A wild-type (wt) E. coli and (Figure 3B) Syn61A3. serU and serT deletion in Syn61A3prevents expression of essential genes that contain TCG and TCA codons (highlighted in red) in the T7 genome. Figure 3C shows a genome map of a T7 phage variant recoded to remove TCA and TCG codons from the genome (rT7). The relative abundance of all serine codons in (Figure 3D) wild-type (wt) T7 and (Figure 3E) recoded T7. Figure 3F shows plaquing efficiency in plaque forming units per mL (PFU / mL) of replication competent wt T7 and rT7 lysates on E. coli, n = 2. Figure 3G shows target cell lysis, as measured by ODeoo, of E. coli incubated with wt T7 or rT7, n = 2.

[0048] Figure 4 shows self-circularizing phage vectors enhancing transduction efficiency in vitro. Figure 4A shows schematics of plasmids used to make T7 phage vectors. These vectors were used to transduce E. coli that either do (SSR+) or do not (SSR-) express the Bxbl site-specific recombinase. Figure 4B shows titers of vectors described in (A). Figure 4C shows the transgene to genome (T:G) packing ratio of the vectors described in Figure 4A. One-way ANOVA with Tukey’s post hoc test, n = 3, **p<0.005, DF = 12. Figure 4D shows the number of E. coli cells stably transduced at an MOI of 0.1 by T7 vectors packed with the DNA described in Figure 4A. Two-way ANOVA with Tukey’s post hoc test, n = 3, ***p<0.0005, DF = 12. Figure 4E shows the percent of target cells that were transduced at an MOI of 0.1. Two-way ANOVA with Tukey’s post hoc test, n = 3, DF = 12. Figure 4F shows target cell transduction as a percent of phage vectors dosed at an MOI of 0.1. Two-way ANOVA with Tukey’s post hoc test, n = 3, ***p<0.0005, DF = 12.

[0049] Figure 5 shows self-circularizing phage vectors efficiently transduce target bacteria in the lumen of the mammalian GI tract. Figure 5 A shows target E. coli are transformed with a plasmid conferring ampicillin resistance (AmpR). Figure 5B shows T7-NRPVs are packed with a phagemid encoding: (i) the T7 packing signal (pac), (ii) Bxbl attP and attB sites flankingClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 constitutive transgene expression cassettes encoding (iii) mScarlet and (iv) aminoglycoside 3'- phosphotransferase (KanR), and (v) an origin of replication. T7-scNRPVs are packed with the same phagmid, where the mScarlet coding sequences has been replaced with that of the Bxbl site-specific recombinase (SSR). Figure 5C shows C57BL / 6 mice were administered an antibiotic cocktail for 3 days to deplete native gut microbiota; mice were then colonized with the target E. coli described in Figure 5A, mice are then kept on drinking water containing ampicillin to maintain colonization; following target cell colonization, mice were dosed with 2x108T7- NRPVs or T7-scNRPVs described in Figure 5B on day 0 and day 1. Feces was collected and plated throughout the study to determine concentrations of both the target cells and cells transduced by the T7-NRPVs. Figure 5D shows ampicillin resistant target bacterial concentrations in feces. Figure 5E shows kanamycin resistant transduced target bacterial concentrations in feces.

[0050] Figure 6 shows phagemid recircularization increases total transgene expression. E. coli expressing i) nothing (cells only and wt), ii) A-RED proteins (HR), or iii) the Bxbl site-specific recombinase (SSR) were co-incubated with T7-NRPVs packed with the phagemid described in Figure 6 A (cells only were co-incubated with a buffer only control). Fluorescence was then measured as a function of time and the area under the curve (AUC) of each group was calculated to determine total transgene (mScarlet) expression levels from the transductants. **** indicates a p-value <0.0005 as determined by one-way ANOVA with Tukey’s post-hoc test, n = 3, DF =8.

[0051] Figure 7 shows nanoluciferase reporter plasmid schematics. Schematics diagrams of plasmids encoding for Nanoluciferase (NanoLuc) expression, repressed by varied repression schemes. Bent arrow - promoter; hemisphere - ribosome binding site; open box - tet operator sequence (tetO), binding site for the tet repressor protein (tetR); inverted triangle - cin operator sequence (cinO), binding site for the cin repressor protein (cinR); white tree - theophylline- inducible hammerhead aptazyme (HHAz), encodes self-cleaving mRNA secondary structure in the presence of theophylline; black tree - theophylline-inducible terminator-forming RNA aptamer sequence (yit J), forms a strong RNA hairpin terminator in the presence of theophylline; U-shape - lac operator sequence (lacO), binding site for the lac repressor protein (lacl).

[0052] Figure 8 shows recoded T7 exhibits slightly smaller plaque morphology in plaque formation assays. Representative plates where wild type (wt) T7 and recoded T7 (rT7) viralClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 lysates were serially diluted and spotted onto lawns of either BW25113, Syn61, or Syn61A3E. coli.

[0053] Figure 9 shows an illustration of T7 NRPV production. A T7 helper phage is produced by knocking out the T7 tail fiber gene (gpl7), rendering progeny from this phage incapable of infection if not propagated on a strain supplying a complementary form of the tail fiber. These helper phage are then used to infect an NRPV production strain, containing i) a helper plasmid encoding gpl7, and ii) an NRPV cargo phagemid. The resulting NRPV progeny are a mixed population of T7 particles packed with either the cargo phagemid (T7-NRPVs) or the replication incompetent helper phage genome.

[0054] Figure 10 shows T7-scNRPV Transduction at MOI = 1. T7-NRPVs (mScarlet | att+and SSR | att ) and T7-scNRPV (SSR | att+) were produced, packing the phagemids indicated in Fig 4A. These vectors were then used to transduced either: IxlO8wt E. coli cells (SSR ) or IxlO8E. coli cells engineered to constitutively express the SSR (SSR+) at a multiplicity of infection (MOI) of 1 phagemid-packed vector per target cell. The percent of target cells transduced by the vectors and able to stably maintain the transduced DNA is shown above. Two-way ANOVA with Tukey’s post hoc test, n = 3, ***p<0.0005, DF = 12.

[0055] Figure 11 shows recircularization of att+phagemids delivered, in vivo, to SSR+target E. coli by T7 NRPVs mediates high-level in situ transduction. Figure 11 A shows target E. coli are transformed with a plasmid conferring ampicillin resistance (AmpR) and encoded the Bxbl sitespecific recombinase (SSR). Figure 1 IB shows T7 -NRPVs are packed with a phagemid encoding: (i) the T7 packing signal (pac), (ii) Bxbl attP and attB sites flanking constitutive transgene expression cassettes encoding (iii) mScarlet and (iv) aminoglycoside 3'- phosphotransferase (KanR), and (v) an origin of replication. Figure 11C shows two independent studies were carried out where C57BL / 6 mice were administered an antibiotic cocktail for 3 days to deplete native gut microbiota; mice were then colonized with the target E. coli described in Figure 11 A, mice are then kept on drinking water containing ampicillin to maintain colonization; following target cell colonization, mice were dosed with 2x108T7 -NRPVs described in Figure 1 IB. Feces was collected and plated throughout the study to determine concentrations of both the target cells and cells transduced by the T7 -NRPVs. The first study was conducted for 8 days post vector dosing. The second study was conducted for 20 days post vector dosing. Figure 1 IDClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 shows ampicillin resistant target bacterial concentrations in feces for the 8 day arm. Figure 1 IE shows ampicillin resistant target bacterial concentrations in feces for the 20 day arm. Figure 1 IF shows kanamycin resistant transduced target bacterial concentrations in feces in the 8 day arm. Figure 11G shows kanamycin resistant transduced target bacterial concentrations in feces in the 20 day arm.

[0056] Figure 12 shows serU (tRNASer(CGA)) decodes TCA codons. The tRNA-codon interaction map of (Figure 12 A) wild-type E. coli (orange line represents the proposed serU decoding of TCA described here), (Figure 12B) Syn61AT, with serT (encodes tRNASer(UGA)) knocked out, (Figure 12C) Syn61AU, with serU (encodes tRNASer(CGA)) knocked out, and (Figure 12D) Syn61A3, with both serT and serU knocked out. In all Syn61 strains, every TCA and TCG codon has been removed from the genome via synonymous mutations, indicated by the lack of codon boxes. Figure 12E shows a plasmid encoding a kanamycin resistance (KanR) gene is transformed into different Syn61 tRNA knockout variants (red bars represent the presence of TCA codons in the KanR coding sequence). Given the widely reported interaction map in (A), transformation of this plasmid into Syn61 AT and Syn61 A3 is expected to not yield transformants. Figure 12F shows a mean + standard deviation of colony forming units (transformants) resulting from this transformation reaction (n=3). Figure 12G shows a representative images of the transformant plates quantified in (F). Syn61A3 containing (H) helper plasmids that encode wild-type serU (with anticodon CGA) or a form of serU where the anticodon has been mutated to that of serV (GCU) were transformed with the same KanR plasmids as in Figures 12F and 12G. Figure 12(1) shows a mean + standard deviation of transformants resulting from this transformation reaction (n=3).

[0057] Figure 13 shows compressed codons mediate repression in select GROs only if sufficient codons are present. Figure 13A shows a mean logio luminescence intensity across a panel of Syn61 tRNA knockout variants (y-axis) transformed with a nanoluciferase gene containing varying numbers of TCA and TCG codons in the coding sequence (x-axis) (n=2). Figure 13B provides an overview of the recombination assay. A panel of plasmids encoding i) the Bxbl sitespecific recombinase with varying numbers of TCA and TCG codons in the coding sequence, and ii) the Bxbl attP and attB sites, which are oriented such that their recombination by the SSR (which forms an attL site) excises ~800 bp from the plasmid, is transformed into Syn61 tRNA knockout variants. Plasmid DNA is purified from the entire population of transformants andClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 sequenced by longread nanopore sequencing. The reads are then analyzed for signatures of recombination and the percent of sequenced plasmid molecules that have undergone recombination is calculated. Figure 13C shows the mean ± std. dev. of percent recombination from the protocol described by Figure 13B (n=2).

[0058] Figure 14 shows SSR-mediated circularization of DNA delivered by Pl -derived phage vectors increases transduction. Figure 14A shows Pl phage vectors were made by transforming an E. coli strain containing the Pl episome (ATCC: BAA- 1001) with a phagemid encoding i) the Pl packing signal and ii) Bxbl SSR attP and attB sites flanking, iii) an mScarlet and KanR reporter cassette and iv) an origin of replication. Upon heat induction of this strain, Pl phage vectors were produced and used to transduce either i) wild-type E. coli BW25113 harboring no plasmid (SSR-) or ii) E. coli BW25113 harboring a plasmid that encodes for constitutive expression of the Bxbl SSR (SSR+). Figure 14B shows he total number of KanR / mScarlet+ cells (transductants) after co-incubation with the phage vectors. After co-incubating the SSR- and SSR+ cells with equal amounts of the Pl vectors (as determined by ddPCR), cells were washed, serially diluted and plated on LB plates supplemented with kanamycin. Figure 14C shows the number of transduced cells as a percentage of Pl vectors added to the transduction co-incubation (the effective transduction efficiency). Statistical analysis: unpaired two-tailed t-test, n = 3 independent biological replicates, ** = p < 0.01.

[0059] Figure 15 provides the sequences of the DNA fragments described in Example 3.DETAILED DESCRIPTION

[0060] While the bacteria that live on and in our bodies play a pivotal role in human health, mechanistic insights into microbiome-induced pathogenesis and methods that can harness the microbiome to improve health with molecular specificity remain limited. Indeed, current therapies targeting the bacterial cells that make up our microbiota are limited to clearance by broad-spectrum antibiotics, or other antimicrobials (including lytic phages), and more recently by ‘wholesale’ replacement via fecal transplantation. Tools that can safely and controllably probe and alter select members of the native microbial communities are sorely needed, in much the same way that engineered systems delivering nucleic acids to specific cells in our body have been harnessed to reinforce the immune system or correct for the underlying root causes of genetic diseases.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0061] An essential hallmark of all approved engineered systems to deliver genetic information (RNA, mRNA, DNA) to human cells is either the complete inability for the systems to replicate, such as viral vectors for gene therapy, or, at a minimum, a greatly diminished capacity to replicate (such as attenuated vaccines). This led to the development of NRPVs that can achieve high transduction efficiencies in vivo, as described herein. Described herein is how poor transduction efficiency of conventional NRPVs is directly attributable to the linear topology of the DNA they deliver. As demonstrated herein, this bottleneck is effectively overcome by enabling self-circularization of the transduced DNA by encoding for the expression of specific recombinases in cis. Indeed, it was found that T7-scNRPV transduced target bacteria with efficiencies that are orders of magnitude greater than prior reported NRPVs, and approach efficiencies previously reported for replicative phages. Two low doses of the T7-scNRPV were sufficient to achieve high levels of transduced bacteria in the mammalian gut for many days. Coupled with their large transgene packing capacities (equal to the size of the underlying phage genome), the lack of risks of horizontal gene transfer and the limited target toxicity, scNRPVs represent an ideal platform for precision microbiome engineering.

[0062] The results provided herein present a generalizable approach for engineering other scNRPVs beyond T7, including phage vectors derived from other lytic phages, as well as lysogenic phages. Phages are the most abundant organisms on earth, and tailed phages with double-stranded DNA (dsDNA) genomes comprise more than 95% of known or putative phages. To date, the evidence suggests the vast majority of, if not all, dsDNA phages — and consequently the vectors derived from them — pack their capsids with linear DNA during progeny formation. Thus, NRPVs derived from nearly all known phages transduce target bacteria with linear DNA, facing the same challenges in generating stable transductants (i.e. target bacterial cells that have been transduced by phage vectors and can maintain the DNA delivered to them). By converting linear dsDNA into circularized plasmid DNA, this DNA can be protected from degradation and take advantage of existing theta, rolling circle, or strand displacement replication, which drive the replication of nearly all plasmids, especially those with broad host ranges. Additionally, by utilizing select SSRs, which require no host factors and mediate att site recombination with exceptional efficiency, scNRPVs can function in a wide range of target prokaryotes.Client Ref No. 24-0106Attny Docket No. UNC2.43224.6011. DEFINITIONS

[0063] The terms "comprise(s)," "include(s)," "having," "has," "can," "contain(s)," and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms "a," "and", and "the" include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments "comprising," "consisting of," and "consisting essentially of," the embodiments or elements presented herein, whether explicitly set forth or not.

[0064] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 69, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0065] The term "about" is utilized herein to specify precise numerical support for the value it precedes, as well as to encompass numbers in close proximity to or approximating the specified value. Certain ranges are delineated in this document with numerical values preceded by the term "about." When determining if a number is near to or approximates a specifically stated value, the unrecited number that closely approximates the stated value, considering the context in which it is presented, may be deemed substantially equivalent to the specified number.

