Cutibacterium acnes recombinant phage, method of production thereof, and use

Abstract

The present invention relates to P. acnes strains carrying a DNA vector for the production of recombinant P. acnes phage. The invention includes P. acnes producer cells carrying a DNA vector having a template suitable for homologous recombination with the P. acnes phage genome, resulting in the insertion of a gene of interest, for the production of recombinant phage capable of conferring transgene expression in P. acnes infected with the recombinant phage. The invention includes P. acnes strains containing the vector, P. acnes recombinant phage, and methods of using the recombinant phage.

Description

[Technical Field]

[0001] This invention relates to a recombinant Cutibacterium acnes phage and a method for producing the same. [Background technology]

[0002] The skin is the largest organ in the human body and the largest interface between our body and the environment. Therefore, the skin also acts as a barrier protecting us from physical threats (e.g., UV rays, trauma), chemical threats (e.g., acids, bases), and microbial threats (viruses, bacteria, fungi). This defense is not merely a result of the passive physical isolation of the skin, which consists of a continuous layer of densely and interconnected dead cells (stratum corneum) surrounded by a lipid matrix. It is also thanks to an active mechanism integrated by diverse types of skin and immune cells that secrete antimicrobial peptides (AMPs), produce cytokines and chemokines to recruit lymphocyte immune cells, sense skin damage, and trigger wound healing mechanisms among other processes. 1 .

[0003] The skin is the first organ we come into contact with microorganisms after birth, and because it contains a vast number of immune cells in close contact with a wide variety of microorganisms, the skin immune system needs to develop the ability to distinguish beneficial microorganisms from pathogenic ones in order to avoid constant immune responses and inflammation. Part of this education occurs when certain bacterial species colonize the skin early in life, and the immune response is modified so that those bacterial species can be tolerated. 2 In that case, this specific bacterial species can stably colonize the skin, establish a community, and become a symbiotic strain.

[0004] The skin is not physiologically and spatially homogeneous throughout the body. That is, depending on the body site, there are fatty skin (e.g., cheeks, back), moist skin (e.g., inguinal crease, interdigital web space, antecubital crease), and dry skin (e.g., volar forearm, hypothenar palm). 3 These different body sites are associated with different physiological conditions and have unique microbiomes, with Cutibacterium acnes (formerly known as Propionibacterium acnes) predominantly colonizing fatty sites and higher amounts of Staphylococcus and Corynebacterium species in moist sites. 4 In addition to these physiological characteristics, the skin is further heterogeneous in spaces with different appendages, namely, sweat glands, hair follicles, and sebaceous glands. Colonization in these appendages has only recently been studied and shows differences compared to the skin surface (stratum corneum). 4~6 .

[0005] These skin appendages are specific anatomical locations as they do not have a stratum corneum. As a result, the microorganisms within these appendages come into contact with living keratinocytes and, due to their proximity to the dermis, can access a more diverse range of immune cells. Hair follicles have specific immunological properties. Hair follicles can recruit specific immune cells, such as monocyte-derived Langerhans cell precursors, 7 and can actively maintain resident memory T cells (TRM). 8 Therefore, hair follicles are potential essential sites for antigen presentation. Hair follicles also have a paucity of effector T cells and a strongly immunosuppressive environment, and thus, hair follicles are immune-privileged regions. 9 .

[0006] Examples in the published literature show that commensal skin bacteria actively participate in host immunity through intact skin barriers and activate specific immune cells in a species- and strain-dependent manner (Chen et al., Nature 2018;555(7697):543 (Non-Patent Document 1)). For example, some, but not all, Staphylococcus epidermidis induce the activation of CD8 T cells (Naik et al., Nature 2015, 520(7545):104-108 (Non-Patent Document 2)). + CD8 + T cells (Naik et al., Nature 2015, 520(7545):104-108 (Non-Patent Document 2)).

[0007] Due to the absence of the stratum corneum, skin appendages are more permeable to chemicals. This is because chemicals only need to cross the tight junction barrier instead of the stratum corneum that usually prevents water exchange, and as a result, all water-soluble substances can diffuse.

[0008] Acne bacteria that mainly thrive in the sebum-rich anaerobic environment are mainly colonized in the pilosebaceous subunit including hair follicles and sebaceous glands. Cutibacterium acnes (formerly Propionibacterium acnes) is a Gram-positive anaerobic bacillus first isolated from the skin in 1897. It belongs to the order Actinomycetales, is part of the family Propionibacteriaceae, and belongs to the genus Cutibacterium. This genus also includes other human skin species, such as Cutibacterium avidum, Cutibacterium granulosum, and Cutibacterium humerusii 10It includes. Cutibacterium acnes is one of the most common and abundant bacteria on human skin and can be found in both the skin surface (stratum corneum) and hair follicles. Cutibacterium acnes is in direct contact with a considerably diverse population of live cells within the hair follicle, such as keratinocytes, stem cells, sebaceous gland cells, and immune cells, which is different from the stratum corneum that mainly contacts dead corneocytes. Cutibacterium acnes is a symbiotic bacterium, and furthermore, it is associated with several skin diseases, such as acne vulgaris 11 or progressive macular hypomelanosis 12~14 is associated with.

[0009] In particular, new discoveries regarding Cutibacterium acnes reveal that certain phylotypes can play a decisive role in the development of acne 11 . Specifically, the role of Cutibacterium acnes phylotype IA1 has been widely emphasized. Fitz-Gibbon and co-researchers demonstrated that loci 1, 2, and 3 of chromosomal regions, which are characteristic of ribotypes RT4 and RT5 (classified within phylotype IA1), are strongly associated with acne 15 . Since this chromosomal region is absent in ribotypes associated with healthy skin (i.e., RT6), this chromosomal region could be a potential target for removing acne-associated Cutibacterium acnes strains.

[0010] For preventing or treating diseases such as acne vulgaris, it is an interesting therapeutic approach to edit the Cutibacterium acnes population by removing specific proinflammatory strains or to utilize its privileged position in the hair follicle sebaceous gland subunit to regulate the host immune response or improve wound healing. To implement such an approach, it is possible either to genetically modify Cutibacterium acnes strains in situ or to provide Cutibacterium acnes that has been genetically modified in vitro. Due to the high microbiome diversity both at the species level and strain level within and between individuals, it seems difficult to provide a single engineered Cutibacterium acnes strain or a cocktail thereof that can colonize the skin of most patients.

Prior Art Documents

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[0013] In situ DNA delivery to *Propionibacterium acnes* populations offers a way to circumvent such difficulties without potentially disrupting the local microbiome, by enabling the use of preliminary established strains. However, in situ delivery of genetic material to *Propionibacterium acnes* presents several challenges. First, to date, there are no genetic elements, such as plasmids, that can replicate robustly and autonomously within *Propionibacterium acnes*. The few described genetic modifications consist of genomic insertions of synthetic DNA via homologous recombination. 16~18This in vitro process has been shown to be very inefficient and to rely on the use of antibiotic selection markers to select such events. Furthermore, this genetic recombination is limited to a few specific strains (KPA17202, RT6 of Propionibacterium acnes) and may not be generalizable to all Propionibacterium acnes strains. Secondly, in situ genetic recombination of Propionibacterium acnes requires the delivery of DNA into the bacteria. The only described method for introducing DNA into Propionibacterium acnes is the use of electroporation. 19、20 This method can only be performed in vitro. [Means for solving the problem]

[0014] This invention solves both the lack of reproducible and stable DNA vectors and their delivery into *Propionibacterium acnes* using phage-derived particles. Furthermore, this invention provides a unique and robust tool for manipulating *Propionibacterium acnes* phages.

[0015] The present invention includes Cutibacterium acnes phagemide, bacterial cells containing the phagemide, recombinant Cutibacterium acnes phage, methods for producing phage-derived particles containing the phagemide, phage-derived particles containing the phagemide, and methods for using the phagemide, particles, and cells in particular in the treatment of Cutibacterium acnes-related disorders and / or diseases.

[0016] The present invention comprises a recombinant DNA phagemide vector, a phage-derived particle containing the vector, and a Cutibacterium acnes that carries the vector, wherein the vector is - Phage packaging signals that enable the packaging of DNA vectors in Cutibacterium acnes phage capsids; and - Target gene Includes.