[0066] Bacterial production strain A bacterial production strain refers to a specific type or strain of bacteria that has been genetically engineered, selected, or optimized to efficiently produce a desired product or compound. This could include various substances such as proteins, enzymes, antibiotics, biofuels, organic acids, or other biotechnologically valuable molecules. These strainsClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 are typically engineered through genetic manipulation or selected based on natural characteristics that enhance their productivity or yield of the target product. Bacterial production strains are crucial in biotechnology and industrial processes where microbial factories are used to synthesize valuable substances economically and sustainably.

[0067] As used herein, the terms “Colitis” and “Ulcerative colitis” refers to a chronic inflammatory disease characterized by diffuse mucosal inflammation of the colon. In some embodiments, the disease is characterized by amongst other features bloody diarrhea, often with symptoms of rectal urgency and tenesmus. In some embodiments, the terms “Colitis” and “Ulcerative colitis used herein includes diverticulitis, pouchitis, proctitis, mucositis, diversion colitis, ischemic colitis, infectious colitis, chemical colitis, radiation-induced colitis, microscopic colitis (including collagenous colitis and lymphocytic colitis), atypical colitis, pseudomembraneous colitis, fulminant colitis, autistic enterocolitis, interdeminate colitis, Behcet's disease, jejunoiletis, ileitis, ileocolitis and granulomatous colitis. The invention contemplates the use of the composition as described herein for the treatment of any of such condition. Also contemplated is use of the composition for the uses described herein for use in the treatment of colitis associated with inflammatory diseases of the gastrointestinal tract, particularly colitis associated with inflammatory diseases affecting mainly the colon; for example colitis associated with primary sclerosing cholangitis or radiation.

[0068] As used herein, the term "essential genes" refers to genes that are indispensable for the growth and division of bacteria when cultured in a nutritionally complete medium. These genes are critical for basic cellular functions, and their absence results in an inability of the bacteria to survive and propagate or replicate under standard laboratory conditions. In some embodiments, essential genes are fundamental to essential metabolic pathways, cell wall synthesis, DNA replication, and other vital cellular processes.

[0069] As used herein, the term "non-essential genes," as used herein, refer to genes whose absence does not impede the growth or division of bacteria when cultured in a nutritionally complete medium. These genes are not required for basic cellular functions under standard laboratory conditions and may include genes involved in accessory functions, environmental adaptation, or secondary metabolic pathways. In some embodiments, non-essential genesClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 indicate that their presence or absence does not critically affect bacterial viability or growth rate under normal culture conditions.

[0070] As used herein, the terms “nucleic acid,” “polynucleotide,” or “oligonucleotide” refer a polymer composed of nucleotides or nucleosides (including ribonucleotides, deoxyribonucleotides, or analogs or modified versions thereof), which have nitrogenous heterocyclic bases or base analogs linked together along a backbone. Oligonucleotides may be isolated from genes, or chemically synthesized by methods known in the art. A nucleic acid “backbone” may be made up of a variety of linkages, including one or more of sugarphosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCT No. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of nucleic acid may be ribose, deoxyribose, or similar compounds with optional substitutions, e.g., methoxy or 2' halide substitutions. Oligonucleotides may be DNA (deoxyribonucleic acid), RNA (ribonucleic acid), or a hybrid. Oligonucleotides may be singlestranded, double-stranded, or multi-stranded. Oligonucleotides may be of a variety of different lengths, depending on the form. Oligonucleotides often range in size from a few monomeric units, e.g. 3-4, to hundreds of monomeric units. Whenever an oligonucleotide is represented by a sequence of letters, such as “ATGCCTG,” it will be understood that the nucleotides are in 5 >3 ' order from left to right and that “A” denotes adenosine, “C” denotes cytosine, “G” denotes guanosine, and “T” denotes thymidine, unless otherwise noted. The letters A, C, G, and T may be used to refer to the bases themselves, to nucleosides, or to nucleotides comprising the bases, as is standard in the art.

[0071]

[0061] The term “DNA (deoxyribonucleic acid)” or “DNA molecule” refers to a chain of nucleotides comprising deoxyribonucleotides that each comprise one of four nucleobases, namely, adenine (A), thymine (T), cytosine (C), and guanine (G). The term “RNA (ribonucleic acid)” or “RNA molecule” refers to a chain of nucleotides comprising four types of ribonucleotides that each comprise one of four nucleobases, namely; A, uracil (U), G, and C. Certain pairs of nucleotides specifically bind to one another in a complementary fashion (called complementary base pairing). In DNA, adenine (A) pairs with thymine (T) and cytosine (C) pairs with guanine (G). In RNA, adenine (A) pairs with uracil (U) and cytosine (C) pairs with guanine (G).Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0072] As used herein, the term "helper phage" refers to a type of bacteriophage (a virus that infects bacteria) that aids in the replication of a recombinant phage vector during the production of recombinant DNA molecules. Helper phages provide essential functions, such as packaging and amplification of the recombinant phage DNA, which is crucial for generating high yields of the recombinant DNA in bacterial host cells. Examples of a helper phase include those derived from T7, Pl, A,, OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, <pl 1 , SEP1, vB_SepS_SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDA , phi-adh, Lv-1, Lu- 1, vB Gva ABl, or combinations thereof.

[0073] As used herein, the term "helper plasmid" refers to a plasmid that serves a critical role in molecular biology experiments. It contains essential genetic elements that assist in the replication, maintenance, or expression of recombinant DNA molecules. Helper plasmids are often used in conjunction with other expression vectors or cloning vectors to enhance the efficiency of gene cloning, protein expression, or other genetic manipulations in bacteria or other host organisms.

[0074] As used herein, the term "origin of replication (ori)" refers to a specific DNA sequence where DNA replication is initiated. In some embodiments, the ori can be a DNA replication processes, serving as the starting point for the assembly of replication machinery and the unwinding of DNA strands. In some embodiments, the ori sequence is recognized by proteins that initiate replication, including helicases and DNA polymerases, which unwind the DNA double helix and synthesize new DNA strands, respectively. In some embodiments, in prokaryotic cells, such as bacteria, the ori can be a relatively short sequence of DNA where replication begins bidirectionally, leading to the formation of two replication forks moving in opposite directions. This process can be essential for duplicating the bacterial chromosome and ensuring the accurate transmission of genetic information to daughter cells during cell division. In some embodiments, in eukaryotic cells, ori are more complex and distributed throughout the genome. This process can also involve specific sequences and regulatory proteins that coordinate replication initiation at multiple sites to ensure efficient and accurate DNA duplication during cell division.

[0075] As used herein, the term "packing signal (pac)” refers to a specific DNA sequence found in bacteriophages (viruses that infect bacteria). The packing signal, also known as pac sequence,Client Ref No. 24-0106Attny Docket No. UNC2.43224.601 is essential for the packaging of viral DNA into pre-formed capsids during the assembly of new viral particles. This sequence can be recognized by viral packaging proteins, initiating the encapsulation of the viral genome into the protein coat (capsid). The presence of the packing signal ensures that each viral particle contains a complete and functional copy of the viral genome, crucial for the virus to infect new host cells and propagate efficiently. Thus, the packing signal can play a critical role in the assembly and replication of bacteriophages.

[0076] As used herein, the term "recombinase" refers to an enzyme that catalyzes the recombination of DNA sequences by facilitating the cutting, exchanging, and rejoining of DNA strands. In some embodiments, recombinases can play a crucial role in genetic recombination processes, including homologous recombination, site-specific recombination, and transposition. In some embodiments, recombinases can be essential for various cellular processes such as DNA repair, genetic diversity, and the regulation of gene expression. Recombinases can be utilized in molecular biology and genetic engineering to manipulate DNA sequences, create gene knockouts, integrate foreign DNA into genomes, and perform site-specific modifications in organisms.

[0077] As used herein, the term "site-specific recombinase (SSR) expression" refers to the process by which a specific enzyme, known as a site-specific recombinase, is produced or activated within a biological system. Site-specific recombinases are enzymes capable of recognizing and binding to specific DNA sequences, termed recombination sites, and catalyzing the rearrangement of DNA between these sites. This enzymatic activity can allow for precise manipulation of genetic material at defined loci in the genome. In some embodiments, the SSR expression can involve the transcription and subsequent translation of the gene encoding the sitespecific recombinase within a cell or organism. Upon expression, the recombinase protein is produced and becomes active, facilitating DNA recombination events at its target sites. In some embodiments, the SSR expression can be crucial in genetic engineering, molecular biology research, and biotechnological applications where controlled DNA rearrangement is desired to achieve specific genetic modifications or gene expression control.

[0078] As used herein, the terms "subject" and "patient" are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms "subject" and "subjects" may refer to any vertebrate, including, but not limited to, aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 mammal (e.g., cow, pig, camel, buffalo, elk, deer, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus or rhesus monkey, chimpanzee, etc.) and a human).

[0079] “Treat,” “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a disease and / or injury, or one or more symptoms of such disease, to which such term applies. Depending on the condition of the subject, the term also refers to preventing a disease, and includes preventing the onset of a disease, or preventing the symptoms associated with a disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. Such prevention or reduction of the severity of a disease prior to affliction refers to administration of a pharmaceutical composition to a subject that is not at the time of administration afflicted with the disease. "Preventing" also refers to preventing the recurrence of a disease or of one or more symptoms associated with such disease. "Treatment" and "therapeutically," refer to the act of treating, as "treating" is defined above.

[0080] As used herein, an “unstable polynucleotide sequence” refers to a polynucleotide, which, when maintained episomally in a vector in a serU / serT-competent bacterial host under non- selective growth (e.g., LB, 37 °C), exhibits a propensity for loss, excision, inversion, rearrangement, or disabling mutation that is measurably greater than a control sequence lacking instability determinants. In certain embodiments, instability is evidenced by >10% of plasmid molecules (population level) undergoing a structural change of >20 bp within <20 generations, as quantified by (i) junction-specific ddPCR or qPCR (e.g., attP / attB vs. attL / attR), (ii) long-read sequencing of purified vector populations, or (iii) loss-of-function of a marker cassette flanked by recombination sites. In some embodiments, "instability determinants" — non-limiting, structurally defined features that confer instability — include a site-specific recombinase (SSR) coding sequence in the same molecule as its cognate recombination sites flanking a segment, wherein basal SSR expression catalyzes excision, inversion, or integration (e.g., Bxbl attP / attB; Cre / lox; Flp / FRT; -Int attP / attB; Gin / gix; Tn3 / res); direct or inverted repeats >20 bp separated by <1 kb; palindromes >15 bp with >60% GC predicted to form hairpins; tandem repeats or microsatellites (>5 units); transposons, insertion sequences, or clustered endonuclease sites that nick or cleave the molecule; self-targeting restriction sites; genes whose expression reduces hostClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 growth or viability (e.g., genes encoding toxins or nucleases such as Bamase, MazF endoribonuclease, RelE, or phage holins or endolysins), thereby driving rapid structural change of the polynucleotide sequence or vector loss unless expression is repressed. In certain embodiments, the same sequence that is “unstable” in a serU / serT-competent host is stably propagated (e.g., <5% recombination over 24 h) in a production strain lacking serU and serT due to translational repression of the SSR or other burdening ORFs by enrichment of TCG / TCA codons in those ORFs. By way of example, Figures 2 and 13 demonstrate that enriching SSRs for TCG / TCA codons and producing in serU / serT-deficient cells prevents premature recombination, enabling stable maintenance of cassettes that are otherwise unstable in standard hosts.2. BACTERIAL PRODUCTION STRAIN

[0081] In one embodiment, the present disclosure relates to a bacterial production strain (also referred to herein as an “engineered bacterial strain”), and methods of producing a bacterial strain. The bacterial production strain or engineered bacterial strain described herein does not contain any serT, serU or any serT and serU tRNA and comprises at least one vector or plasmid. The at least one vector or plasmid: (a) encodes a site-specific recombinase (SSR); (b) comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR; and (c) comprises in the 5’ to 3’ direction: (i) a nucleic acid sequence encoding a first attachment (att) site; (ii) one or more nucleic acids or transgenes of interest (which may encode one or more proteins of interest); and (iii) a nucleic acid nucleic acid sequence encoding a second att site.

[0082] In some embodiments, the bacterial production strain exhibits no expression of serT, serU or serT and serU tRNA. In some embodiments, the bacterial production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene. In some embodiments, the essential genes are genes that may confer either a significant survival disadvantage or a slowdown in their replication kinetics.

[0083] The vector or plasmid used in the bacterial production strain is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises 1 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 50 TCA, TCG, or TCA and TCG codonsClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 12 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0084] In some embodiments, the vector comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 6 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 7 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 8 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 9 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 10 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 11 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 12 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 13 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 14 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 15 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 16 TCA, TCGClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 17 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 18 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 19 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 20 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 21 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 22 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 23 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 24 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 25 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0085] In some embodiments, the vector or plasmid further comprises at least one origin of replication (ori). In some embodiments, the ori is located between the nucleic acid sequence encoding a first and second att site. In further embodiments, the ori is located outside of the nucleic acid sequence encoding a first and second att site. In some embodiments, the vector further comprises a nucleic acid sequence encoding a packing signal (pac).

[0086] In still further embodiments, the vector or plasmid contains one or more nucleic acids or transgenes of interest. In some embodiments, the one or more nucleic acids or transgenes of interest encode one or more proteins of interest. In some embodiments, the one or more proteins of interest may not be expressed in the bacterial production strain. In some embodiments, the one or more proteins of interest may affect bacterial cell growth kinetics or viability.

[0087] The one or more nucleic acids or transgenes of interest included in the vector are not critical. For example, in some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short-chain fatty acid, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronicclient Ref No. 24-0106Attny Docket No. UNC2.43224.601 acid, or any combination thereof. In other embodiments, the nucleic acid or transgene of interest encodes one or more toxins, such as, for example, Pseudomonas exotoxin A, cholera toxin, diptheria toxin, E. coli toxins, botulinum toxins, anthrax toxins, pertussin toxins, shiga toxins, ricin, tetanus toxins, Staphylococcal toxins, toxin-antitoxin systems, cedB toxins, or any combinations thereof. In other embodiments, the nucleic acid or transgene of interest encodes a toxin gene or a toxin protein that can be used to limit or prevent the spread of the nucleic acid or transgene of interest to another organism (e.g., an unintended organism, such as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a nontarget animal species), such that expression of the toxin (e.g. Bamase, Colicin E3, Gelonin, Ricin Chain A) in the unintended organism reduces viability, growth, or function of that organism, thereby restricting the persistence or transfer of the genetic material outside of desired target contexts.

[0088] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more toxic nucleic acid sequences. Examples of one or more toxic nucleic acid sequences are: HIV-1 env protein, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes including endonucleases and exonucleases, and / or toxin-antitoxin systems. In some aspects, when the vector or plasmid contains multiple nucleic acids or transgenes of interest (e.g., a first nucleic acid or transgene of interest and a second nucleic acid or transgene of interest), the use of one or more nucleic acids or transgenes that encode one or more toxic nucleic acid sequences can be used to prevent the spread of one or more other nucleic acids or transgenes of interest into an untended organism (e.g., as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a non-target animal species).

[0089] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more unstable polynucleotide sequences. Examples of unstable polynucleotide sequences include: (i) a Bxbl SSR coding sequence bearing >1 codons selected from TCG / TCA and, in cis, Bxbl attP and attB sites flanking a >20 bp segment containing (optionally) an ori and / or pac; (ii) Cre or Flp coding sequences with lox or FRT sites in cis; (iii) recombinase / invertase systems Gin / gix or Tn3 / res; (iv) plasmids containing >2 inverted repeats of >25 bp within 1 kb (hairpinClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 prone); and / or (v) coding sequences for growth-inhibitory proteins (e.g., Bamase, MazF endoribonuclease, RelE, phage holins or endolysins).

[0090] In some further aspects, the one or more nucleic acids or transgenes of interest can incorporate or include one or more TCA, TCG, or TCA and TCG codons.

[0091] In further aspects, one or more nucleic acid sequences or transgenes of interest are contained within the vector or plasmid, or are integrated into the genome of the vector or plasmid using techniques known in the art.

[0092] In some embodiments, the vector or plasmid used in the bacterial production system is non-replicative. In other embodiments, the vector or plasmid used in the bacterial production system is self-replicating.

[0093] The vector or plasmid enriched with one or more TCA, TCG, or TCA and TCG codons can be delivered or transferred to one or more bacterial cells to create the bacterial production strain using any techniques known in the art. As mentioned previously, the bacterial cells should be derived from a bacterial strain that is deficient in one or more tRNA genes such as serU and / or serT. In some aspects, the one or more tRNA genes are translationally repressed in the bacterial strain. In some aspects, the one or more translationally repressed tRNA genes are not required for mobilization or transfer of the vector to a recipient bacterial cells or strain.

[0094] In some embodiments, the vector or plasmid contains a conjugation system capable or suitable for use in transferring the vector or plasmid to one or more recipient bacterial cells.

[0095] In some embodiments, the bacteria used in the bacterial production system is E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In some embodiments, the bacteria are E. coli. In some embodiments, the production strain is Syn61. In some embodiments, the recombinase is Flp, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0096] In some embodiments, the bacterial production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCGClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 codons in any essential gene. In some embodiments, the helper phage exists in the bacterial production strain temporarily or transiently. In still other embodiments, the helper phage is prepared separately from the bacterial production strain. In still other embodiments, the bacterial production strain is infected with at least one helper phage. In still other embodiments, the helper phage is introduced into the bacterial production strain as an episome. In still another embodiment, the helper phage is integrated into a chromosome of the bacterial production strain using routine techniques known in the art. In some embodiments, the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0097] In some embodiments, the helper phage is derived from T7, Pl, , OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, (pl 1, SEP1, vB_SepS_SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDA , phi-adh, Lv-1, Lu-1, vB_Gva_ABl, or any combination thereof.

[0098] The bacterial production strain or engineered bacterial strain, which lacks serT and serU tRNA and contains the vector or plasmid described herein, can be used directly as a delivery vehicle. Specifically, the engineered bacterial strain can be used to directly transfer nucleic acids, directly to one or more recipient bacterial cells using routine techniques known in the art. Specifically, the engineered bacterial strain (also referred to as a “donor strain”), can be used to deliver nucleic acids encoded by the vector to one or more recipient bacterial cells using bacterial conjugation. In some aspects, the vector or plasmid, encodes one or more polypeptides or proteins of interest that contain one or more TCA, TCG, or TCA and TCG codons, which greatly reduces their expression in the donor strain. In some aspects, the donor strain comprises a conjugation system capable of transferring the vector or plasmid to one or more recipient bacterial cells. Examples of bacterial conjugation systems that can be used include the F plasmid transfer system, the RK2 / RP4 broad-host-range conjugation system, the IncP-type conjugation system, the R388 plasmid transfer system, the pKMIOl or POX38 derivatives, or any combinations thereof. The recipient bacterial cells used to receive the vector or plasmid from the production strain are not critical. In some aspects, recipient bacterial cells are from E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In other aspects, the recipient bacterial cells are part of a microbial community, such as in a host organism or within an environmental microbiome.3. A SELF-CIRCULARIZING NON-REPLICATIVE PHAGE VECTOR (scNRPV) PRODUCTION SYSTEM

[0099] In another embodiment, the present disclosure relates to a self-circularizing non- replicative phage vector (scNRPV) production system or non-replicative scNRPV system, and methods for creating such a scNRPV production system. The scNRPV system comprises: a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene and a bacterial production strain (also referred to as an “engineered bacterial strain”). The bacterial production strain or engineered bacterial strain described herein does not contain any serT and serU tRNA and comprises at least one vector. The at least one vector: (a) encodes a site-specific recombinase (SSR); (b) comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR; and (c) comprises in the 5’ to 3’ direction: (i) a nucleic acid sequence encoding a first attachment (att) site; (ii) one or more nucleic acids or transgenes of interest (which may encode one or more proteins of interest); and (iii) a nucleic acid nucleic acid sequence encoding a second att site.

[0100] In some embodiments, the bacterial production strain exhibits no expression of serT and serU tRNA. In some embodiments, the bacterial production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene. In some embodiments, the essential genes are genes that may confer either a significant survival disadvantage or a slowdown in their replication kinetics.

[0101] The vector used in the bacterial production strain is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the vector comprises 1 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of theClient Ref No. 24-0106Attny Docket No. UNC2.43224.601SSR. In some embodiments, the vector comprises 5 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 12 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0102] In some embodiments, the vector comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 6 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 7 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 8 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 9 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 10 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 11 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 12 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 13 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 14 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 15 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 16 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 17 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 18 TCA, TCG or TCA and TCG codons in theClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 coding sequence of the SSR. In some embodiments, the vector comprises at least 19 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 20 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 21 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 22 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 23 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 24 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 25 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

[0103] In some embodiments, the vector further comprises at least one origin of replication (ori). In some embodiments, the ori is located between the nucleic acid sequence encoding a first and second att site. In further embodiments, the ori is located outside of the nucleic acid sequence encoding a first and second att site. In some embodiments, the vector further comprises a nucleic acid sequence encoding a packing signal (pac).

[0104] In still further embodiments, the vector contains one or more nucleic acids or transgenes of interest. In some embodiments, the one or more nucleic acids or transgenes of interest encode one or more proteins of interest. In some embodiments, the one or more proteins of interest may not be expressed in the bacterial production strain. In some embodiments, the one or more proteins of interest may affect bacterial cell growth kinetics or viability.

[0105] The one or more nucleic acids or transgenes of interest included in the vector are not critical. For example, in some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short-chain fatty acid, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof. In other embodiments, the nucleic acid or transgene of interest encodes one or more toxins, such as, for example, Pseudomonas exotoxin A, cholera toxin, diptheria toxin, E. coli toxins, botulinum toxins, anthrax toxins, pertussin toxins, shiga toxins, ricin, tetanus toxins, Staphylococcal toxins, toxin-antitoxin systems, cedB toxins, or any combinations thereof. In other embodiments, the nucleic acid or transgene of interest encodes aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 toxin gene or a toxin protein that can be used to limit or prevent the spread of the nucleic acid or transgene of interest to another organism (e.g., an unintended organism, such as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a nontarget animal species), such that expression of the toxin (e.g. Bamase, Colicin E3, Gelonin, Ricin Chain A) in the unintended organism reduces viability, growth, or function of that organism, thereby restricting the persistence or transfer of the genetic material outside of desired target contexts.

[0106] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more toxic nucleic acid sequences. Examples of one or more toxic nucleic acid sequences are: HIV-1 env protein, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes including endonucleases and exonucleases, and / or toxin-antitoxin systems. In some aspects, when the vector or plasmid contains multiple nucleic acids or transgenes of interest (e.g., a first nucleic acid or transgene of interest and a second nucleic acid or transgene of interest), the use of one or more nucleic acids or transgenes that encode one or more toxic nucleic acid sequences can be used to prevent the spread of one or more other nucleic acids or transgenes of interest into an untended organism (e.g., as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a non-target animal species).

[0107] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more unstable polynucleotide sequences. Examples of unstable polynucleotide sequences include: (i) a Bxbl SSR coding sequence bearing >1 codons selected from TCG / TCA and, in cis, Bxbl attP and attB sites flanking a >20 bp segment containing (optionally) an ori and / or pac; (ii) Cre or Flp coding sequences with lox or FRT sites in cis; (iii) recombinase / invertase systems Gin / gix or Tn3 / res; (iv) plasmids containing >2 inverted repeats of >25 bp within 1 kb (hairpin- prone); and / or (v) coding sequences for growth-inhibitory proteins (e.g., Bamase, MazF endoribonuclease, RelE, phage holins or endolysins).

[0108] In some further aspects, the one or more nucleic acids or transgenes of interest can incorporate or include one or more TCA, TCG, or TCA and TCG codons.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0109] In further aspects, one or more nucleic acid sequences or transgenes of interest are contained within the vector, or are integrated into the genome of the vector using techniques known in the art.

[0110] In some embodiments, the vector used in the bacterial production system is non- replicative. In other embodiments, the vector used in the bacterial production system is selfreplicating.

[0111] The vector enriched with one or more TCA, TCG, or TCA and TCG codons can be delivered or transferred to one or more bacterial cells to create the bacterial production strain using any techniques known in the art. As mentioned previously, the bacterial cells should be derived from a bacterial strain that is deficient in one or more tRNA genes such as serU and / or serT. In some aspects, the one or more tRNA genes are translationally repressed in the bacterial strain. In some aspects, the one or more translationally repressed tRNA genes are not required for mobilization or transfer of the vector to a recipient bacterial cells or strain.

[0112] In some embodiments, the vector contains a conjugation system capable or suitable for use in transferring the vector or plasmid to one or more recipient bacterial cells.

[0113] In some embodiments, the bacteria used in the bacterial production system is E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIVA, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In some embodiments, the bacteria is E. coli. In some embodiments, the production strain is Syn61. In some embodiments, the recombinase is Flp, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0114] The bacterial production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCG codons in any essential gene. In some embodiments, the helper phage exists in the bacterial production strain temporarily or transiently. In still other embodiments, the helper phage is prepared separately from the bacterial production strain. In still other embodiments, the production strain is infected with at least one helper phage. In still other embodiments, the helper phage is introduced into the bacterial production system as an episome. In still another embodiment, the helper phage isClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 integrated into a chromosome of the bacterial production strain using routine techniques known in the art. In some embodiments, the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0115] Once the bacterial production strain (which can be referred to as the “donor strain”) has been created, the vector can be delivered to one or more recipient bacterial cells using routine techniques known in the art. The recipient bacterial cells used to receive the vector from the production strain are not critical. In some aspects, recipient bacterial cells are from E. coll, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In other aspects, the recipient bacterial cells are part of a microbial community, such as in a host organism or within an environmental microbiome.4. A SELF-CIRCULARIZING NON-REPLICATTVE PHAGE VECTOR (scNRPV)

[0116] In another embodiment, the present disclosure relates to a self-circularizing non- replicative phage vector (scNRPV) that: (a) encodes a site-specific recombinase (SSR); (b) comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR; and (c) comprises in the 5’ to 3’ direction: (i) a nucleic acid sequence encoding a first attachment (att) site; (ii) one or more nucleic acids or transgenes of interest (which may encode one or more proteins of interest); and (iii) a nucleic acid nucleic acid sequence encoding a second att site. Moreover, the scNRPV does not contain any TCG, TCA or TCG and TCA codons in any essential gene.

[0117] The scNPRV is enriched with one or more TCA, TCG or TCA and TCG codons. In some embodiments, the scNPRV comprises 1 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 1 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 1 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 1 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 5 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In someClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 embodiments, the scNPRV comprises 5 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 5 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 6 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises 6 to12 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0118] In some embodiments, the scNPRV comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 6 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 7 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 8 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 9 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 10 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 11 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 12 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least13 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 14 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 15 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 16 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 17 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at leastClient Ref No. 24-0106Attny Docket No. UNC2.43224.60118 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 19 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 20 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 21 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 22 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 23 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 24 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the scNPRV comprises at least 25 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

[0119] In some embodiments, the scNPRV further comprises at least one origin of replication (ori). In some embodiments, the ori is located between the nucleic acid sequence encoding a first and second att site. In further embodiments, the ori is located outside of the nucleic acid sequence encoding a first and second att site. In some embodiments, the scNPRV further comprises a nucleic acid sequence encoding a packing signal (pac).

[0120] In still further embodiments, the scNPRV contains one or more nucleic acids or transgenes of interest. In some embodiments, the one or more nucleic acids or transgenes of interest encode one or more proteins of interest. In some embodiments, the one or more proteins of interest may affect bacterial cell growth kinetics or viability.

[0121] The one or more nucleic acids or transgenes of interest included in the vector are not critical. For example, in some embodiments, the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short-chain fatty acid, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini -protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof. In other embodiments, the nucleic acid or transgene of interest encodes one or more toxins, such as, for example, Pseudomonas exotoxin A, cholera toxin, diptheria toxin, E. coli toxins, botulinum toxins, anthrax toxins, pertussin toxins, shiga toxins, ricin, tetanus toxins, Staphylococcal toxins, toxin-antitoxin systems, cedB toxins, or any combinations thereof. In other embodiments, the nucleic acid or transgene of interest encodes aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 toxin gene or a toxin protein that can be used to limit or prevent the spread of the nucleic acid or transgene of interest to another organism (e.g., an unintended organism, such as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a nontarget animal species), such that expression of the toxin (e.g. Bamase, Colicin E3, Gelonin, Ricin Chain A) in the unintended organism reduces viability, growth, or function of that organism, thereby restricting the persistence or transfer of the genetic material outside of desired target contexts.

[0122] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more toxic nucleic acid sequences. Examples of one or more toxic nucleic acid sequences are: HIV-1 env protein, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes including endonucleases and exonucleases, and / or toxin-antitoxin systems. In some aspects, when the vector or plasmid contains multiple nucleic acids or transgenes of interest (e.g., a first nucleic acid or transgene of interest and a second nucleic acid or transgene of interest), the use of one or more nucleic acids or transgenes that encode one or more toxic nucleic acid sequences can be used to prevent the spread of one or more other nucleic acids or transgenes of interest into an untended organism (e.g., as a commensal microbe, a beneficial environmental bacterium, a human cell, an agricultural crop plant, or a non-target animal species).