[0017] In one embodiment, the DNA vector is - A phage packaging signal that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76, enabling the packaging of a DNA vector within a Cutibacterium acnes phage capsid; and - Target gene Includes.

[0018] In one embodiment, the DNA vector is - Phage packaging signaling that enables the packaging of DNA vectors within Cutibacterium acnes phage capsids; - The target gene; and - Selection markers enabling selection of DNA vectors in Cutibacterium acnes Includes.

[0019] In one embodiment, the DNA vector is - A phage packaging signal that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76, enabling the packaging of a DNA vector within a Cutibacterium acnes phage capsid; - The target gene; and - Selection markers enabling selection of DNA vectors in Cutibacterium acnes Includes.

[0020] In one embodiment, the DNA vector is - Phage packaging signaling that enables the packaging of DNA vectors within Cutibacterium acnes phage capsids; - The target gene; - A replication origin that enables replication within Cutibacterium acnes; and - In some cases, a selection marker that allows selection of DNA vectors in Cutibacterium acnes. Includes.

[0021] In one embodiment, the DNA vector is - A phage packaging signal that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76, enabling the packaging of a DNA vector within a Cutibacterium acnes phage capsid; - The target gene; - A replication origin that enables replication within Cutibacterium acnes; and - In some cases, a selection marker that allows selection of DNA vectors in Cutibacterium acnes. Includes.

[0022] In one embodiment, the phage packaging signal sequence is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to a phage packaging signal sequence selected from the group consisting of the sequences of SEQ ID NO: 76, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

[0023] In one embodiment, the DNA vector further includes a replication origin for a *Propionibacterium acnes* phage.

[0024] In one embodiment, the DNA vector further includes a replication origin of a Propionibacterium acnes phage whose sequence is at least 75%, 77%, 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 67.

[0025] In one embodiment, the target gene is DNA that codes for an antigen.

[0026] The present invention includes Cutibacterium acnes-producing cells that carry a recombinant DNA vector for the production of Cutibacterium acnes phage-derived particles containing a recombinant DNA vector.

[0027] DNA vectors are typically packaged within proteins produced from the Cutibacterium acnes phage genome or helper phage. The Propionibacterium acnes phage genome can be introduced into Propionibacterium acnes-producing cells, for example, by transformation or transduction using the Propionibacterium acnes phage, while helper phages can be introduced into Propionibacterium acnes-producing cells, for example, by transformation or conjugation before or after the introduction of the DNA vector into the cells (Figure 1).

[0028] Cutibacterium acnes-producing cells that carry recombinant DNA vectors typically contain the Cutibacterium acnes phage genome, leading to the production of phage-derived particles that carry the DNA vector.

[0029] In one embodiment, the Cutibacterium acnes phage genome is the unmanipulated / wild-type genome.

[0030] In other embodiments, the Cutibacterium acnes phage genome is manipulated.

[0031] In one embodiment, the DNA vector includes an origin of replication that can only be replicated in Cutibacterium acnes-producing cells and cannot be replicated in Cutibacterium acnes-receiver cells.

[0032] In one embodiment, the DNA vector is - Phage packaging signaling that enables the packaging of DNA vectors within Cutibacterium acnes phage capsids; - At least one target gene; - A replication origin that enables replication only within Cutibacterium acnes-producing cells; and - In some cases, a selection marker that allows selection of DNA vectors in Cutibacterium acnes. Includes.

[0033] In one embodiment, the selection marker is a nutritional requirement marker, and the proliferation of Cutibacterium acnes-producing cells depends on that nutritional requirement marker.

[0034] In one embodiment, the selection marker is an antibiotic resistance marker.

[0035] In one embodiment, the DNA vector further comprises a CRISPR-Cas system.

[0036] In one embodiment, the CRISPR-Cas system targets a *Propionibacterium acnes* locus. Preferably, the target gene locus is not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the locus.

[0037] In one embodiment, the CRISPR-Cas system targets multiple *Propionibacterium acnes* chromosomal loci. Preferably, the target gene loci are not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the chromosomal loci.

[0038] In one embodiment, the CRISPR-Cas system targets a *Propionibacterium acnes* plasmid locus. Preferably, the target locus is not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the plasmid locus.

[0039] In one embodiment, the CRISPR-Cas system targets multiple *Propionibacterium acnes* plasmid loci. Preferably, the target loci are not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the plasmid loci.

[0040] In one embodiment, the CRISPR-Cas system is not expressed in acne-producing cells. Preferably, the CRISPR-Cas system is suppressed in acne-producing cells.

[0041] In one embodiment, the CRISPR-Cas system targets pro-inflammatory sequences associated with host disease.

[0042] In one embodiment, the CRISPR-Cas system targets pro-inflammatory sequences associated with acne vulgaris.

[0043] In one embodiment, the DNA vector includes a CRISPR-Cas system that targets the DNA vector itself.

[0044] In one embodiment, the DNA vector contains a template suitable for homologous recombination within acne bacterium phages.

[0045] In one embodiment, the DNA vector includes a template suitable for homologous recombination within the acne bacterium chromosome.

[0046] In one embodiment, the DNA vector contains a template suitable for homologous recombination within an endogenous plasmid of Propionibacterium acnes.

[0047] In one embodiment, the DNA vector comprises a template suitable for homologous recombination and a CRISPR-Cas system that targets the DNA vector itself outside the template region.

[0048] In one embodiment, the DNA vector comprises a template suitable for homologous recombination and a CRISPR-Cas system that targets the DNA vector itself outside the template region, wherein the RNA guide (crRNA or sgRNA) derived from the CRISPR-Cas system does not perfectly match the DNA target.

[0049] In one embodiment, the DNA vector includes an integrase gene expression cassette and a site-specific recombination site, enabling the integration of the DNA vector within a chromosome.

[0050] In one embodiment, the DNA vector comprises a prime editor gene expression cassette and one or more pegRNAs.

[0051] In one embodiment, the DNA vector comprises a base-editor gene expression cassette and one or more crRNAs or sgRNAs.

[0052] In one embodiment, the selection marker is catA.

[0053] In one embodiment, the selection marker is ermE.

[0054] In one embodiment, the selection marker is hygB.

[0055] The present invention includes particles derived from *Propionibacterium acnes* phage containing any of the DNA vectors of the present invention.

[0056] The present invention includes Propionibacterium acnes, particularly manipulated Propionibacterium acnes, which comprises any of the DNA vectors of the present invention.

[0057] In one particular embodiment, the manipulated Propionibacterium acnes comprises at least one, two, or three or more DNA vectors, in particular the DNA vector of the present invention.

[0058] In one particular embodiment, the manipulated Propionibacterium acnes contains the DNA vector of the present invention, which includes DNA encoding an antigen.

[0059] The present invention comprises Propionibacterium acnes manipulated according to transduction by phage-derived particles according to one of the vectors of the present invention.

[0060] The present invention includes manipulated Propionibacterium acnes, whose genome is modified in accordance with transduction by phage-derived particles containing one of the vectors of the present invention.

[0061] The present invention includes manipulated acne bacteria produced by transduction of acne bacteria using any of the vectors of the present invention, modification of acne bacteria with the target gene carried by the vector, and selection of the modification.

[0062] The present invention includes manipulated acne bacteria produced by transducing acne bacteria with any of the vectors of the present invention, modifying acne bacteria with the target gene carried by the vector, selecting the modification, and curing the vector from the manipulated acne bacteria.

[0063] In one embodiment, manipulated Propionibacterium acnes is modified by a CRISPR-Cas system carried by a vector and transduced by phage-derived particles containing any vector of the present invention.

[0064] In one embodiment, manipulated Propionibacterium acnes is modified by inserting an exogenous gene into the Propionibacterium acnes chromosome.

[0065] In one embodiment, manipulated Propionibacterium acnes is modified by inserting a foreign gene into a Propionibacterium acnes plasmid.

[0066] In one embodiment, the manipulated Propionibacterium acnes is modified by deletion or mutation of an endogenous gene sequence in the Propionibacterium acnes chromosome.

[0067] In one embodiment, the manipulated Propionibacterium acnes is modified by the deletion, insertion, or substitution of one or more nucleotides in the Propionibacterium acnes chromosome.

[0068] In one embodiment, the manipulated Propionibacterium acnes is modified by the deletion, insertion, or substitution of one or more nucleotides in the Propionibacterium acnes plasmid.