[0123] In some aspects, the one or more nucleic acids or transgenes of interest encode one or more unstable polynucleotide sequences. Examples of unstable polynucleotide sequences include: (i) a Bxbl SSR coding sequence bearing >1 codons selected from TCG / TCA and, in cis, Bxbl attP and attB sites flanking a >20 bp segment containing (optionally) an ori and / or pac; (ii) Cre or Flp coding sequences with lox or FRT sites in cis; (iii) recombinase / invertase systems Gin / gix or Tn3 / res; (iv) plasmids containing >2 inverted repeats of >25 bp within 1 kb (hairpin- prone); and / or (v) coding sequences for growth-inhibitory proteins (e.g., Bamase, MazF endoribonuclease, RelE, phage holins or endolysins).

[0124] In some further aspects, the one or more nucleic acids or transgenes of interest can incorporate or include one or more TCA, TCG, or TCA and TCG codons.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0125] In further aspects, one or more nucleic acid sequences or transgenes of interest are contained within the scNPRV, or are integrated into the genome of the scNPRV using techniques known in the art.

[0126] The scNPRV enriched with one or more TCA, TCG, or TCA and TCG codons can be delivered or transferred to one or more bacterial cells to create a bacterial production strain or donor bacterial cells using any techniques known in the art. The bacterial cells should be derived from a bacterial strain that is deficient in one or more tRNA genes such as serU and / or serT. In some aspects, the one or more tRNA genes are translationally repressed in the bacterial strain.In some aspects, the one or more translationally repressed tRNA genes are not required for mobilization or transfer of the scNPRV to a recipient bacterial cells or strain.

[0127] In some embodiments, the scNPRV contains a conjugation system capable or suitable for use in transferring the vector or plasmid to one or more recipient bacterial cells.

[0128] In some embodiments, the bacteria cells are from E. coli, a Proteobacteria, a Fusobacteria, Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a. Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In some embodiments, the bacteria cells are E. coli. In some embodiments, the production strain is Syn61. In some embodiments, the recombinase is Flp, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0129] After the scNPRV is delivered or transferred to the bacterial production strain (which can be referred to as the “donor strain”), the scNPRV can be delivered to one or more recipient bacterial cells using routine techniques known in the art. The recipient bacterial cells used to receive the vector from the production strain are not critical. In some aspects, recipient bacterial cells are from E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0130] After the scNPRV is delivered to one or more recipient bacterial cells, it can be recovered using routine techniques known in the art. In some embodiments, the scNPRV can be purified after recovery using routine techniques known in the art.5. A METHOD OF PRODUCING A SELF-CIRCULARIZING NON-REPLICATIVE PHAGE VECTOR

[0131] The presently disclosed subject matter, in some embodiments, provides a method of producing a self-circularizing non-replicative phage vector (scNRPV) containing one or more nucleic acids or transgenes of interest. In some embodiments, the method involves infecting a bacterial production strain with a helper phage (e.g., non-replicative or replicative) that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene. The bacterial production strain used in the method does not contain any serT or serU tRNA and contains a vector that: (a) encodes a site-specific recombinase (SSR); (b) comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR; and (c) comprises in the 5’ to 3’ direction: (i) a nucleic acid sequence encoding a first attachment (att) site; (ii) one or more nucleic acids or transgenes of interest (which may encode one or more proteins of interest); and (iii) a nucleic acid nucleic acid sequence encoding a second att site. In some embodiments, the helper phage that infects the bacterial production strain can be a replicative helper phage. In other embodiments, the helper phage is a non-replicative helper phase. In still other embodiments, the production strain may contain a temperate phage lysogen and be induced by heat, UV, or chemical stress to initiate phage vector production.

[0132] In some embodiments, the bacterial production strain exhibits no expression of serT and serU tRNA. In some embodiments, the bacterial production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene. In some embodiments, the essential genes are genes that may confer either a significant survival disadvantage or a slowdown in their replication kinetics.

[0133] In some embodiments, the vector comprises 1 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 1 to 25 TCA, TCG, or TCA and TCG codonsClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 100 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 75 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 50 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 5 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 25 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises 6 to 12 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0134] In some embodiments, the vector comprises at least 1 TCA, TCG or TCA and TCG codon in the coding sequence of the SSR. In some embodiments, the vector comprises at least 2 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 3 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 4 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 6 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 7 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 8 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 9 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 10 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 11 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 12 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 13 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 14 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 15 TCA, TCG or TCA and TCG codons in the coding sequence. In some embodiments, the vector comprises at least 16 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 17 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vectorClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 comprises at least 18 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 19 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 20 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 21 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 22 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 23 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 24 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector comprises at least 25 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR. In some embodiments, the vector farther comprises at least one origin of replication (ori). In some embodiments, the ori is located between the nucleic acid sequence encoding a first and second att site. In farther embodiments, the ori is located outside of the nucleic acid sequence encoding a first and second att site. In some embodiments, the vector farther comprises a nucleic acid sequence encoding a packing signal (pac).

[0135] In some embodiments, the bacteria used in the bacterial production system is E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof. In some embodiments, the bacteria is E. coli. In some embodiments, the production strain is Syn61. In some embodiments, the recombinase is Flp, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0136] After infection of the bacterial production system with the helper phage, the vector, now referred to as “scNPRV,” can be delivered to one or more recipient bacterial cells using routine techniques known in the art. The recipient bacterial cells used to receive the vector from the production strain are not critical. In some aspects, recipient bacterial cells are from E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0137] After the scNPRV is delivered to one or more recipient bacterial cells, it can be recovered using routine techniques known in the art. In some embodiments, the scNPRV can be purified after recovery using routine techniques known in the art.6. A METHOD OF MODIFYING BACTERIA

[0138] In some embodiments, the present disclosure relates to methods of modifying bacteria. In some aspects, the bacteria to be modified can be found at any location in a subject (e.g., mammal) where bacteria reside (e.g, gastrointestinal tract, respiratory tract, urogenital system (e.g., vaginal microbiome, urinary tract, or upper reproductive tract), oral cavity, skin, and / or ocular system (e.g., conjunctiva and ocular surface)). In some aspects, the bacteria to be modified can be found at any location in a human or a non-human mammal, such as, the gastrointestinal tract, respiratory tract, urogenital system (e.g., vaginal microbiome, urinary tract, or upper reproductive tract), oral cavity, skin, and / or ocular system (e.g., conjunctiva and ocular surface). In some aspects, the bacteria to be modified can be found at any location in an environment (e.g., bacteria located on a plant or bacteria located in the soil, air, or water).

[0139] In some aspects, the method relates to modifying bacteria residing at one or more locations in a human or non-human mammal by administering a therapeutically effective amount of a composition to the human or non-human mammal in need thereof to modify bacteria at one or more locations where the bacteria reside in the human or non-human mammal. The one or more locations in which the bacteria reside may be the gastrointestinal tract, respiratory tract, urogenital system (e.g., vaginal microbiome, urinary tract, or upper reproductive tract), oral cavity, skin, ocular system (e.g., conjunctiva and ocular surface) or any combination thereof. In some aspects, the composition comprises at least one self-circularizing non-replicative phage vector as described in Sections 4 and 5 herein. The term “therapeutically effective” as used herein refers to a benefit, including modifying one or more bacteria in or on a subject to reduce, alleviate, and / or prevent one or more conditions and / or diseases caused or resulting from the presence of one or more bacteria in or on a subject. It will be appreciated that the therapeutically effective amount will vary depending upon the intended application, subject, and / or type of bacteria to be modified.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0140] In some embodiments, the non-human mammal is a cow, pig, horse, goat, dog, cat, monkey, rabbit, sheep, buffalo, elk, deer, or any combination thereof.

[0141] In some aspects, the bacteria to be modified can be found at any location in an environment or environmental microbiome (e.g., located in or on a plant and / or located in or on the soil, air, or water (e.g., fresh water, salt or sea water, sewage water, pond, lake and / or river water, rain water, or any combination thereof). In such methods, the method involves applying an effective amount of a composition to an environment or environmental microbiome in which bacteria are residing to modify bacteria present in the environment. In some aspects, the composition comprises at least one self-circularizing non-replicative phage vector as described in Sections 4 and 5 herein.EXAMPLES

[0142] The following examples are for the purposes of illustration only and are not intended to limit the scope of the claims.EXAMPLE 1: Materials and Methods for Example 2

[0143] Experimental models

[0144] E. coli strains: All Syn61 variants were cultured at 37°C with shaking in 2xYT media supplemented with streptomycin (50 pg / mL). All remaining strains were cultured at 37°C with shaking in LB media. All mice were 4-6 week old female C57BL / 6 mice kept in cage groups of 5 and housed at the UNC Mouse core.

[0145] Cloning

[0146] All plasmids were cloned through Gibson HiFi (New England Biolabs, cat. no. E2621L) assembly of synthesized DNA fragments (IDT) or via Golden Gate assembly (New England Biolabs, cat. no. E1601L) from synthesized DNA fragments (Twist & IDT).

[0147] ddPCR

[0148] Phage DNA was purified using the Norgen Phage DNA Isolate Kit (Norgen Biotek Corp.# 46800) following the manufacturers recommended protocol. Purified DNA was digested with Sca-I (NEB cat. no. 3122) and 3 10-fold dilutions were made of the completed digestion reaction. ddPCR was then performed on these dilutions using the primers and probes in Table AClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 and the following reaction conditions: annealing temperature, 63 °C; primer concentration, 5.1 pM; probe concentration, 2.25 pM; ramp rate, 1 °C / s; cycles, 50. Transgene and genome-packed vector titers were then calculated based on positive droplet counts.

[0149] Table A

[0150] Colony formation transduction assay

[0151] E. coli NEB5a were cultured for 16 hrs overnight. Overnight cultures were passaged1 : 100 into fresh media and cultures until an ODeoo of 0.6-0.8 was reached. 500 pL of these cells at a concentration varying concentrations were then mixed with 500 uL of phage vectors diluted to achieve the specified MOI dilution in a final volume of 1 mL. These transduction reactions were incubated at 37°C for 90 min with shaking. Cells were then pelleted at 16,000 x G for 5 min and resuspended in fresh LB. Serial dilutions of the transduced cells were made and plated on LB plates supplemented with no antibiotics, kanamycin (50 pg / mL), and carbenicillin (100 pg / mL). Plates were incubated at 37°C until colonies were visible and colonies were counted to calculate the number of transduced cells (transductants) per mL. To calculate percent transduction, the following formula was used: transductants percent transduction = - ; - — ■ 100% total target cells

[0152] where transductants are the number of kanamycin resistant CFUs and the total target cells are the number of CFUs that formed on either the LB or LB + carbenicillin plates. To calculate effective transduction efficiency, the following formula was used: transductants effective transduction efficiency = - - — ; - 100% transgene packed vectorsClient Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0153] where the number of transgene packed vectors added to the transduction reaction was determined by ddPCR.

[0154] Fluorescence kinetics assay

[0155] E. coli NEB5a were cultured for 16 hrs overnight. Overnight cultures were passaged 1 : 100 into fresh media and cultures until an ODeoo of 0.6-0.8 was reached. 500 pL of these cells at a concentration of 1x108cells / mL were then mixed with of phage vectors, diluted to achieve the specified MOI dilution in a final volume of 1 mL. These transduction reactions were incubated at 37°C for 90 min with shaking. Cells were then pelleted at 16,000 x G for 5 min and resuspended in fresh LB. Transduced cells were then transferred to a microplate and mScarlet fluorescence was read every 10 minutes for 12 hours on a Biotek Synergy Hl plate reader (excitation: 550 nm, emission: 594 nm, gain: 100) with established incubation conditions44.

[0156] Nanoluciferase assay

[0157] Marionette strains (sAJM.1504)24and Syn61A3were grown to an ODeoo of 0.5-0.6 and made electrocompetent by 4 washes with cold 10% glycerol. These cells were electroporated (1.8 kV, 200 Q, 25 pF, 1 mm gap cuvette) with nanoluciferase expression plasmids and recovered in SOC at 37°C for 1 hr. before plating on either LB or 2xYT plates supplemented with kanamycin (50 pg / mL). Single colonies were then cultured for 16 hrs in LB or 2xYT supplemented with kanamycin (50 pg / mL), after which ODeoo was taken. The cultures were pelleted at 16,000 x g for 3 min and supernatants from the cultures were then mixed with an equal volume of Nano-Gio reagent (Promega, cat. no. N1130) Luminescence was quantified via a Biotek Synergy Hl plate reader (gain: 100, integration time: Is). The following formula was used to calculate corrected luminescence values:

[0158] Recombination assay

[0159] The same transformation procedure described for the Nanoluciferase assay was used to transform the (sAJM.1504) and Syn61A3with each SSR expression plasmid. After recovery, allClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 cells were plated on LB agar supplemented with kanamycin (50 pg / mL). Plates were incubated at 37°C until colonies were visible. 1 mL of LB was added to the plates and all colonies were resuspended by scraping. Plasmid DNA was then purified from the resuspended transformant population (Zymo Research, cat. no. D4036). Nanopore sequencing was performed on the purified plasmid DNA by Plasmidsaurus.

[0160] A python script (DOI: 10.5281 / zenodo.l0525174) was then used to determine the percent of reads that had undergone recombination. Briefly, this script filters reads by aligning them to the plasmid reference sequence, if the read is > 70% the length of the reference and has > 80% identity, the sequence flanked by the attP and attB sites and the attL sequence are both aligned to the read. If the flanked sequence is aligned with > 75% identity and the attL sequence is not present, the read is considered to have not undergone recombination. If the flanked sequence is not present and the attL sequence is aligned with > 99% identity, the read is considered to have undergone recombination.

[0161] T7 genome recoding, assembly, and rebooting

[0162] TCA, TCG, and TAG codons, as well as Bsal restriction sites, were removed from the T7 genome in silico using a custom python script (DOI: 10.5281 / zenodo.10869476). Briefly, each annotated coding sequence was searched for TCA, TCG, TAG, and the Bsal site. If TCA, TCG, or TAG codons were found and the codon was not in an out of frame overlap with another coding sequence, a random synonymous mutation was made, weighted by the codon usage frequencies of E. coli. If the Bsal recognition site was found, a silent single bp mutation was made. The resulting recoded genome was divided into 24 separate fragments using NEB SplitSet. B sal-compatible adapter sequences were added to each fragment and they synthesized by Twist Bioscience.

[0163] Synthesized recoded T7 genome fragments were Golden Gate assembled (New England Biolabs, cat. no. E1601L) and rebooted via transformation according to an established protocol45. Briefly, after assembly, the recoded T7 genome was electroporated (1.8 kV, 200 Q, 25 pF, 1 mm gap cuvette) into DH10B (Invitrogen cat. no. 18290015). Cells were then mixed with 1 mL of SOC media and allowed to recover at 37°C for 5 min. cells were then mixed with 4 mL ofClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 molten soft agar, plated on prewarmed LB agar plates, and incubated at 37°C until an opaque lawn formed. Plaques were then picked to isolate recoded T7.