[0069] The present invention provides a method for producing particles derived from *Propionibacterium acnes* phage containing any of the vectors of the present invention, comprising the steps of introducing any of the DNA vectors of the present invention into *Propionibacterium acnes* producing cells, and contacting the producing cells with the *Propionibacterium acnes* phage genome.

[0070] The present invention includes a method for manipulating Propionibacterium acnes, comprising introducing one of the DNA vectors of the present invention into Propionibacterium acnes. The method may further include a step of selecting modified having an insertion of an exogenous gene into the Propionibacterium acnes chromosome or endogenous plasmid. The method may further include a step of selecting modified Propionibacterium acnes having one or more deletions, insertions, or substitutions of one or more nucleotides within the Propionibacterium acnes chromosome or endogenous plasmid.

[0071] This invention includes phage-derived particles produced by any of the methods of the present invention.

[0072] The present invention includes a method for treating a disorder or disease related to Propionibacterium acnes. In one embodiment, the method includes the step of administering the phage-derived particles of the present invention, or a bacterium that produces such phage-derived particles, to a target. The present invention further relates to the phage-derived particles of the present invention, or a bacterium that produces such phage-derived particles, for use in a method for treating a disorder or disease related to Propionibacterium acnes.

[0073] The present invention includes a method for modifying Propionibacterium acnes for the treatment of disorders, diseases, or skin conditions, or for cosmetic purposes. In one embodiment, the method includes a step of administering the phage-derived particles of the present invention, or bacteria that produce such phage-derived particles, to a target. The present invention further relates to the phage-derived particles of the present invention or bacteria that produce such phage-derived particles for use in a method for treating disorders, diseases, or skin conditions.

[0074] In one embodiment, this method is performed ex-situ.

[0075] In one embodiment, this method is performed in situ.

[0076] In one embodiment, this method is performed ex-situ using a strain of Propionibacterium acnes isolated from the target organism.

[0077] To enhance the understanding of the subject matter disclosed herein and to illustrate methods that can be actually carried out, embodiments are described in conjunction with the accompanying drawings as non-limiting examples. In particular with respect to the drawings, it is emphasized that the illustrated details are illustrative and for the purpose of describing embodiments of the present invention. [Brief explanation of the drawing]

[0078] [Figure 1] This diagram shows acne-producing cells infected with acne phages carrying a DNA vector containing a packaging signal and a transgene; then, phage-derived particles carrying the DNA vector are produced, and upon binding to acne-receiving cells, they transduce the DNA vector, replicate the transgene, and bring about its expression. Alternatively, acne-producing cells are not infected by phages and carry helper phages that are induced to stimulate the production of phage-derived particles. [Figure 2] This shows the host range determination of isolated *Propionibacterium acnes* bacteriophages. 1 indicates strain infection by full spot lysis; 0.5 indicates lower efficiency in strain infection, where single plaques were observed instead of full spot lysis. [Figure 3] The gel is shown. Individual colonies were streaked from titration of phage-derived particles, and PCR was performed on the individual colonies using primers IC208 (SEQ ID NO: 99) / IC310 (SEQ ID NO: 100) to confirm the presence of phagemids. Figures 1 and 2 relate to transders derived from independent production and titration of phage-derived particles carrying the same phagemid. Figures B and W are PCR results on phagemid extract (positive control) and strain ATCC11828 (negative control), respectively. The presence of plasmids after re-streaking confirms that the transders carry replicated phagemids. [Figure 4]This describes a method for manipulating the *Propionibacterium acnes* genome using a non-replicating vector that carries a recombination template. Figure 4A shows a vector (pEB_HR01) containing a single homology arm (HA) to the *Propionibacterium acnes* chromosome, which is conjugated into *Propionibacterium acnes* cells. Because this vector is non-replicating in *Propionibacterium acnes*, only *Propionibacterium acnes* cells that undergo a single recombination event can grow on an antibiotic plate while stably maintaining the antibiotic marker. Cells that do not undergo the first recombination event, or cells that undergo both the first and second recombination events, cannot grow on the antibiotic plate (erythromycin). Figure 4B shows a vector (pEB_HR02) containing two homology arms to the *Propionibacterium acnes* chromosome, which is conjugated into *Propionibacterium acnes* cells. Selection of the final recombinant is performed using antibiotic selection (ErmE) and counter-selection (SacB). [Figure 5] This describes a method for manipulating the *Propionibacterium acnes* genome using a replication CRISPR-Cas system selection vector that carries a recombinant template. The replication CRISPR-Cas system selection vector, containing a template suitable for homologous DNA recombination with chromosomes, is conjugated into *Propionibacterium acnes*. The template contains two homology arms (LHA and RHA), leading to homologous recombination within the *Propionibacterium acnes* chromosome and removal of the target sequence of the CRISPR-Cas system. Therefore, by selecting the presence of a vector expressing the CRISPR-Cas system, only recombinant *Propionibacterium acnes* can grow in the presence of erythromycin. [Figure 6]This describes a method for manipulating the *Propionibacterium acnes* genome using a self-targeted replication vector carrying a CRISPR-Cas system and a recombinant template. Figure 6A shows a vector conjugated into *Propionibacterium acnes* containing a CRISPR-Cas system with two homology arms targeting an adjacent antibiotic selection marker and the vector outside the homology region. The CRISPR-Cas system cleaves the vector, resulting in linearization of the template and plasmid loss. Therefore, only recombinant cells can grow in the presence of antibiotics. Figure 6B shows a vector conjugated into *Propionibacterium acnes* containing a CRISPR-Cas system with two homology arms targeting an adjacent mutant allele and the vector and a non-mutant allele of the *Propionibacterium acnes* chromosome outside the homology region. The CRISPR-Cas system cleaves the vector, resulting in linearization of the template and plasmid loss, and also cleaves the *Propionibacterium acnes* chromosome. Therefore, only recombinant cells can grow in the presence of erythromycin. [Figure 7] This document describes a two-step method for manipulating and selecting *Propionibacterium acnes* phages using the CRISPR-Cas system. Figure 7A shows the first step of this method. The first step consists of the generation of a mutant phage (mt PAC7) by infecting a strain (*Propionibacterium acnes* pEB-PRECOMB) containing a plasmid with a recombinant template with a first phage (wt PAC7). The recombinant template contains a left homology arm (LHA) and a right homology arm (RHA) adjacent to the mutant allele. The two homology arms recombine in vivo between the plasmid (pEB-PRECOMB) and the phage genome. The suspension obtained from the infection contains a mixture of the initial phage (wt PAC7) and the novel mutant phage (mt PAC7). Figure 7B shows the second step of this method. To select between the two phage particles, the suspension is brought into contact with a strain of *Propionibacterium acnes* (pEB-PSCREEN) containing a plasmid expressing a CRISPR-Cas system that targets only wt PAC7 phages and not mt PAC7. Therefore, infection with *Propionibacterium acnes* pEB-PSCREEN results in selective replication of mt PAC7. [Figure 8] This paper shows *Propionibacterium acnes* transdermal phage-derived particles carrying DNA vectors containing phage packaging signals (cos) of different sizes. Each suspension of phage-derived particles, also containing phages, was mixed with *Propionibacterium acnes* ATCC11828 pseudolysogen. The mixture was incubated at room temperature for 1 hour, diluted, and 4 μL of each dilution was plated onto a Brucella plate in the presence of erythromycin (5 μg / mL). For each phage-derived particle containing the same DNA vector, two suspensions derived from independent productions were used (e.g., pIC400.1 and pIC400.2). [Figure 9] This report shows titrations of the supernatant, called Sup-253, obtained after infection with *Propionibacterium acnes* (Ca0s18206) containing the editing / targeting vector pIC253, using *Propionibacterium acnes* ATCC11828 wt, *Propionibacterium acnes* ATCC11828 (Ca0s18233) containing pIC238, and *Propionibacterium acnes* ATCC11828 (Ca0s18234) containing pIC240. *Propionibacterium acnes* ATCC11828 strain is susceptible to both wild-type phage PAC7 and any mutant phage obtained by homologous recombination. Strain Ca0s18233 is resistant to wild-type phage PAC7 and susceptible to mutant phage obtained by homologous recombination with the pIC253 template. Strain Ca0s18234 is resistant to wild-type phage PAC7 and resistant to mutant phage obtained by homologous recombination with pIC253. [Figure 10]This report shows titrations of the supernatant, called Sup-257, obtained after infection with *Propionibacterium acnes* (Ca0s18208) containing the editing / targeting vector pIC257, using *Propionibacterium acnes* ATCC11828 wt, *Propionibacterium acnes* ATCC11828 (Ca0s18234) containing pIC240, and *Propionibacterium acnes* ATCC11828 (Ca0s18233) containing pIC238. *Propionibacterium acnes* ATCC11828 strain is susceptible to both wild-type phage PAC7 and any mutant phage obtained by homologous recombination. Strain Ca0s18234 is resistant to wild-type phage PAC7 and susceptible to mutant phage obtained by homologous recombination with pIC257. Strain Ca0s18233 is resistant to wild-type phage PAC7 and resistant to mutant phage obtained by homologous recombination with pIC257. [Figure 11] This shows titrations of the supernatant, called Sup-350, obtained after infection with *Propionibacterium acnes* (Ca0s18379) containing pIC350, using *Propionibacterium acnes* ATCC11828 wt and *Propionibacterium acnes* ATCC11828 (Ca0s18233) containing pIC238. *Propionibacterium acnes* ATCC11828 strain is susceptible to both wild-type phage PAC7 and mutant phages obtained by homologous recombination with pIC350. Strain Ca0s18233 is resistant to wild-type phage PAC7 and susceptible to mutant phages obtained by homologous recombination with pIC350. [Figure 12] This section shows titrations of the supernatant, called Sup-351, obtained after infection with *Propionibacterium acnes* (Ca0s18381) containing pIC351, using *Propionibacterium acnes* ATCC11828 wt and *Propionibacterium acnes* ATCC11828 (Ca0s18233) containing pIC238. *Propionibacterium acnes* ATCC11828 strain is susceptible to both wild-type phage PAC7 and mutant phages obtained by homologous recombination with pIC351. *Ca0s18233* strain is resistant to both wild-type phage PAC7 and mutant phages obtained by homologous recombination with pIC351. [Figure 13]This shows the structure of the *Propionibacterium acnes* phage genome. Five different regions encode different proteins involved in various functions, namely packaging, head assembly, tail assembly, lysis, and DNA replication (Brown et al. 28). [Figure 14] (A and B) Absorbance values ​​from ELISA for the presence of chicken ovalbumin (OVA) protein in different 1 / 10 diluted Propionibacterium acnes culture supernatants are shown. Figures 14A and 14B represent two independent replicas. The bar graph represents the average of three technical replicates of the same supernatant culture. Propionibacterium acnes strain ATCC11828 (WT) was used as a negative control. [Figure 15] This shows ovalbumin-specific Western blots using culture supernatants from different Propionibacterium acnes strains engineered to secrete ovalbumin. From left to right: (1) Pageruler ladder, (2) Supernatant from strain Ca0s22120, (3) Supernatant from strain Ca0s22122, (4) Supernatant from strain Ca0s22126, (5) Supernatant from strain Ca0s22128, (6) Supernatant from strain Ca0s22130, (7) Supernatant from strain Ca0s22132, (8) Supernatant from strain Ca0s16973, (9) Ovalbumin. [Figure 16] The gel is shown. The first well on the left (1) corresponds to the GeneRuler 1kbDNA ladder. The other wells correspond to PCR performed on plaques using primer IC443 / IC290. From left to right: (2) Plaque number 5 from the bacterial flora of a suspension of Ca0s20472 strain + infected Ca0s20855, (3) Plaque number 7 from the bacterial flora of a suspension of Ca0s20472 strain + infected Ca0s20855, (4) Plaque number 1 from the bacterial flora of a suspension of Ca0s20472 strain + PAC7, (5) Plaque number 2 from the bacterial flora of a suspension of Ca0s20472 strain + PAC7, (6) Plaque number 3 from the bacterial flora of a suspension of Ca0s20472 strain + infected Ca0s20857, (7) Plaque number 3 from the bacterial flora of a suspension of Ca0s20472 strain + PAC7. [Figure 17]The gel is shown. The first well on the left (1) corresponds to the GeneRuler 1kbDNA ladder. The other wells correspond to PCR using primers IC619 / AL219. From left to right: (wells 2-8) seven plaques isolated from plaque number 28, (wells 9-15) seven plaques isolated from plaque number 28, (well 16) PCR on wt PAC7 (positive control), (well 17) negative control on Ca0s22235 flora. [Figure 18] This shows the DNA alignment between the PAC7 wt genome (SEQ ID NO: 113) and sequencing of three different plaques isolated from PAC7-m28-gp45 using IC619 (SEQ ID NO: 78; SEQ ID NO: 85; SEQ ID NO: 114). The alignment was performed using Clustal Omega (Sievers, F. et al., Mol Syst Biol 7, pp. 539-539 (2011) (Non-Patent Literature)). [Modes for carrying out the invention]