[0164] Phage vector production

[0165] E. coli NEB5a was transformed with a vector packing phagemid and pT7gpl7. These cells were cultured for 16 hrs overnight. Overnight cultures were passaged 1 :100 into fresh media and cultures until an ODeoo of 0.6 was reached. Unless otherwise specified, these cultures were spiked with IxlO8gp 17 -deficient barcoded T7 helper phage. Infection was allowed to proceed for 4 hrs at 37°C with shaking. Infections were then centrifuged at 10,000 x G for 10 min and supernatant was 0.2 pm filtered to isolate phage vectors.

[0166] Plaque formation assays

[0167] E. coli strains were cultured for 16 hrs overnight. Overnight cultures were passaged1 : 100 into fresh media and cultures until an ODeoo of 0.6 was reached. 800 pL of these cells were mixed with 8 mL of molten soft agar and overlaid on prewarmed LB agar plates supplemented with ampicillin (100 pg / mL). 12 10-fold dilutions of phage vector were made and spotted onto the lawn plates. Plates were incubated for 16 hrs at 37°C and plaques were counted to calculate the number of plaque forming units (PFU) per mL.

[0168] Phage infection kinetic assay

[0169] E. coli strains were cultured for 16 hrs overnight. Overnight cultures were passaged 1 : 100 into fresh media and cultures until an ODeoo of 0.6 was reached. 2xl07PFU of phage were add to a total of 200 pL of cells in microtiter plates and absorbance at 600 nm was read on a Biotek Synergy Hl plate reader every 10 min for 8 hrs.

[0170] In vivo transduction

[0171] Mice were obtained from Jackson Laboratory and allowed to acclimate to a 12 hour light / dark cycle for 2 weeks. After 2 weeks, mice were started on sweetened (30 mg / mL sucrose) drinking water supplemented with ampicillin (1 mg / mL). Mice were administered 200 pL of antibiotic cocktail (vancomycin (25 mg / mL), gentamicin (50 mg / mL), neomycin (50 mg / mL), ampicillin (50 mg / mL), and metronidazole (12.5 mg / mL) via oral gavage for 3 days. After a 24 hr washout period, mice were then treated with 200 pL of target cells grown for 16 hrs toClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 stationary phase via oral gavage. Mice were then dose with 200 pL of phage vector treatments via oral gavage (LB was used as a vehicle control). Each day, feces was collected and suspended in PBS to 1 g / mL. Fecal solids were pelleted at 300 x g for 20 s and serial dilutions of the supernatant were made and then plated on LB plates supplemented with either kanamycin (50 pg / mL) or carbenicillin (100 pg / mL).

[0172] Statistical Analyses

[0173] All measurements were taken from biologically independent samples. Statistical analyses were performed using GraphPad Prism (version 10.4.2). For one-way and two-way ANOVAs, Tukey’s post hoc test was used to correct for multiple comparisons. To model expression kinetics by transduced cells, an exponential growth model was fit, yielding an R2value of 0.9829. To model lysis kinetics by wt T7 and rT7, one-phase decay models were fit, yielding R2values of 0.9155 and 0.9482, respectively.EXAMPLE 2: Inefficient transduction by conventional NRPVs is due to the innate linear topology of their DNA

[0174] As a proof of concept, the focus was on modifying T7, an obligate lytic phage capable of infecting select strains of E. coli16,17, as T7 has been among the most common phages used to generate NRPVs to date16-21. Notably, all genes in the 40 kb T7 genome are oriented in a single direction with no overlap between the reading frames of any essential genes22. This size and simplicity make T7 well-suited for genome-scale perturbations.

[0175] The transduction efficiency of conventional T7 NRPVs was established by producing vectors packed with a phagemid encoding (i) the T7 packing signal (pac), (ii) Bxbl attP and attB sites flanking constitutive transgene expression cassettes encoding (iii) mScarlet and (iv) aminoglycoside 3'-phosphotransferase (KanR), and (v) a high copy origin of replication (Figure 1 A). When these T7-NRPVs were used to transduce E. coli at a range of MOIs (0.3-0.003), it was found that transductant formation was ~200-fold lower than the number of transgene-packed vectors dosed (Figure IB). This exceptionally low T7-NRPV transduction efficiency is consistent with earlier publications for T7 and other NRPV systems, where transduction efficiencies can be up to 6 order of magnitude lower than the theoretical maximum14.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0176] To assess whether the linear topology of NRPV-packed DNA contributes to the observed poor transduction efficiency (Figure 1C), target E. coli were first transformed with a plasmid encoding A.RED23, which enhances homologous recombination (HR)28. When T7-NRPVs were added to the HR+ E. coli at a multiplicity of infection (MOI) of 1 (i.e. 1 NRPV per target bacteria), a ~20% increase in the transductants was seen as compared to wild-type (wt) E. coli, with the fraction of stably transduced E. coli modestly increasing from 0.22% to 0.34%. E. coli expressing the Bxbl site specific recombinase (SSR) was tested instead, which recombines the Bxbl attP and attB (att sites) — present in the phagemid — to generate a new plasmid (indicated by the dotted lines in Figure 1 A). In contrast to HR, a dramatic increase in stable transductants was seen with SSR+ E. coli (~65-fold more than wt E. coli) (Figure ID), with the fraction of stably transduced E. coli reaching -15% (Figure IE). Overall, the amount of transduced target E. coli relative to the number of transgene-packed T7-NRPVs dosed (herein defined as the effective transduction efficiency) was 0.060%, 0.075%, and 3.6% for wt, HR+,+ and SSR+ E. coli, respectively (Figure IF). To evaluate expression kinetics of transgenes encoded in the phagemid and predict how these vectors may perform in the context of in situ transduction, target cells were co-incubated with T7 NRPVs packed with the phagemid described in Figure 1 A. The cells were then grown under non-selective conditions — to recapitulate the natural conditions in the intestinal lumen — and mScarlet signal of the transductants was measured as a function of time. It was found that doubling time of the fluorescent signal for SSR+ cells was 12 -fold faster than wt cells (2.6 vs. 32.2 hrs) and total transgene expression was >11 -fold higher (Figure 1G and Figure 6), indicating that SSR-mediated recircularization of transduced DNA protects this DNA and allows it to replicate and persist at rates more than an order of magnitude higher than when the transduced DNA is not recircularized. Altogether, these data suggest site-specific recombination of linear DNA into circularized phagemid DNA, after injection of the linear phagemid DNA into target bacteria, dramatically improves NRPV transduction efficiency.

[0177] Production of scNRPVs requires stringent recombinase repression

[0178] Since the vast majority of bacteria do not naturally express SSRs, it is essential to design NRPVs such that they are capable of self-catalyzed, host-independent phagemid recircularization in order to capture the potent increase in transduction efficiency that SSR-mediated circularization confers. Specifically, it sought to do so by encoding in the phagemid a Bxbl SSRClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 expression cassette in cis to the Bxbl SSR att sites that flank the transgenic DNA of interest. This design allows expression of the phagemid-encoded SSR in target bacteria, enabling rapid recombination of the att sites and culminating in the formation of a circularized phagemid containing only the transgene cassette (Figure 2A). However, this design faces a critical constraint: since the SSR-mediated recombination reaction is irreversible, ablates the att sites, and excises any DNA outside of the att sites (e.g. the packing signal), this reaction must only occur after scNRPVs inject their linear phagemid DNA cargo into the target bacteria, and never prematurely in the bacterial strain used to produce the scNRPVs. Premature recombination of phagemid DNA in the production strain prevents functional scNRPV production due to irreversible ablation of the att sites (Figure 2B). This underscores the importance of having exceptionally stringent control over SSR expression in the production strain.

[0179] To determine how the necessary level of repression of SSR expression can be achieved in the production strain, a panel of reporter plasmids was tested, in which Nanoluciferase (NanoLuc) expression is controlled by different combinations of transcriptional and translational repression elements. These constructs include combinations of operator sequences that are repressed by tetR, cinR, or lacl, as well as a theophylline-inducible riboswitch (yitJ) and hammerhead ribozyme (HHaz) (Figure 2C and Figure 7). Notably, none of the individual repressors or repressor combinations were able to achieve sufficiently strong repression: even with the most stringent repression achieved through the combination of tet / yit J, luminescence was only reduced ~70-fold (to -700 RLU) and remained markedly above background (Figure 2D). To determine how this level of repression translates to the prevention of SSR-mediated recombination in the scNRPV production strain, phagemids were cloned with and without attP / B sites that encode, in cis, the Bxbl SSR under tet repression. Consistent with the observed limited NanoLuc repression, when these phagemids were transformed into an E. coli strain expressing tetR, cinR, and lacl24, 93% of the attP / B-i- phagemid molecules detected in the transformed E. coli had undergone (Figure 2E)).

[0180] Next, whole genome codon compression and tRNA-deficiency25was examined in an attempt to achieve more stringent repression (Figure 2C). To compare this strategy to the previously evaluated conventional methods of repression, a constitutively expressed variant of the NanoLuc plasmid was engineered where, critically, the NanoLuc coding sequence contains 6 TCG codons (NanoLucTCG). This was then transformed into the E. coli strain Syn61A3, a serTClient Ref No. 24-0106Attny Docket No. UNC2.43224.601(encodes tRNASer(UGA)), serU (encodes tRNASer(CGA)) and RF1 (recognized the TAG stop codon) triple knockout wherein all TCA, TCG, and TAG codons have been removed from its genome26. In this setup25, luminescence from NanoLucTCG-transformed Syn61A3cultures was effectively reduced to background levels (5 RLU, on average; not statistically different from background). This level of repression is around a 150-fold improvement over the best repressor strategyevaluated earlier (tetO / yitJ) (Figure 2D). The dynamic range of NanoLucTCG(expression in Syn61A3vs. DH5a) was more than 3 orders of magnitude greater than that achieved by any of the repressor combination constructs (Figure 2F). These results underscore whole genome codon compression coupled with select tRNA knockout as an effective strategy to achieve absolute repression in the production strain. Importantly, tRNA-deficiency does not impact expression in natural bacteria, and thus linear phagemid DNA encoding the SSR can be replicated and packed into scNRPVs in the production strain while still being readily expressed once the SSR-encoding phagemid is injected from the scNRPV into target bacteria.

[0181] Recoded T7 phage infect a tRNA-deficient vector production E. coli strain

[0182] The wt T7 genome cannot effectively function in Syn61A3, as the presence of TCA and TCG serine codons in the coding sequences within the genome preclude expression of essential T7 proteins in Syn61A3(Figure 3A and 3B). In order to produce T7 scNRPVs, a T7 variant with a Syn61A3-compatible genome, i.e. recoded to remove TCA, TCG, and TAG codons in all essential genes (rT7) (Figure 3C) was engineered. The codons were reassigned to TCT, TCC, AGT, and AGC based on E. coli codon usage rates to minimize the risk of overburdening any one tRNA. Through recoding, 161 TAG, TCA, and TCG, codons were removed while leaving 1 TAG, 5 TCA, and 8 TCG codons unmodified in the rT7 genome. These codons appeared in non- essential genes that overlapped out of frame with essential genes, and compatible synonymous recoding mutations in the non-essential genes that resulted in silent mutations in the essential genes could not be identified. Overall, the recoding effort shifted the amount of TCA and TCG codons, as a percent of total serine codons, from 14.6% and 7.2% in native T7 to 0.59% and 0.95% in rT7, respectively (Figure 3D and 3E).

[0183] Upon synthesis and rebooting of rT7, plaque formation assays were performed using three E. coli strains: BW25113 (wt E. coli), Syn61(TCA, TCG, and TAG recoded E. coli), and Syn61A3(Syn61 serT n serU double knockout). As expected, both wt T7 and rT7 were able toClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 infect BW25113 and Syn61, while only rT7 was able to infect Syn61A3, with the assay indicating both viruses had comparable plaquing efficiency and titer (Figure 3F), although rT7 did exhibit smaller plaques than wt T7 on BW25113 (Figure 8).

[0184] To further understand the fitness of these recoded T7 variants, liquid cultures of BW25113 were infected in their exponential growth phase with wt T7 and rT7 and measured their optical density over time to understand the kinetics of infection by these viruses. Similar to the plaque formation assay, wt T7 and rT7 both cleared the wt strain (Figure 3G). However, the lag times before lysis was observed was 54 min for wt T7 and 109 min for rT7. This corresponded to lysis-induced half-lives of the cultures of 0.30 and 0.63 hrs, respectively, indicating that the recoding effort did impact viral fitness by prolonging the T7 lifecycle. Despite this, the overall titer achievable with rT7, as compared to the wild-type virus, is not significantly decreased.

[0185] scNRPVs greatly enhance transduction efficiency in vitro and in vivo

[0186] To create the scNRPV biomanufacturing platform, a ‘production strain’ was generated by transforming Syn61A3with a T7 phagemid encoding transgenes of interest together with the SSR / att-sites necessary for self-circularization, as well as a plasmid encoding the T7 tail fiber gene, gpl7. This second plasmid complements the rT7 gpl7 knockout variant (rT7Agp17) helper phage used to inoculate the production strain culture. Upon infection with the helper phage, production strain cells produce T7 -scNRPV progeny packed with the phagemid (Figure 9). To determine how these vectors compare to conventional NRPVs, three phagemid variants were generated: one expressing mScarlet, one where the mScarlet CDS was replaced with that of the SSR, and one that encodes the SSR but lacks att-sites. The resulting vectors were used to transduce wt E. coli and E. coli engineered to constitutively express the SSR (Figure 4A). While the titers of these vectors did not differ appreciably (ranging from 1.2x109to 1.9x109; Figure 4B), it was found a transgene-to-genome (T:G) packing ratio (indicative of vector purity) of 14 was observed for the mScarlet NRPVs, while the att“ NRPVs and the scNRPVs did not significantly differ, with T:G ratios of 3.4 and 2.9, respectively (Figure 4C). When each vector was added to IxlO8E. coli at an MOI of 0.1, control scNRPVs (att ) resulted in only 3.3xl04E. coli that stably maintained the phagemid, whereas the scNRPVs transduced 3.4xl06E. coli, i.e. a ~100-fold improvement (Figure 4D). When normalized to the total number of target cells, theseClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 data corresponded to 0.08% of the total target cells transduced by NRPVs vs. 10% for the scNRPVs, a 116-fold difference (Figure 4E), with effective transduction efficiencies of 0.3% for NRPVs (or 3 out of every 1000 vectors dosed transducing a target) vs. 33% for the scNRPVs (Figure 4F). Similar improvements were observed across MOIs, where scNRPVs transduced >70% of target cells at an MOI of 1, while NRPV controls only transduced ~2% (Figure 10).

[0187] T7-scNRPV transduction in situ in the mammalian gut was compared to that of NRPVs, using mice colonized with ampicillin resistant target E. coli (Figure 5A). T7 -NRPVs were packed with a phagemid encoding mScarlet and KanR, whereas T7-scNRPVs were packed with a phagemid encoding the Bxbl SSR and KanR (Figure 5B). Upon target cell engraftment, we administered either 2xl08(per dose) NRPVs or scNRPVs, on 2 consecutive days via oral gavage. The fecal concentrations of target bacteria and transduced bacteria were monitored over 20 days by quantifying the number of ampicillin and kanamycin resistant bacteria detected in the stool, respectively (Figure 5C). Target E. coli colonization was maintained at ~ 5xlO8- IxlO9CFU / g feces over the course of the study by supplementation of the drinking water with ampicillin (Figure 5D).

[0188] One day after vector treatment, it was found 3.3 xlO4CFU / g feces in mice receiving T7- NRPV, while groups that did not receive target cells or vectors showed no detectable transduction. The amount of transduced E. coli increased to a maximum of 3.28x105CFU / g feces at 2 days post treatment, after which transduced cells began to quickly decline and were no longer detected at Day 7 following the final NRPV dose (Figure 5E). In contrast, in mice receiving T7-scNRPVs, an average of 3.5x107CFU / g feces 3 days after the initial dose were found, representing a > 100-fold increase in target bacteria transduced compared to conventional NRPVs. This high level of in situ recircularization-mediated transduction shows robust reproducibility, with an average of 3.7x107transduced CFU / g feces observed (and with transductants persisting up to 20 days post dosing) when this study was independently repeated twice using SSR expressing target cells (Figure 11).Example 3: Materials and Methods used for Example 4

[0189] Experimental Models

[0190] E. coli strains: All Syn61 variants were cultured at 37°C with shaking in 2xYT media supplemented with streptomycin (100 pg / mL). All remaining strains were cultured at 37°C withClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 shaking in LB media. Strains harboring pKD46 were cultured at 30°C with shaking until curing was required.

[0191] Method Details

[0192] Plasmid cloning

[0193] Plasmids PV260, PV480, and PV476 were cloned through Gibson HiFi (New England Biolabs, cat. no. E2621L) assembly of synthesized DNA fragments (IDT) (See, Figure 15). tRNA, Nanoluciferase, and Bxbl recombinase expression plasmids were cloned via Golden gate assembly (New England Biolabs, cat. no. El 60 IL) from synthesized DNA fragments (Twist & IDT) into PV480 or PV476 backbones according to Tables B and C below and Figure 15.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601_

[0194] Serine tRNA Knock out

[0195] Recombineering was performed to knock out both serU and serT66. Briefly doublestranded DNA knock out templates consisting of 350-500 bp regions homologous to the Syn61 genome immediately up and downstream of the serT and serU genes, flanking a chloramphenicol resistance cassette, were synthesized (IDT). Syn61 cells harboring pKD46 were grown at 30°C in 2xYT + 10 pg / mL L-arabinose to an ODeoo of 0.5-0.6 and made electrocompetent by 4 washes with cold 10% glycerol and electroporated (1.8 kV, 200 Q, 25 pF, 1 mm gap cuvette) with 1-1.5 pg of knock out template. Transformed cells were recovered in SOC at 37°C for 1 hr, plated on 2xYT plates supplemented with chloramphenicol (5 pg / mL), and incubated at 37 °C until colony growth was visible.

[0196] Colonies were isolated and cultured at 42°C, followed by gDNA purification (New England Biolabs, cat. no. T3010L). Roughly 1 pg of purified gDNA was sent for whole genome nanopore sequence by Plasmidsaurus to confirm knock out.

[0197] Kanamycin resistance transformation assays

[0198] All Syn61 strains were made electrocompetent as in the above section. For the serU rescue experiments, electrocompetent Syn61 3 was first transformed with the tRNA expression plasmids PV516 or PV518 and then made electrocompetent again. The cells were electroporated as above with 250 ng of either PV260 or PV480 plasmid DNA in three independent replicates and recovered for 1 hr in SOC media. 1 :5 serial dilutions were made of the recovered cells which were then plated on 2xYT plates supplemented with kanamycin (50 pg / mL). Dilution points with 10-100 colonies were used to quantify the number of transformants / mL.

[0199] Nanoluciferase assay

[0200] Two independent replicates of Syn61 variants harboring each of the Nanoluciferase expression plasmids were cultured for 16 hrs in 2xYT supplemented with kanamycin (50 pg / mL), after which ODeoo was taken. Supernatant from the culture was then mixed with an equal volume of Nano-Gio reagent (Promega, cat. no. N1130) and luminescence was quantifiedClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 via a Biotek Synergy Hl plate reader (gain: 100, integration time: Is). The following formula was used to calculate corrected luminescence values:

[0201] Average luminescence values were then normalized on a 0-100 scale in Graphpad Prism.

[0202] Recombination assay

[0203] The same transformation procedure as for the kanamycin resistance transformation assay was used to transform the Syn61 variants with each of the recombinase plasmids. After recovery, all cells were plated on 2xYT plates supplemented with kanamycin (50 pg / mL). Plates were incubated at 37°C until colonies were visible. 1 mL of LB was added to the plate and all colonies were resuspended by scraping. Plasmid DNA was then purified from the resuspended transformant population (Zymo Research, cat. no. D4036). Nanopore sequencing was performed on the purified plasmid DNA by Plasmidsaurus.

[0204] A python script (DOI: 10.5281 / zenodo.l0525174) was then used to determine the percent of reads that had undergone recombination. Briefly, this script filters reads by aligning them to the plasmid reference sequence, if the read is > 70% the length of the reference and has > 80% identity, the sequence flanked by the attP and attB sites and the attL sequence are both aligned to the read. If the flanked sequence is aligned with > 75% identity and the attL sequence is not present, the read is considered to have not undergone recombination. If the flanked sequence is not present and the attL sequence is aligned with > 99% identity, the read is considered to have undergone recombination.EXAMPLE 4: serU (tRNASer(CGA)) is a weak decoder of TCA codons

[0205] It is widely reported that serU (tRNASer(CGA)) only decodes TCG, and that TCA is only decoded by serT (tRNASc^UGA))46-50. Given that genetic firewall effects in Syn61 are expected to be driven by these non-redundant interactions, Syn61 serU (Syn61AU) and serT (Syn61 AT) single knockout strains (Figure 12B & 12C) were generated, and compared against Syn61 and Syn61 A3 (the serU, serT, and prfA triple knockout strain) (Figure 12D). Based on the widely reported serine codon interaction map, only strains with a copy of serT should be susceptible to transformation by a plasmid encoding a kanamycin resistance gene that contains 7 TCA codonsClient Ref No. 24-0106Attny Docket No. UNC2.43224.601 out of 16 total serine codons (KanRTCA+) (Figure 12E). As expected, no Syn61A3 transformants were observed on kanamycin plates. However, surprisingly, it was found that Syn61 AT was actually susceptible to transformation by this plasmid, albeit with low efficiency (a ~98% reduction in colonies relative to the same plasmid lacking TCA codons, KanRTCA) (Figure 12F & 12G).

[0206] To explore what allowed Syn61A3 to resist transformation compared to Syn61 AT, a set of serU expression plasmids was cloned. Both plasmids encode the E. coli serU gene under control of the natural serU promoter, and differ only by their anticodon, where one possesses the wild type serU anticodon (serUCGA), and the other possesses the serV (present in both Syn61 AT and Syn61A3) anticodon (serUGCU) (Figure 12H). When Syn61A3 strains containing these plasmids were then transformed with the same KanR plasmids, it was found found that the plasmid encoding wildtype serU (serUCGA), but not the serU anticodon mutant plasmid (serUGCU), rescued the transformation of Syn61A3 by KanRTCA+(Figure 121). Indeed, consistent with the low efficiency observed in Syn61 AT, transformation by the KanRTCA+plasmid, Syn61 A3 containing serUCGAwas susceptible to low-levels of transformation by KanRTCA+, at ~2% the efficiency of the KanRTCA‘ plasmid. These results directly indicate that the tRNA encoded by serU in Syn61, tRNASer(CGA), is a decoder of TCA codons. Despite the seemingly poor efficiency of this interaction, the presence of serU was sufficient to circumvent the genetic firewall imposed by serT knockout in Syn61, allowing biologically relevant levels of expression of TCA-containing transgenes.

[0207] Compressed Codon Content in Genes Repressed by GROs

[0208] With this unexpected observation, the compressed codon thresholds that govern gene repression in tRNA knockout GROs was next examined. A set of nine plasmids containing the gene encoding Nanoluciferase (NanoLuc) were generated, a 192 aa protein with 6 serine residues designed to be encoded by a differing number of TCA and / or TCG codons. When transformed into the tRNA knockout GRO panel (Syn61, Syn61AT, Syn61AU, and Syn61A3), the presence of TCG codons in combination with serT and serU double knockout resulted in the tightest repression. With at least 3 TCG codons, it was found Syn61 A3 exhibited the greatest repression, with over 4 orders of magnitude reduced NanoLuc expression. In contrast, luminescence from Syn61 AT with NanoLuc containing as many as 6 TCA codons was only reduced by less than 2Client Ref No. 24-0106Attny Docket No. UNC2.43224.601 orders of magnitude, consistent with the observations above, while there was no change in expression observed with any TCA or TCG combination in Syn61AU (Figure 13A). How these recoded codon thresholds extend beyond a small reporter gene was next investigated.

[0209] Site-specific recombinases (SSRs) are commonly used elements in gene circuits, but their exceptionally high recombination efficiency means that even minimal leaky expression can profoundly impact gene circuit fimction51,52. A panel of plasmids were again engineered containing (i) the Bxbl SSR gene (500 aa with 25 serine residues)53with varying numbers of TCA and TCG codons, and (ii) attP and attB sites (the sequences the SSR acts upon) oriented in such a manner that their recombination by the SSR (which forms a distinct attL site) will excise ~800 bp from the plasmid. This panel of Bxbl SSR expression plasmids in the same tRNA knockout GRO panel was examined by performing longread nanopore sequencing of plasmid DNA purified from the whole transformant population to quantify the number of plasmid molecules that had undergone recombination (Figure 13B). As expected, 100% of the sequenced plasmid molecules were recombined in Syn61 serU and serT competent). Consistent with the NanoLuc results, no recombination was seen with plasmids containing 6 to 25 TCG codons in Syn61A3, suggesting complete repression with as few as 6 TCG codons. In Syn61 AT, plasmids with 6-12 TCA codons in the SSR gene exhibited low to moderate levels of repression, while the plasmid with 25 TCA codons in the SSR gene yielded full repression. These results suggest a much larger TCA codon threshold is needed to effectively shut off expression in GROs lacking serT. Interestingly, less than 100% recombination was observed in Syn61 AT when all 25 serines in the SSR gene were encoded by TCG codons. In Syn61 AU, little to no recombination was observed with the presence of 6-25 TCA codons and 6-12 TCG codons. However, similar to Syn61 AT, less than half the sequenced plasmid molecules were recombined with 25 TCG codons present (Figure 13C).

[0210] The recently developed GRO, Syn61, is built in part on the longstanding assumption that serU (tRNASer(CGA)) decodes only TCG, and TCA is only decoded by serT (tRNASer(UGA)) (Figure 12A). Thus, serine codon compression that eliminates all TCA and TCG codons would allow for removal of both serU and serT. In this context, knocking out serT alone from Syn61 should also prevent the decoding and expression of proteins encoded by genes containing any number ofClient Ref No. 24-0106Attny Docket No. UNC2.43224.601TCA codons. Here, it is shown that, contrary to this prevailing understanding46-50, serU can actually decode TCA.

[0211] While translation of genes containing TCA codons in Syn61 AT was detectable in all assays used here (including the serU rescue assay in Syn61 A3 (Figure 121)), indicating decoding of TCA by serU, it should be noted that the translation efficiency with this underappreciated tRNA interaction appears relatively low. In the antibiotic resistance assay, there was a >98% reduction in the number of Syn61 AT colonies that formed when the KanR CDS contained TCA. In the Nanoluc assay, there was >95% reduction in the amount of luminescence the Syn61 AT sample produced compared to the Syn61 sample when the Nanoluc CDS contained 6 TCA codons. Finally, in the recombination assay, there was a >87% reduction in the amount of recombination observed in Syn61 AT when the recombinase CDS contained 25 TCA codons. These results clearly underscore that serT deletion can offer meaningful reductions in protein expression. It is, thus, only when exceptionally stringent repression is needed that serT deletion alone is inadequate. This also likely explains why the decoding of TCA by serU was not previously discovered.

[0212] Recent developments in the understanding of decoding indicate that the ribosome restricts the geometry of the first and second base pairs during codon-anticodon pairing in the decoding center of the ribosome, such that they must be canonical Watson-Crick base pairs and maintain a Watson-Crick-like helix54. The geometry of the third pair is not constrained, however. Thus, the data put forth in this Example suggests that, while not a canonical Watson-crick pair, the A«C wobble pair — which can occur through trans Watson-Crick / Watson-Crick, trans Watson-Crick / Hoogsteen, and trans Watson-Crick / sugar-edge base pairing55— observed at the third position of the TCA-CGA interaction when serU decodes TCA is likely tolerated and sufficient to allow decoding. Despite this, perfect Watson-Crick pairing of the first two bases of the TCA codon only has moderate thermodynamic favorability (-2.2 kcal / mol vs. -3.1 kcal / mol when the first two bases contain only G / C). By further decreasing favorability through an A«C wobble pair, TCA-CGA pairing may not be kinetically favored. While this may help explain both why it is so often misreported that TCA codons cannot be decoded by serU, and why dramatically reduced expression is seen in the data when TCA codons are present in a Syn61 AT-Client Ref No. 24-0106Attny Docket No. UNC2.43224.601 translated transcript, further experiments are needed to mechanistically valid the interactions between serU and TCA.

[0213] Beyond the unexpected serU and TCA interactions, this data demonstrate the parameters by which complete repression of gene expression can be achieved in Syn61. Specifically, within the sensitivity of the assays, it was found that knocking out both serU and serT affords complete repression of proteins encoded with as few as 6 TCG codons in its gene. In contrast, when only serU is knocked out, expression levels do not change, as serT can adequately decode TCA and TCG codons. More importantly, when only serT is knocked out, expression is only markedly reduced (though still not completely prevented) when as many as 25 TCA codons are present in a gene. This observed reduction in expression is likely attributed to multiple factors. In addition to the potential contribution of the unfavorable kinetics of the TCA-serU interaction during decoding, the more dramatic reduction in expression was observed in the recombination assay when the gene contained 25 TCA codons may also be due, in part, to a bottleneck caused by the size of the serU tRNA pool. SerU is the lowest abundance serine tRNA, with only ~25% the amount of serT molecules46, and it is possible that such a high number of TCA codons in a constitutively expressed transcript driven by a strong promoter may over burden this relatively small resource pool. Importantly, this data also indicates that TCG codons yield superior levels of repression in Syn61 A3, compared to the same number of TCA codons (by roughly an order of magnitude, on average). Together, these results provide a clear blueprint for a method of blocking transgene expression with exquisite control.