[0079] The inventors have for the first time demonstrated the introduction of recombinant replicating DNA in Propionibacterium acnes by transduction using phage-derived particles.

[0080] The inventors have also demonstrated for the first time the production of *Propionibacterium acnes* phage-derived particles from a *Propionibacterium acnes* strain that carry a recombinant self-replicating DNA vector.

[0081] The present invention relates to a strain of Propionibacterium acnes that carries a DNA vector containing a phage packaging signal and a gene of interest, the production of phage-derived particles containing the DNA vector, the use of the phage-derived particles for transduction of Propionibacterium acnes in vitro or in situ, and the expression of the gene of interest in the transductioned Propionibacterium acnes cells. The present invention further relates to modified Propionibacterium acnes, which may or may not contain a DNA vector, obtained by transduction of a DNA vector with phage-derived particles.

[0082] Propionibacterium acnes phages are naturally occurring in human skin and have been isolated numerous times since their initial isolation in 1964. Only recently, sequencing of Propionibacterium acnes phages has revealed an exceptionally high level of nucleotide conservation, with approximately 85% identity. All Propionibacterium acnes phages described to date belong to the Siphoviridae family, sharing a similar genome size constraint of approximately 30 kb and a similar genomic structure. Despite their limited genetic diversity, most Propionibacterium acnes phages are considered broad-host organisms, possessing the ability to infect multiple Propionibacterium acnes phyllotypes. Their in-situ infectivity and broad host range make Propionibacterium acnes phages suitable platforms for manipulation for transgene delivery into Propionibacterium acnes populations.

[0083] The inventors are for the first time demonstrating that phage-derived particles can be produced by the simultaneous presence of a wild-type or manipulated *Propionibacterium acnes* phage genome and a recombinant DNA vector having a packaging signal in *Propionibacterium acnes* cells ("producing cells"). These phage-derived particles can transduce the DNA vector into "receiver" *Propionibacterium acnes* cells to express transgenes, such as antibiotic resistance genes, and enable the selection of transdextrins. This greatly expands the possibilities for directly manipulating *Propionibacterium acnes* populations on the skin and facilitates many applications (industrial, therapeutic, cosmetic, environmental). The present invention includes *Propionibacterium acnes* "producing" cells that carry DNA vectors, particularly phagemids, and methods for generating phage-derived particles, and the use of phage-derived particles for modifying or killing *Propionibacterium acnes*.

[0084] DNA vector The present invention includes a recombinant DNA vector for use with Cutibacterium acnes. Preferably, the DNA vector is a recombinant DNA vector that is not integrated into the chromosome of Propionibacterium acnes. The vector enables transmission to progeny cells. The vector is preferably a phagemide. The DNA vector preferably includes a replication origin that enables replication within Propionibacterium acnes, and a phage packaging signal.

[0085] In various embodiments, the DNA vector includes any combination of a phage packaging signal, an origin of replication that enables replication within *Propionibacterium acnes*, a selection marker that enables selection of the DNA vector within *Propionibacterium acnes*, a gene of interest, and an origin of replication that enables replication within *Propionibacterium acnes*-producing cells but does not enable replication within *Propionibacterium acnes* receiver cells.

[0086] In one embodiment, the DNA vector comprises a phage packaging signal, a replication origin that enables replication within *Propionibacterium acnes*, a first selection marker that enables selection of the DNA vector within *Propionibacterium acnes*, and the gene of interest.

[0087] In one embodiment, the DNA vector includes a phage packaging signal, a replication origin that enables replication within *Propionibacterium acnes* but not within *Propionibacterium acnes* receiver cells, a first selection marker that enables selection of the DNA vector within *Propionibacterium acnes*, and the gene of interest.

[0088] Preferably, the target gene is foreign to *Propionibacterium acnes*, that is, it is a gene not naturally found in *Propionibacterium acnes*.

[0089] In one embodiment, the DNA vector comprises a phage packaging signal which is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76; a replication origin which enables replication within Propionibacterium acnes; a selection marker which enables selection of the DNA vector within Propionibacterium acnes; and the gene of interest.

[0090] In one embodiment, the DNA vector comprises a phage packaging signal which is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76; a replication origin which enables replication within Propionibacterium acnes or a closely related species; a selection marker which enables selection of the DNA vector within Propionibacterium acnes; and the gene of interest.

[0091] In one embodiment, the DNA vector comprises a phage packaging signal which is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of Sequence ID No. 76; an origin of replication which enables replication within Propionibacterium acnes or a closely related species; a selection marker which enables selection of the DNA vector within Propionibacterium acnes; a selection marker which enables selection within a first bacterium which is Escherichia coli; an origin of replication which enables replication within the first bacterium which is Escherichia coli; and the gene of interest.

[0092] In one embodiment, a DNA vector can be efficiently introduced into acne-producing cells and stably replicated using electroporation, electroporation of protoplasts, chemical transformation, conjugation, natural competency, or transduction.

[0093] In one embodiment, a DNA vector can be efficiently transformed into acne-producing cells and stably replicated using a physical method, such as electroporation of acne bacteria cells or acne bacteria protoplasts.

[0094] In one embodiment, *Propionibacterium acnes* protoplasts are generated by resuspension in a hypotonic media following treatment with mutanoridine or lysozyme, mutanoridine-lysozyme, or mutanoridine-lysozyme bead crushing.

[0095] In one embodiment, a mixture of acne bacteria protoplasts can be used together with a DNA vector, or with a DNA vector plus glass beads, to efficiently transform the acne bacteria-producing cells and stably replicate the DNA vector.

[0096] In one embodiment, the delivery of the DNA vector into *Propionibacterium acnes* is by transduction. In one embodiment, the DNA vector includes a packaging signal selected from the group consisting of the following *Propionibacterium acnes* phages: PAC7 (typically sequence of SEQ ID NO: 68); PAC1 (typically sequence of SEQ ID NO: 69); PAC9 (typically sequence of SEQ ID NO: 70); PAC2 (typically sequence of SEQ ID NO: 71); PAC10 (typically sequence of SEQ ID NO: 72); PAC22 (typically sequence of SEQ ID NO: 73); PAC13 (typically sequence of SEQ ID NO: 74); and PAC263 (typically sequence of SEQ ID NO: 75), and phage P The DNA vector is packaged within a protein expressed from the genome of a Propionibacterium acnes phage selected from the group consisting of AC7 (typically the sequence of SEQ ID NO: 68); PAC1 (typically the sequence of SEQ ID NO: 69); PAC9 (typically the sequence of SEQ ID NO: 70); PAC2 (typically the sequence of SEQ ID NO: 71); PAC10 (typically the sequence of SEQ ID NO: 72); PAC22 (typically the sequence of SEQ ID NO: 73); PAC13 (typically the sequence of SEQ ID NO: 74); and PAC263 (typically the sequence of SEQ ID NO: 75), enabling transduction of the DNA vector into Propionibacterium acnes.

[0097] In one embodiment, the DNA vector includes a packaging signal whose sequence is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to any of the sequences of the packaging signals.

[0098] In one embodiment, the phage packaging signal has a sequence that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to a phage packaging signal sequence selected from the group consisting of SEQ ID NOs. 76, 86, 87, 88, 89, and 90.

[0099] In one embodiment, the delivery of the DNA vector into the acne bacteria is by conjugation.

[0100] In one embodiment, the DNA vector is oriT_pMRC01 (typically sequence of SEQ ID NO: 1); oriT_RSF1010 (typically sequence of SEQ ID NO: 2); oriT_pRS01 (typically sequence of SEQ ID NO: 3); oriT_pMV158 (typically sequence of SEQ ID NO: 4); oriT_pTF1 (typically sequence of SEQ ID NO: 5); oriT_pSC101 (typically sequence of SEQ ID NO: 6); oriT_pBTK445 (typically sequence of SEQ ID NO: 7); oriT_pBBR1 (typically sequence of SEQ ID NO: 8); oriT_R721 (typically sequence of SEQ ID NO: 1); Array of 9);oriT_pRmeGR4a(typically array of sequence number 10);oriT_ColE1(typically array of sequence number 11);oriT_pTiC58(typically array of sequence number 12);oriT_pMdT1(typically array of sequence number 13);oriT_R1(typically array of sequence number 14);oriT_Tn5520(typically array of sequence number 15);oriT_QKH54(typically array of sequence number 16);oriT_R64(typically array of sequence number 17);oriT_R751(typically array of sequence number 18);or iT_RP4 (typically the sequence of sequence number 19); oriT_pKL1 (typically the sequence of sequence number 20); oriT_RK2 (typically the sequence of sequence number 21); oriT_R1162 (typically the sequence of sequence number 22); oriT_Tn4555 (typically the sequence of sequence number 23); oriT_pHT (typically the sequence of sequence number 24); oriT_Tn4399 (typically the sequence of sequence number 25); oriT_Tn916 (typically the sequence of sequence number 26); oriT_pST12 (typically the sequence of sequence number 27); oriT_pCU1 (typically (This is an array with sequence number 28); oriT_pSU233 (typically an array with sequence number 29); oriT_F (typically an array with sequence number 30); oriT_pMAB01 (typically an array with sequence number 31); oriT_R388 (typically an array with sequence number 32); oriT_pS7a (typically an array with sequence number 33); oriT_pS7b (typically an array with sequence number 34); oriT_R702 (typically an array with sequence number 35); oriT_pMUR274 (typically an array with sequence number 36); oriT_R100 (typically an array with sequence number 37);It includes an origin of transfer selected from the group consisting of oriT_pVCR94deltaX (typically the sequence of sequence number 38); oriT_R46 (typically the sequence of sequence number 39); oriT_pGO1 (typically the sequence of sequence number 40); and oriT_pIP501 (typically the sequence of sequence number 41).