[0214] It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the disclosure, which is defined solely by the appended claims and their equivalents.

[0215] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the disclosure, may be made without departing from the spirit and scope thereof.

[0216] For reasons of completeness, various aspects of the disclosure are set out in the following numbered clauses:Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0217] Clause 1. A bacterial production strain, wherein the production strain does not contain any serT and serU tRNA and further wherein the production strain comprises a vector encoding a sitespecific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) one or more nucleic acids or transgenes of interest; and (iii) a nucleic acid sequence encoding a second att site.

[0218] Clause 2. The bacterial production strain of clause 1, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0219] Clause 3. The bacterial production strain of clause 1 , wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

[0220] Clause 4. The bacterial production strain of clause 3, wherein the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0221] Clause 5. The bacterial production strain of clause 1 , wherein the vector further comprises at least one origin of replication (ori).

[0222] Clause 6. The bacterial production strain of clause 5, wherein the ori is located between the a nucleic acid sequence encoding a first and second att site.

[0223] Clause 7. The bacterial production strain of any of clauses 1-6, wherein the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0224] Clause 8. The bacterial production strain of clause 1, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidennidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0225] Clause 9. The bacterial production strain of clause 8, wherein the bacteria is E. coli.

[0226] Clause 10. The bacterial production strain of clause 9, wherein the production strain is Syn61.

[0227] Clause 11. The bacterial production strain of any of clauses 1-10, wherein the recombinase is Flp, KD, X-INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0228] Clause 12. The bacterial production strain of any of clauses 1-11, wherein the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA, TCG or TCA and TCG codons in any essential gene.

[0229] Clause 13. The bacterial production strain of clause 12, wherein the helper phage is derived from T7, Pl, , OcrAssOOl, Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, <pl 1, SEP1, vB _SepS_SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDAO, phi-adh, Lv- 1, Lu-1, vB Gva ABl.

[0230] Clause 13. The bacterial production strain of clause 12, wherein the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0231] Clause 14. The bacterial production strain of any of clauses 1-13, wherein the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini-protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0232] Clause 15. A bacterial production strain, wherein the production strain does not contain any serT and serU tRNAs and further wherein the production strain comprises a vector encoding a protein, wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the protein.

[0233] Clause 16. The bacterial production strain of clause 15, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0234] Clause 17. The bacterial production strain of clause 15, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the protein.

[0235] Clause 18. The bacterial production strain of clause 17, wherein the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence of the protein.

[0236] Clause 19. The bacterial production strain of clause 15, wherein the vector further comprises at least one origin of replication (ori).

[0237] Clause 20. The bacterial production strain of clause 19, wherein ori is located between the nucleic acid sequence encoding a first and second att site.

[0238] Clause 21. The bacterial production strain of clause 15, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, aClient Ref No. 24-0106Attny Docket No. UNC2.43224.601Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0239] Clause 22. The bacterial production strain of clause 21, wherein the bacteria is E. coli.

[0240] Clause 23. The bacterial production strain of any of clauses 15-23, wherein the protein is a HIV-1 env gene, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes including endonucleases and exonucleases, toxin-antitoxin systems, eukaryotic RNase A Bamase, MazF endoribonuclease, RelE, phage holins or endolysins, and / or ccdB toxin, and the human apoptosis modulator protein Bax.

[0241] Clause 24. A self-circularizing non-replicative phage vector (scNRPV) production system comprising:

[0242] a. a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene; and

[0243] b. a bacterial production strain wherein the production strain does not contain any serT and serU tRNA and further wherein the production strain comprises a vector encoding a site- specific recombinase (SSR) wherein the vector comprises one or more TCA or TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or transgene of interest; and (iii) a nucleic acid sequence encoding a second att site.

[0244] Clause 25. The system of clause 24, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0245] Clause 26. The system of clause 24, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

[0246] Clause 27. The system of clause 26, wherein the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0247] Clause 28. The system of clause 24, wherein the vector further comprises at least one origin of replication (ori).

[0248] Clause 29. The system of clause 28, wherein ori is located between the nucleic acid sequence encoding a first and second att site.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0249] Clause 30. The system of any of clauses 24-29, wherein the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0250] Clause 31. The system of clause 24, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0251] Clause 32. The system of clause 31, wherein the bacteria is E. coli.

[0252] Clause 33. The system of clause 32, wherein the production strain is Syn61.

[0253] Clause 34. The system of any of clauses 24-33, wherein the recombinase is F1P, KD, X- INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0254] Clause 35. The system of any of clauses 24-33, wherein the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA or TCG codons in any essential gene.

[0255] Clause 35A. The system of clause 35, wherein the helper phage is derived from T7, Pl, X, crAssOOl , Hankyphage, B40-8, B56-3, Brigit, Oengus, phiCD27, K, <pl 1, SEP1, vB _SepS_SEP9, PAD20, M102AD, FNU1, Dp-1, Cp-1, HP1, MDA<b, phi-adh, Lv-1, Lu-1, vB Gva ABl.

[0256] Clause 36. The system of clause 35 or clause 35 A, wherein the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0257] Clause 37. The system of any of clauses 24-36, wherein the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini-protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0258] Clause 38. A self-circularizing non-replicative phage vector (scNRPV) comprising: a vector that encodes a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG, or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and further wherein the scNRP does not contain any TCG or TCA codons in any essential gene.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0259] Clause 39. The scNRPV of clause 38, wherein the recombinase is F1P, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0260] Clause 40. The scNRPV of clause 38 or clause 39, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

[0261] Clause 41. The scNRPV of clause 40, wherein the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in the coding sequence of the SSR.

[0262] Clause 42. The scNRPV of any of clauses 38-41, wherein the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini-protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0263] Clause 43. A composition comprising a scNRPV of any of clauses 38-42.

[0264] Clause 44. A method of producing a self-circularizing non-replicative phage comprising a nucleic acid or transgene of interest, the method comprising the steps of:

[0265] a. infecting a bacterial production strain with a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene,

[0266] wherein the production strain does not contain any serT or serU tRNA, and

[0267] further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and fiirther comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and

[0268] still further wherein the vector is incorporated into progeny of helper phage producing a self-circularizing non-replicative phage vector; and

[0269] a. recovering the self-circularizing non-replicative phage produced by the bacterial production strain.

[0270] Clause 45. The method of clause 44, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0271] Clause 46. The method of clause 44, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in a coding sequence.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0272] Clause 47. The method of clause 46, wherein the vector comprises 5 to 15 TCA, TCG, or TCA and TCG codons in a coding sequence.

[0273] Clause 48. The method of clause 44, wherein the vector further comprises at least one origin of replication (ori).

[0274] Clause 49. The method of clause 48, wherein ori is located between the nucleic acid sequence encoding a first and second att site.

[0275] Clause 50. The method of any of clauses 44-49, wherein the vector further comprises a polynucleotide sequence encoding a packing signal (pac).

[0276] Clause 51. The method of any of clauses 44-50, wherein the bacteria is E. coll, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0277] Clause 52. The method of clause 51, wherein the bacteria is E. coli.

[0278] Clause 53. The method of clause 52, wherein the production strain is Syn61.

[0279] Clause 54. The method any of clauses 44-53, wherein the recombinase is F1P, KD, X-INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

[0280] Clause 55. The method of any of clauses 44-54, wherein the production strain further comprises at least one helper plasmid or at least one helper phage which does not contain any TCA or TCG codons in any essential gene.

[0281] Clause 56. The method of clause 55, wherein the at least one helper plasmid or at least one helper phage further comprises at least one tail fiber gene.

[0282] Clause 57. The method of any of clauses 44-56, wherein the nucleic acid or transgene of interest encodes insulin, somatropin, an interferon, tumor necrosis factor, an interleukin, an immunoglobulin, a vitamin, a short chain fatty acid, a bacteriocin, an antiviral, an antifungal agent, an immunogen, a hormone, a GLP-1 agonist, an enzyme, a mini-protein binder, a biopolymer such as collagen or hyaluronic acid, or any combination thereof.

[0283] Clause 58. The method of any of clauses 44-57, wherein the method further comprises purifying the self-circularizing non-replicative phage vector produced in step b).Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0284] Clause 59. A self-circularizing non-replicative phage produced by the method of any one of clauses 44-58.

[0285] Clause 60. A method of modifying bacteria located in or on a subject, the method comprising the step of: administering a therapeutically effective amount of a composition of claim 43 to a subject in need thereof to modify the bacteria residing in or on the subject.

[0286] Clause 60A. The method of clause 60, wherein the bacteria reside in or on a gastrointestinal tract, respiratory tract, urogenital system (e.g., vaginal microbiome, urinary tract, or upper reproductive tract), oral cavity, skin, and / or ocular system (e.g., conjunctiva and ocular surface).

[0287] Clause 61. The method of clause 60 or clause 61 , wherein the subject is a human or nonhuman animal.

[0288] Clause 62. A method of modifying bacteria located in an environment, the method comprising the step of: applying an effective amount of a composition to an environment in which bacteria reside to modify the bacteria located in the environment.

[0289] Clause 62A. The method of clause 62, wherein the bacteria reside in or on a plant or in or on soil, water, and / or air.

[0290] Clause 63. A method of producing a self-circularizing non-replicative phage comprising a nucleic acid or transgene of interest, the method comprising the steps of:

[0291] a. introducing into a bacterial production strain a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene,

[0292] wherein the production strain does not contain any serT or serU tRNA, and

[0293] further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and

[0294] still further wherein, the vector is incorporated into progeny of helper phage producing a self-circularizing non-replicative phage vector; and

[0295] b. recovering the self-circularizing non-replicative phage produced by the bacterial production strain.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0296] Clause 64. The method of clause 63, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

[0297] Clause 65. The method of clauses 63 or clause 64, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in a coding sequence.

[0298] Clause 66. The method of any of clauses 63-65, wherein the helper phage is introduced into the bacterial production strain: (a) by infecting the bacterial production strain with the helper phage; (b) as an episome; or (c) integrating the helper phage into a chromosome of the bacterial production strain.

[0299] Clause 67. The method of any of clauses 63-66, wherein the bacteria is E. coll, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

[0300] Clause 68. A self-circularizing non-replicative phage produced by the method of any of clauses 63-68.

[0301] Clause 69. A bacteria production system comprising: a bacteria comprising at least one vector, wherein the vector comprises one or more nucleic acids or transgenes of interest that encode one or more toxic nucleic acids or one or more unstable polynucleotide sequences, and further wherein the bacteria does not contain any serT and serU tRNA.

[0302] Clause 70. The bacterial production system of clause 69, where the one or more toxic nucleic acid and / or unstable nucleic acid sequence encode a HIV-1 env protein, Pseudomonas exotoxin A, cholera toxin, diphtheria toxin, E. coli toxins, botulinum toxin, anthrax toxin, pertussis toxin, shiga toxin, ricin, tetanus toxin, Staphylococcal toxins, restriction enzymes, such as, endonucleases and exonucleases, toxin-antitoxin systems, eukaryotic RNase A, Bamase, MazF endoribonuclease, RelE, phage holins or endolysins, ccdB toxin, and a human apoptosis modulator protein Bax.

[0303] Clause 71. A method of producing an unstable polynucleotide sequence or toxic nucleic acid, the method comprising the steps of:Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0304] a. introducing into the bacteria production strain of clause 23 one or more nucleic acids or transgenes of interest, wherein the nucleic acids or transgenes of interest are one or more toxic nucleic acids or unstable polynucleotide sequences;

[0305] b. growing the bacterial production strain; and

[0306] c. recovering the toxic DNA or unstable polynucleotide sequence from the bacterial production strain.Clause 72. A method of producing a replicative phage vector, the method comprising the steps of:

[0307] a. infecting the bacterial production strain of clause 23 with a helper phage or phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential genes, wherein the bacterial production strain does not contain any serT or serU tRNA, and further wherein the helper phage or phage comprises at least one nucleic acid sequences or transgenes of interest that comprise one or more TCA, TCG or TCA and TCG codons in the coding sequence in a 5’ to 3’ direction; and

[0308] b. recovering the phage produced by the bacterial production strain.

[0309] Clause 73. An engineered bacterial strain comprising: a bacteria comprising at least one plasmid, wherein the plasmid comprises: (a) one or more nucleic acids or transgenes of interest; and 0?) one or more nucleic acids or transgenes that encode one or more toxins incorporating one or more TCA, TCG or TCA and TCG codons, wherein: (i) the one or more nucleic acids or transgenes that encode one or more toxins prevent the spread of the nucleic acids or transgenes of interest into an unintended organism; and (ii) the bacterial strain is deficient in serU and serT.

[0310] Clause 74. The engineered bacterial strain of clause 73, wherein the one or more nucleic acids or transgenes of interest are integrated into the genome of the plasmid.

[0311] Clause 75. The engineered bacterial strain of clause 73 or clause 74, wherein the one or more nucleic acids or transgenes that encode one or more toxins is adjacent to the one or more nucleic acids or transgenes of interest.

[0312] Clause 76. A method of delivering a plasmid to one or more recipient bacterial cells via bacterial conjugation, the method comprising:Client Ref No. 24-0106Attny Docket No. UNC2.43224.601

[0313] a. providing a donor bacterial strain that is deficient in one or more tRNA genes selected from serU and serT and comprises a plasmid comprising one or more nucleic acids or transgenes of interest enriched in codons TCA, TCG or TCA and TCG; and

[0314] b. delivering the plasmid from the donor bacterial strain to one or more recipient bacterial cells using bacterial conjugation.

[0315] Clause 77. The method of clause 76, wherein the one or more nucleic acids or transgenes of interest are translationally repressed in the donor bacterial strain.

[0316] Clause 78. The method of clause 76 or clause 77, wherein the plasmid in the donor strain is a non-replicative plasmid or a self-replicating plasmid.

[0317] Clause 79. The method of any of clauses 76-78, wherein the bacterial conjugation is a F plasmid transfer system, a RK2 / RP4 broad-host-range conjugation system, a IncP-type conjugation system, a R388 plasmid transfer system, the pKMIOl or pOX38 derivatives, or combinations thereof.

[0318] Clause 80. The method of any of clauses 76-79, wherein the recipient bacterial cells reside on or in an environment.

[0319] Clause 81. The method of clause 80, where the environment is on or in a plant or on or in soil, air, or water.

[0320] Clause 82. The method of claims 81, wherein the water is fresh water, salt or sea water, sewage water, pond, lake and / or river water, rainwater, or any combination thereof.

[0321] Clause 83. The method of any of claims 76-79, wherein the recipient bacterial cells reside at one or more locations in a human or non-human mammal.

[0322] Clause 84. A composition comprising: a bacteria strain, wherein the bacterial strain comprises at least one plasmid, wherein the plasmid comprises: (a) one or more nucleic acids or transgenes of interest that incorporate one or more TCA, TCG or TCA and TCG codons; and (b) a conjugation system capable of transferring the plasmid to one or more recipient bacterial cells, wherein: (i) one or more nucleic acids or transgenes are translationally repressed in the donor strain; and (ii) the bacterial strain is deficient in serU and serT.Client Ref No. 24-0106Attny Docket No. UNC2.43224.601References:1. Cani, P. D. Human gut microbiome: hopes, threats and promises. Gut 67, 1716-1725 (2018).2. Lynch, S. V. & Pedersen, O. The human intestinal microbiome in health and disease. New England Journal of Medicine vol. 375 2369-2379 Preprint at https: / / d0i.0rg / l 0.1056 / NEJMral 600266 (2016).3. Gilbert, J. A. et al. Microbiome-wide association studies link dynamic microbial consortia to disease. Nature vol. 535 94—103 Preprint at https: / / doi.org / 10.1038 / naturel8850 (2016).4. Gilbert, J. A. et al. Current understanding of the human microbiome. Nat Med 24, 392- 400 (2018).5. Lemire, S., Yehl, K. M. & Lu, T. K. Phage-Based Applications in Synthetic Biology. Annu Rev Virol 5, 453^176 (2018).6. Mimee, M., Citorik, R. J. & Lu, T. K. Microbiome therapeutics — Advances and challenges. Advanced Drug Delivery Reviews vol. 105 44 54 Preprint at https: / / d0i.0rg / l 0.1016 / j .addr.2016.04.032 (2016).7. Pires, D. P., Cleto, S., Sillankorva, S., Azeredo, J. & Lu, T. K. Genetically Engineered Phages: a Review of Advances over the Last Decade. Microbiology and Molecular Biology Reviews 80, 523-543 (2016).8. Lenneman, B. R., Fembach, J., Loessner, M. J., Lu, T. K. & Kilcher, S. Enhancing phage therapy through synthetic biology and genome engineering. Curr Opin Biotechnol 68, 151-159 (2021).9. Hsu, B. B., Way, J. C. & Silver, P. A. Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut. mSystems 5, (2020).10. Hsu, B. B. et al. In situ reprogramming of gut bacteria by oral delivery. Nature Communications 202011 : 1 11, 1-11 (2020).11. Pires, D. P. et al. Designing P. aeruginosa synthetic phages with reduced genomes. Scientific Reports 2021 11:1 11, 1-10 (2021).12. Tridgett, M., Ababi, M., Osgerby, A., Ramirez Garcia, R. & Jaramillo, A. Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery. ACS Synth Biol 10, 107- 114 (2021).13. Lam, C. N. et al. A Tail Fiber Engineering Platform for Improved Bacterial Transduction- Based Diagnostic Reagents. ACS Synth Biol (2021) doi:10.1021 / ACSSYNBI0.1C00036.14. Damelin, L. H., Mavri-Damelin, D., Klaenhammer, T. R. & Tiemessen, C. T. Plasmid Transduction Using Bacteriophage <badh for Expression of CC Chemokines by Lactobacillus gasseri ADH. Appl Environ Microbiol 76, 3878 (2010).15. Lovett, S. T. The DNA exonucleases of Escherichia coli. EcoSal Plus 4, (2011).16. Yosef, I., Goren, M. G., Globus, R., Molshanski-Mor, S. & Qimron, U. Extending the Host Range of Bacteriophage Particles for DNA Transduction. Mol Cell 66, 721-728.e3 (2017).17. Auster, O., Globus, R., Yosef, I. & Qimron, U. Optimizing DNA transduction by selection of mutations that evade bacterial defense systems. RNA Biol 16, 595 (2019).18. Holtzman, T. et al. A continuous evolution system for contracting the host range of bacteriophage T7. SciRep 10, 1-8 (2020).19. Chung, Y. B. & Hinkle, D. C. Bacteriophage T7 DNA Packaging. I. Plasmids containing a T7 replication origin and the T7 concatemer junction are packaged into transducing particles during phage infection. J Mol Biol 216, 911-926 (1990).Client Ref No. 24-0106Attny Docket No. UNC2.43224.601 Yue, H. et al. T7 Phage as an Emerging Nanobiomaterial with Genetically Tunable Target Specificity. Advanced Science 9, 2103645 (2022). Chung, Y. B. & Hinkle, D. C. Bacteriophage T7 DNA Packaging. II. Analysis of the DNA sequences required for packaging using a plasmid transduction assay. J Mol Biol 216, 927 938 (1990). Dunn, J. J., Studier, F. W. & Gottesman, M. Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J Mol Biol 166, 477-535 (1983). Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U SA 97, 6640-6645 (2000). Meyer, A. J., Segall- Shapiro, T. H., Glassey, E., Zhang, J. & Voigt, C. A. Escherichia coli “Marionette” strains with 12 highly optimized small -molecule sensors. Nature Chemical Biology 2018 15:2 15, 196-204 (2018). Voorhees, P. J., Chang, X. & Lai, S. K. Defining serine tRNA knockout as a strategy for effective repression of gene expression in organisms with a recoded genome. Nucleic Acids Res 53, 1266 (2025). Fredens, J. et al. Total synthesis of Escherichia coli with a recoded genome. Nature 2019 569:7757569, 514-518 (2019). Cheng, Y. W. & Fischer, M. Fecal Microbiota Transplantation. Clin Colon Rectal Surg 36, 151-156 (2023). Khoruts, A. Targeting the microbiome: From probiotics to fecal microbiota transplantation. Genome Med 10, 1-3 (2018). Brussow, H. & Hendrix, R. W. Phage Genomics: Small Is Beautiful. Cell 108, 13 16 (2002). Yuan, S., Shi, J., Jiang, J. & Ma, Y. Genome-scale top-down strategy to generate viable genome-reduced phages. Nucleic Acids Res 50, 13183-13197 (2022). Black, L. W. DNA packaging in dsDNA bacteriophages. Annu Rev Microbiol 43, 267- 292 (1989). Roos, W. H., Ivanovska, I. L., Evilevitch, A. & Wuite, G. J. L. Viral capsids: Mechanical characteristics, genome packaging and delivery mechanisms. Cellular and Molecular Life Sciences 200764:12 64, 1484-1497 (2007). Earnshaw, W. C. & Casjens, S. R. DNA packaging by the double-stranded DNA bacteriophages. Cell 21, 319-331 (1980). Hendrix, R. W. Bacteriophage DNA packaging: RNA gears in a DNA transport machine. Cell 94, 147-150 (1998). Gameau, J. R., Depardieu, F., Fortier, L. C., Bikard, D. & Monot, M. PhageTerm: A tool for fast and accurate determination of phage termini and packaging mechanism using next-generation sequencing data. Sci Rep 7, (2017). Solar, G. del, Giraldo, R., Ruiz-Echevarria, M. J., Espinosa, M. & Diaz-Orejas, R. Replication and Control of Circular Bacterial Plasmids. Microbiology and Molecular Biology Reviews 62, 434 (1998). Jain, A. & Srivastava, P. Broad host range plasmids. FEMS Microbiol Lett 348, 87 96 (2013).Client Ref No. 24-0106Attny Docket No. UNC2.43224.601 Xu, Z. et al. Accuracy and efficiency define Bxbl integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome. BMC Biotechnol 13, 1-17 (2013). Casjens, S. R. & Hendrix, R. W. Bacteriophage lambda: Early pioneer and still relevant. Virology vols 479 480 310 330 Preprint at https: / / doi.Org / 10.1016 / j.virol.2015.02.010 (2015). Fiess Michael & Catalano, C. E. Bacteriophage Lambda Terminase and the Mechanism of Viral DNA Packaging. Madame Curie Bioscience Database (2013). Brodel, A. K. et al. In situ targeted base editing of bacteria in the mouse gut. Nature 2024 632:8026632, 877-884 (2024). Boeke, J. D. et al. The genome project-write we need technology and an ethical framework for genome-scale engineering. Science (1979) 353, 126-127 (2016). Zhao, Z., Anselmo, A. C. & Mitragotri, S. Viral vector-based gene therapies in the clinic. Bioeng Transl Med 7, (2022). Rogers, A. T., Bullard, K. R., Dod, A. C. & Wang, Y. Bacterial Growth Curve Measurements with a Multimode Microplate Reader. Bio Protoc 12, (2022). Pryor, J. M., Potapov, V., Bilotti, K., Pokhrel, N. & Lohman, G. J. S. Rapid 40 kb Genome Construction from 52 Parts through Data-optimized Assembly Design. ACS Synth Biol 11, 2036-2042 (2022). dos Reis, M., Savva, R. & Wemisch, L. Solving the riddle of codon usage preferences: a test for translational selection. Nucleic Acids Res 32, 5036-5044 (2004). Wang, K. et al. Defining synonymous codon compression schemes by genome recoding. Nature 2016539:7627539, 59-64 (2016). Cui, Z., Johnston, W. A. & Alexandrov, K. Cell-Free Approach for Non-canonical Amino Acids Incorporation Into Polypeptides. Front Bioeng Biotechnol 8, 574140 (2020). Cui, Z., Stein, V., Tnimov, Z., Mureev, S. & Alexandrov, K. Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis. J Am Chem Soc 137, 4404—4413 (2015). Dong, H., Nilsson, L. & Kurland, C. G. Co-variation of tRNA abundance and codon usage in Escherichia coli at different growth rates. J Mol Biol 260, 649-663 (1996). Landau, J., Cuba Samaniego, C., Giordano, G. & Franco, E. Computational characterization of recombinase circuits for periodic behaviors. iScience 26, 105624 (2023). Kim, T., Weinberg, B., Wong, W. & Lu, T. K. Scalable recombinase-based gene expression cascades. Nature Communications 2021 12:1 12, 1-9 (2021). Singh, S., Ghosh, P. & Hatfull, G. F. Attachment Site Selection and Identity in Bxbl Serine Integrase-Mediated Site-Specific Recombination. PLoS Genet 9, el 003490 (2013). Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. A new understanding of the decoding principle on the ribosome. Nature 2012 484:7393 484, 256-259 (2012). Leontis, N. B., Stombaugh, J. & Westhof, E. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res 30, 3497-3531 (2002). atsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U SA 97, 6640-6645 (2000).

Claims

Client Ref No. 24-0106Attny Docket No. UNC2.43224.601WHAT IS CLAIMED IS:

1. A bacterial production strain, wherein the production strain does not contain any serT and serU tRNA and further wherein the production strain comprises a vector encoding a sitespecific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) nucleic acid or a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site.

2. The bacterial production strain of claim 1, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

3. The bacterial production strain of claim 1, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

4. The bacterial production strain of claim 1 , wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacteroides, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, aPropionibacterium acnes, a Corynebacterium or any combination thereof.

5. A bacterial production strain of claim 1 , wherein the production strain does not contain any serT and serU tRNAs and further wherein the production strain comprises a vector encoding a protein, wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the protein.

6. The bacterial production strain of claim 5, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

7. The bacterial production strain of claim 5, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the protein.

8. The bacterial production strain of claim 5, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIVA, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, aPropionibacterium acnes, a Corynebacterium or any combination thereof.Client Ref No. 24-0106Attny Docket No. UNC2.43224.6019. A self-circularizing non-replicative phage vector (scNRPV) production system comprising: a. a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene; and b. a bacterial production strain wherein the production strain does not contain any serT and serU tRNA and further wherein the production strain comprises a vector encoding a site-specific recombinase (SSR) wherein the vector comprises one or more TCA or TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site.

10. The system of claim 9, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

11. The system of claim 9, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in the coding sequence of the SSR.

12. The system of claim 9, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

13. A self-circularizing non-replicative phage vector (scNRPV) comprising: a vector that encodes a site-specific recombinase (SSR) wherein the vector comprises one or more TCA, TCG, or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and further wherein the scNRPV does not contain any TCG or TCA codons in any essential gene.

14. The scNRPV of claim 13, wherein the recombinase is F1P, KD, -INT, Cre, Bxbl, Gin, Tn3, B2, or Vika.

15. A composition comprising a scNRPV of claim 13.Client Ref No. 24-0106Attny Docket No. UNC2.43224.60116. A method of producing a self-circularizing non-replicative phage comprising a nucleic acid or transgene of interest, the method comprising the steps of: a. introducing into a bacterial production strain a helper phage that does not contain any TCG, TCA, or TCG and TCA codons in any essential gene, wherein the production strain does not contain any serT or serU tRNA, and further wherein the production strain comprises a vector encoding a sitespecific recombinase (SSR) wherein the vector comprises one or more TCA, TCG or TCA and TCG codons in a coding sequence of the SSR and further comprises in the 5’ to 3’ direction: (i) polynucleotide sequence encoding a first attachment (att) site; (ii) a nucleic acid or a transgene of interest; and (iii) a nucleic acid sequence encoding a second att site, and still further wherein the vector is incorporated into progeny of helper phage producing a self-circularizing non-replicative phage vector; and b. recovering the self-circularizing non-replicative phage produced by the bacterial production strain.

17. The method of claim 16, wherein the production strain does not contain any TCG, TCA, or any TCG or TCA codons in any essential gene.

18. The method of claim 16, wherein the vector comprises at least 5 TCA, TCG or TCA and TCG codons in a coding sequence.

19. The method of claim 16, wherein the helper phage is introduced into the bacterial production strain: (a) by infecting the bacterial production strain with the helper phage; (b) as an episome; or (c) integrating the helper phage into a chromosome of the bacterial production strain.

20. The method of claim 16, wherein the bacteria is E. coli, a Proteobacteria, a Fusobacteria, a Bacterodies, an E. Rectale, a Ruminococcus gnavus, a Clostridium perfringens, a Clostridia in groups IV and XIV A, a Faecalibacterium prausnitzii, a Bifidobacteria, a Lacatobacilli, a Lactococcus lactis, a Staphylococcus epidermidis, a Streptococcus mitis, a Micrococcus luteus, a Propionibacterium acnes, a Corynebacterium or any combination thereof.

21. A self-circularizing non-replicative phage produced by the method of claim 16.Client Ref No. 24-0106Attny Docket No. UNC2.43224.60122. A method of modifying a bacteria located in or on a subject, the method comprising administering a therapeutically effective amount of a composition of claim 15 to a subject in need thereof to modify the bacteria residing in or on the subject.

23. The method of claim 22, wherein the bacteria reside in or on a gastrointestinal tract, a respiratory tract, a urogenital system, an oral cavity, skin, and / or ocular system.

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