[0101] In one embodiment, the DNA vector includes the transmission origin oriT_pMRC01 (typically the sequence of SEQ ID NO: 1). In one embodiment, the DNA vector includes the transmission origin oriT_RSF1010 (typically the sequence of SEQ ID NO: 2). In one embodiment, the DNA vector includes the transmission origin oriT_pRS01 (typically the sequence of SEQ ID NO: 3). In one embodiment, the DNA vector includes the transmission origin oriT_pMV158 (typically the sequence of SEQ ID NO: 4). In one embodiment, the DNA vector includes the transmission origin oriT_pTF1 (typically the sequence of SEQ ID NO: 5). In one embodiment, the DNA vector includes the transmission origin oriT_pSC101 (typically the sequence of SEQ ID NO: 6). In one embodiment, the DNA vector includes the transmission origin oriT_pBTK445 (typically the sequence of SEQ ID NO: 7). In one embodiment, the DNA vector includes the transmission origin oriT_pBBR1 (typically the sequence of SEQ ID NO: 8). In one embodiment, the DNA vector includes the transmission origin oriT_R721 (typically the sequence of SEQ ID NO: 9). In one embodiment, the DNA vector includes the transmission origin oriT_pRmeGR4a (typically the sequence of SEQ ID NO: 10). In one embodiment, the DNA vector includes the transmission origin oriT_ColE1 (typically the sequence of SEQ ID NO: 11). In one embodiment, the DNA vector includes the transmission origin oriT_pTiC58 (typically the sequence of SEQ ID NO: 12). In one embodiment, the DNA vector includes the transmission origin oriT_pMdT1 (typically the sequence of SEQ ID NO: 13). In one embodiment, the DNA vector includes the transmission origin oriT_R1 (typically the sequence of SEQ ID NO: 14). In one embodiment, the DNA vector includes the transmission origin oriT_Tn5520 (typically the sequence of SEQ ID NO: 15). In one embodiment, the DNA vector includes the transmission origin oriT_QKH54 (typically the sequence of SEQ ID NO: 16). In one embodiment, the DNA vector includes the transmission origin oriT_R64 (typically the sequence of SEQ ID NO: 17). In one embodiment, the DNA vector includes the transmission origin oriT_R751 (typically the sequence of SEQ ID NO: 18). In one embodiment, the DNA vector includes a transmission origin oriT_RP4 (typically the sequence of SEQ ID NO: 19).In one embodiment, the DNA vector includes the transmission origin oriT_pKL1 (typically the sequence of SEQ ID NO: 20). In one embodiment, the DNA vector includes the transmission origin oriT_RK2 (typically the sequence of SEQ ID NO: 21). In one embodiment, the DNA vector includes the transmission origin oriT_R1162 (typically the sequence of SEQ ID NO: 22). In one embodiment, the DNA vector includes the transmission origin oriT_Tn4555 (typically the sequence of SEQ ID NO: 23). In one embodiment, the DNA vector includes the transmission origin oriT_pHT (typically the sequence of SEQ ID NO: 24). In one embodiment, the DNA vector includes the transmission origin oriT_Tn4399 (typically the sequence of SEQ ID NO: 25). In one embodiment, the DNA vector includes the transmission origin oriT_Tn916 (typically the sequence of SEQ ID NO: 26). In one embodiment, the DNA vector includes the transmission origin oriT_pST12 (typically the sequence of SEQ ID NO: 27). In one embodiment, the DNA vector includes the transmission origin oriT_pCU1 (typically the sequence of SEQ ID NO: 28). In one embodiment, the DNA vector includes the transmission origin oriT_pSU233 (typically the sequence of SEQ ID NO: 29). In one embodiment, the DNA vector includes the transmission origin oriT_F (typically the sequence of SEQ ID NO: 30). In one embodiment, the DNA vector includes the transmission origin oriT_pMAB01 (typically the sequence of SEQ ID NO: 31). In one embodiment, the DNA vector includes the transmission origin oriT_R388 (typically the sequence of SEQ ID NO: 32). In one embodiment, the DNA vector includes the transmission origin oriT_pS7a (typically the sequence of SEQ ID NO: 33). In one embodiment, the DNA vector includes the transmission origin oriT_pS7b (typically the sequence of SEQ ID NO: 34). In one embodiment, the DNA vector includes the transmission origin oriT_R702 (typically the sequence of SEQ ID NO: 35). In one embodiment, the DNA vector includes the transmission origin oriT_pMUR274 (typically the sequence of SEQ ID NO: 36). In one embodiment, the DNA vector includes the transmission origin oriT_R100 (typically the sequence of SEQ ID NO: 37). In one embodiment, the DNA vector includes the origin of transmission oriT_pVCR94deltaX (typically the sequence of SEQ ID NO: 38).In one embodiment, the DNA vector includes the transmission origin oriT_R46 (typically the sequence of SEQ ID NO: 39). In one embodiment, the DNA vector includes the transmission origin oriT_pGO1 (typically the sequence of SEQ ID NO: 40). In one embodiment, the DNA vector includes the transmission origin oriT_pIP501 (typically the sequence of SEQ ID NO: 41).

[0102] In one embodiment, the DNA vector includes an oriT whose sequence is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to the sequence of any of the aforementioned origins of transmission (oriT).

[0103] In one embodiment, the donor bacteria, for example, Escherichia coli, include pMRC01, RSF1010, pRS01, pMV158, pTF1, pSC101, pBTK445, pBBR1, R721, pRmeGR4a, ColE1, pTiC58, pMdT1, R1, Tn5520, QKH54, R64, R751, RP4, pKL1, RK2, R1162, Tn4555, pH These devices are used to carry conjugation plasmids, conjugation transposons, or integrative and conjugative elements (ICEs), selected from the group consisting of T, Tn4399, Tn916, pST12, pCU1, pSU233, F, pMAB01, R388, pS7a, pS7b, R702, pMUR274, R100, pVCR94deltaX, R46, pGO1, and pIP501, and to efficiently deliver DNA vectors into acne bacterium recipient cells. In one embodiment, the DNA vector is pMRC01;RSF1010;pRS01;pMV158;pTF1;pSC101;pBTK445;pBBR1;R721;pRmeGR4a;ColE1;pTiC58;pMdT1;R1;Tn5520;QKH54;R64;R751;RP4;pKL1;RK2;R1162;Tn4555;pHT;Tn4399;Tn9 It contains conjugation plasmids, conjugation transposons, and transmission origins and associated relaxases for integration and conjugation elements (ICE), selected from the group consisting of 16;pST12;pCU1;pSU233;F;pMAB01;R388;pS7a;pS7b;R702;pMUR274;R100;pVCR94deltaX;R46;pGO1, and pIP501.

[0104] In one preferred embodiment, the DNA vector is one of the following: pMRC01;RSF1010;pRS01;pMV158;pTF1;pSC101;pBTK445;pBBR1;R721;pRmeGR4a;ColE1;pTiC58;pMdT1;R1;Tn5520;QKH54;R64;R751;RP4;pKL1;RK2;R1162;Tn4555;pHT;Tn The group comprises conjugation plasmids, conjugation transposons, and origins and relaxases for integration and conjugation elements (ICE), selected from the group consisting of 4399;Tn916;pST12;pCU1;pSU233;F;pMAB01;R388;pS7a;pS7b;R702;pMUR274;R100;pVCR94deltaX;R46;pGO1, and pIP501.

[0105] In one preferred embodiment, the DNA vector is oriT_pMRC01(sequence number 1);oriT_RSF1010(sequence number 2);oriT_pRS01(sequence number 3);oriT_pMV158(sequence number 4);oriT_pTF1(sequence number 5);oriT_pSC101(sequence number 6);oriT_pBTK445(sequence number 7);oriT_pBBR1(sequence number 8);oriT_R721(sequence number 9);oriT_pRmeGR4a( Array No. 10);oriT_ColE1(Array No. 11);oriT_pTiC58(Array No. 12);oriT_pMdT1(Array No. 13);oriT_R1(Array No. 14);oriT_Tn5520(Array No. 15);oriT_QKH54(Array No. 16);oriT_R64(Array No. 17);oriT_R751(Array No. 18);oriT_RP4(Array No. 19);oriT_pKL1(Array No. 20);oriT_RK2(Array No. No. 21);oriT_R1162(Sequence ID 22);oriT_Tn4555(Sequence ID 23);oriT_pHT(Sequence ID 24);oriT_Tn4399(Sequence ID 25);oriT_Tn916(Sequence ID 26);oriT_pST12(Sequence ID 27);oriT_pCU1(Sequence ID 28);oriT_pSU233(Sequence ID 29);oriT_F(Sequence ID 30);oriT_pMAB01(Sequence ID 31);oriT_R388( The transmission origin is selected from the group consisting of (SEQ ID NO: 32);oriT_pS7a(SEQ ID NO: 33);oriT_pS7b(SEQ ID NO: 34);oriT_R702(SEQ ID NO: 35);oriT_pMUR274(SEQ ID NO: 36);oriT_R100(SEQ ID NO: 37);oriT_pVCR94deltaX(SEQ ID NO: 38);oriT_R46(SEQ ID NO: 39);oriT_pGO1(SEQ ID NO: 40); and oriT_pIP501(SEQ ID NO: 41).

[0106] In one embodiment, the DNA vector includes an origin of transmission (oriT) that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to any of those ICEs.

[0107] In one embodiment, the present invention includes a DNA vector comprising a replication origin that enables replication within Propionibacterium acnes, an oriT that enables conjugation within Propionibacterium acnes, a selection marker that enables selection within a transconjugant Propionibacterium acnes, and a selection marker that enables selection within a donor organism. In another embodiment, the present invention includes a DNA vector comprising a replication origin that enables replication within Propionibacterium acnes, and the oriT defined above that enables conjugation within Propionibacterium acnes.

[0108] In one embodiment, the replication origins that enable replication within *Propionibacterium acnes* are R6K (typically sequence of SEQ ID NO: 42); RK2 (typically sequence of SEQ ID NO: 43); pBBR1 (typically sequence of SEQ ID NO: 44); pRO1600 (typically sequence of SEQ ID NO: 45); RSF1010 (typically sequence of SEQ ID NO: 46); pAMβ1 (typically sequence of SEQ ID NO: 47); pLME106 (typically sequence of SEQ ID NO: 48); pTZC1 (typically sequence of SEQ ID NO: 49); pBC1 (typically sequence of SEQ ID NO: 50); pEP2 (typically sequence of SEQ ID NO: 51); pWVO1 (typically sequence of SEQ ID NO: 52); pAP1 (typically sequence of SEQ ID NO: 53) The sequence is selected from the group consisting of: pWKS1 (typically the sequence of sequence number 54); pLME108 (typically the sequence of sequence number 55); pLS1 (typically the sequence of sequence number 56); pUB6060 (typically the sequence of sequence number 57); p545 (typically the sequence of sequence number 58); pJD4 (typically the sequence of sequence number 59); pIJ101 (typically the sequence of sequence number 60); pSN22 (typically the sequence of sequence number 61); pGP01 (typically the sequence of sequence number 62); pIP501 (typically the sequence of sequence number 63); pCU1 (typically the sequence of sequence number 64); and pBAV1K-T5 (typically the sequence of sequence number 65). In one embodiment, the replication origin that enables replication within Propionibacterium acnes is R6K (typically the sequence of sequence number 42). In one embodiment, the replication origin that enables replication within Propionibacterium acnes is RK2 (typically the sequence of sequence number 43). In one embodiment, the replication origin that enables replication within Propionibacterium acnes is pBBR1 (typically the sequence of sequence number 44). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pRO1600 (typically the sequence of SEQ ID NO: 45). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is RSF1010 (typically the sequence of SEQ ID NO: 46). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pAMβ1 (typically the sequence of SEQ ID NO: 47). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pLME106 (typically the sequence of SEQ ID NO: 48).In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pTZC1 (typically the sequence of SEQ ID NO: 49). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pBC1 (typically the sequence of SEQ ID NO: 50). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pEP2 (typically the sequence of SEQ ID NO: 51). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pWVO1 (typically the sequence of SEQ ID NO: 52). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pAP1 (typically the sequence of SEQ ID NO: 53). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pWKS1 (typically the sequence of SEQ ID NO: 54). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pLME108 (typically the sequence of SEQ ID NO: 55). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pLS1 (typically the sequence of SEQ ID NO: 56). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pUB6060 (typically the sequence of SEQ ID NO: 57). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is p545 (typically the sequence of SEQ ID NO: 58). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pJD4 (typically the sequence of SEQ ID NO: 59). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pIJ101 (typically the sequence of SEQ ID NO: 60). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pSN22 (typically the sequence of SEQ ID NO: 61). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pGP01 (typically the sequence of SEQ ID NO: 62). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pIP501 (typically the sequence of SEQ ID NO: 63). In one embodiment, the replication origin that enables replication within *Propionibacterium acnes* is pCU1 (typically the sequence of SEQ ID NO: 64). In one embodiment, the replication origin that enables replication within Propionibacterium acnes is pBAV1K-T5 (typically the sequence of SEQ ID NO: 65).

[0109] In one embodiment, the DNA vector includes a replication origin that enables replication within the bacteria *Propionibacterium acnes*. In one embodiment, the DNA vector includes a replication origin selected from the group consisting of R6K (sequence number 42); RK2 (sequence number 43); pBBR1 (sequence number 44); pRO1600 (sequence number 45); RSF1010 (sequence number 46); pAMβ1 (sequence number 47); pLME106 (sequence number 48); pTZC1 (sequence number 49); pBC1 (sequence number 50); pEP2 (sequence number 51); pWVO1 (sequence number 52); pAP1 (sequence number 53); pWKS1 (sequence number 54); pLME108 (sequence number 55); pLS1 (sequence number 56); pUB6060 (sequence number 57); p545 (sequence number 58); pJD4 (sequence number 59); pIJ101 (sequence number 60); pSN22 (sequence number 61); pGP01 (sequence number 62); pIP501 (sequence number 63); pCU1 (sequence number 64); and pBAV1K-T5 (sequence number 65).

[0110] Preferably, the replication origin has a sequence that is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 97%, 98%, 99%, or 100% identical to any of the sequences of the replication origins.

[0111] In various embodiments, the selection marker is chosen from ermE, catA, hygB, ermX, tetW, erm(50), and other high-GC antibiotic resistance genes. In one embodiment, the selection marker is not ermE. In one embodiment, the selection marker is catA. In one embodiment, the selection marker is hygB.

[0112] In one embodiment, the DNA vector further comprises a CRISPR-Cas system. Typically, the CRISPR-Cas system comprises a CRISPR array. Typically, the CRISPR-Cas system comprises an RNA guide (crRNA or sgRNA).

[0113] In one embodiment, the CRISPR-Cas system targets a *Propionibacterium acnes* locus. Preferably, the target gene locus is not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the locus.

[0114] In one embodiment, the CRISPR-Cas system targets multiple *Propionibacterium acnes* chromosomal loci. Preferably, the target gene loci are not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the chromosomal loci.

[0115] In one embodiment, the CRISPR-Cas system targets a *Propionibacterium acnes* plasmid locus. Preferably, the target locus is not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the plasmid locus.

[0116] In one embodiment, the CRISPR-Cas system targets multiple *Propionibacterium acnes* plasmid loci. Preferably, the target loci are not present in *Propionibacterium acnes*-producing cells. Preferably, a CRISPR array derived from the CRISPR-Cas system expresses one or more crRNAs that target the plasmid loci.

[0117] In one embodiment, the CRISPR-Cas system is not expressed in acne-producing cells. Preferably, the CRISPR-Cas system is suppressed in acne-producing cells.

[0118] In one embodiment, the CRISPR-Cas system targets pro-inflammatory sequences associated with host disease.

[0119] In one embodiment, the CRISPR-Cas system targets pro-inflammatory sequences associated with acne vulgaris.

[0120] In one embodiment, the DNA vector contains a template suitable for homologous recombination, and the CRISPR-Cas system targets the DNA vector itself.

[0121] In one embodiment, the DNA vector contains a template suitable for homologous recombination within acne bacterium phages.

[0122] In one embodiment, the DNA vector includes a template suitable for homologous recombination within the acne bacterium chromosome.

[0123] In one embodiment, the DNA vector contains a template suitable for homologous recombination within an endogenous plasmid of Propionibacterium acnes.

[0124] In one embodiment, the DNA vector comprises a template suitable for homologous recombination and a CRISPR-Cas system that targets the DNA vector itself outside the template region.

[0125] In one embodiment, the DNA vector comprises a template suitable for homologous recombination and a CRISPR-Cas system that targets the DNA vector itself outside the template region, wherein the RNA guide (crRNA or sgRNA) derived from the CRISPR-Cas system does not perfectly match the DNA target.

[0126] In one embodiment, the DNA vector includes an integrase gene expression cassette and a site-specific recombination site, enabling the integration of the DNA vector within a chromosome.

[0127] In one embodiment, the DNA vector comprises a base-editor gene expression cassette and one or more crRNAs or sgRNAs.

[0128] In one embodiment, a base editor is used to inactivate the expression of a gene by editing one or more nucleotides involved in the transcription or translation of the gene. In particular, the base editor targets one or more nucleotides of the promoter, RBS, or start codon.

[0129] In one embodiment, a base editor is used to introduce an immature stop codon.

[0130] In one embodiment, a base editor is used to introduce one or more rare codons.

[0131] In other embodiments, a base editor is used to regulate the expression of a gene by editing one or more nucleotides involved in the transcription or translation of the gene. In particular, the base editor targets one or more nucleotides of the promoter, RBS, or start codon, resulting in an increase or decrease in gene expression.

[0132] In other embodiments, a base editor is used to reverse mutations that result in inactivation, decreased activity, or increased activity of a gene or pathway.

[0133] In other embodiments, a base editor is used to reverse mutations that result in increased pathogenicity.

[0134] In one embodiment, a base editor is used to modify the regulation of a gene by editing one or more nucleotides involved in gene regulation, such as operator sequences, transcription factor binding sites, riboswitches, RNAse recognition sites, protease cleavage sites, methylation sites, or post-translational modification sites (phosphorylation, glycosylation, acetylation, pupylation, etc.).

[0135] In one embodiment, the DNA vector comprises a prime editor gene expression cassette and one or more pegRNAs.

[0136] In one embodiment, a prime editor is used to introduce one or more immature stop codons.

[0137] In one embodiment, a prime editor is used to introduce one or more rare codons.

[0138] In one embodiment, a prime editor is used to introduce or delete nucleotides and induce a frameshift in the reading frame.

[0139] In other embodiments, a prime editor is used to regulate the expression of a gene by replacing, deleting, or inserting one or more nucleotides involved in the transcription or translation of the gene. In particular, a prime editor replaces, deletes, or inserts one or more nucleotides in the promoter, RBS, or start codon, resulting in an increase or decrease in gene expression.

[0140] In other embodiments, a prime editor is used to reverse mutations that result in the inactivation or reduced activity of a gene or pathway.

[0141] In other embodiments, a prime editor is used to reverse mutations that result in increased pathogenicity.

[0142] In one embodiment, the vector is a plasmid containing an E. coli replicon and an E. coli resistance marker, enabling extraction of the plasmid from Propionibacterium acnes, as well as transformation and replication in E. coli.

[0143] In one embodiment, the vector is a plasmid containing an E. coli replicon and an E. coli resistance marker, enabling extraction of the plasmid from E. coli and transformation and replication in Propionibacterium acnes.

[0144] In one embodiment, the vector includes two origins of replication, one of which allows replication only within Propionibacterium acnes or Propionibacterium acnes-producing cells, and a second origin of replication that allows replication within other bacteria.

[0145] In one embodiment, a vector containing template DNA suitable for homologous recombination enables the expression of genes that increase the recombination rate.

[0146] In one embodiment, a mold suitable for homologous recombination includes homology arms upstream and downstream of the recombination point. The homology arms may have a size of at least 50 bp, 100 bp, 500 bp, or at least 1000 bp.

[0147] In one embodiment, the target gene contained in the DNA vector may be an introduced gene that is foreign to *Propionibacterium acnes*. The introduced gene is - DNA encoding a fluorescent protein (e.g., UnaG) that, once a specific substrate is added, causes the cells to become fluorescent (e.g., UnaG); - DNA encoding an enzyme reporter (e.g., LacZ) that leads to the production of chromogenic compounds by *Propionibacterium acnes* colonies; - DNA that codes for human proteins (e.g., interleukins); - DNA encoding an antigen (e.g., tumor antigen, viral antigen, bacterial antigen, fungal antigen, autoantigen, allergen, or graft-specific antigen); - CRISPR-Cas system; - Prime editing system; or - Base editor system This includes, but is not limited to, those items.

[0148] In one particular embodiment, the gene of interest encoded by the DNA vector is DNA encoding an antigen, in particular, DNA encoding an antigen selected from the group consisting of tumor antigens, viral antigens, bacterial antigens, fungal antigens, autoantigens, allergens, and graft-specific antigens, as defined below.

[0149] Propionibacterium acnes strains containing DNA vectors, manipulated Propionibacterium acnes strains The present invention comprises *Propionibacterium acnes* containing any of the DNA vectors of the present invention. The present invention further comprises *Propionibacterium acnes* produced by any of the methods of the present invention. Accordingly, the present invention comprises *Propionibacterium acnes* modified according to transduction of any of the DNA vectors of the present invention by phage-derived particles, regardless of whether the DNA vector is retained or subsequently removed from *Propionibacterium acnes* (i.e., cured).

[0150] Accordingly, the present invention includes acne bacteria produced by a method comprising the steps of: producing phage-derived particles from acne bacteria-producing cells containing the DNA vector of the present invention; contacting the phage-derived particles with acne bacteria receiver cells to transduce the DNA vector into the acne bacteria receiver cells and to cause modification of the acne bacteria receiver cells by the target gene (e.g., the CRISPR-Cas system) and / or an exogenous gene inserted into the acne bacteria chromosome carried by the vector; selecting the modification; and curing the vector from acne bacteria.

[0151] The present invention includes a modified form of Propionibacterium acnes, which is transduced by a CRISPR-Cas system using phage-derived particles carrying the vector of the present invention.

[0152] The present invention includes modified Propionibacterium acnes, which are obtained by transduction of a DNA vector and subsequent insertion of a foreign gene into the Propionibacterium acnes chromosome.

[0153] The present invention includes modified Propionibacterium acnes obtained by transduction of a DNA vector, followed by deletion or mutation of endogenous genes in the Propionibacterium acnes chromosome or endogenous plasmid of Propionibacterium acnes.

[0154] This invention includes *Propionibacterium acnes* produced by transduction of the DNA vector of the present invention.

[0155] The present invention includes manipulated Propionibacterium acnes modified by plasmid delivery, particularly by conjugation. In one particular embodiment, the plasmid comprises a CRISPR-Cas system. In another particular embodiment, the plasmid comprises a foreign gene. In another particular embodiment, the plasmid enables insertion of the foreign gene into the Propionibacterium acnes chromosome. In another particular embodiment, the plasmid enables deletion or mutation of an endogenous gene into the Propionibacterium acnes chromosome or an endogenous plasmid of Propionibacterium acnes. In one particular embodiment, the plasmid comprises a replication origin and / or a transfer origin as defined above, enabling replication within Propionibacterium acnes.

[0156] Cutibacterium acnes, formerly known as Propionibacterium acnes, was historically classified into three major phyllotypes: IA, IB, II, and III, based on recA and tly sequences. These phyllotypes were further subdivided into IA1, IA2, IB, II, and III using different multilocus sequence typing (MLST) schemes. More recently, Fitz-Gibbon et al. (Fitz-Gibbon, S. et al. (2013) J Invest Dermatol 133, pp. 2152-2160 (Non-Patent Literature 3)) introduced a new classification based on 16S rRNA gene sequence diversity (ribotyping), as well as a refined classification of phyllotypes: IA-1, IA-2, IB-1, IB-2, IB-3, IC, II, and III. This disclosure relates to this classification, but the agreement between this classification and other classifications is known to those skilled in the art and can be derived from the following overview (Dreno, B. et al. (2018). Journal of the European Academy of Dermatology and Venereology 32, pp. 5-14 (Non-Patent Literature 4)). Accordingly, in a particular embodiment, *Propionibacterium acnes* may be derived from a phylotype selected from the group consisting of phylotypes IA-1, IA-2, IB-1, IB-2, IB-3, IC, II, and III.

[0157] By comparing the whole-genome sequences of strains isolated from acne and healthy subjects, Fitz-Gibbon and collaborators were able to identify acne-associated strains (IA-2 and IB-1) and healthy-associated strains (II), consistent with previous studies. Interestingly, specific loci (locus 1, locus 2, and locus 3) were found to be present in the acne-associated strains but absent in the neutral strain and healthy strains. Similar loci were found in subsequent metagenomic analyses, confirming the association between the presence of these loci and acne vulgaris (Barnard, E. et al. (2016) Scientific Reports 6, srep39491 (Non-Patent Literature 5)).

[0158] The ability of specific strain phylotypes to induce immune responses has recently been investigated (Yu et al. (2016) Journal of Investigative Dermatology 136: pp. 2221-2228 (Non-Patent Literature 6)). Yu et al. demonstrated that different *Propionibacterium acnes* phylotypes induce different cytokine profiles when incubated with peripheral blood mononuclear cells (PBMCs). In particular, Yu et al. showed that acne-associated phylotypes IA-2 p+ (i.e., containing large plasmids associated with acne), IB-1, and IC induce high levels of inflammatory IFN-γ and IL-17, but low levels of IL-10, suggesting that these specific phylotypes can induce both Th1 and Th17 responses. Furthermore, phylotypes IB-3, II, and III induced lower levels of IL-17 (and IFN-γ in phylotype III) but higher levels of IL-10, suggesting induction of a Treg response. Additionally, phylotypes IA-1, IA-2 p- (i.e., those without the large plasmid associated with acne), and IB-2 induced lower levels of IFN-γ and IL-10, as well as higher levels of IL-17, suggesting induction primarily of a Th17 response.

[0159] Therefore, when using the manipulated Propionibacterium acnes of the present invention for a specific indication, the use of a predetermined Propionibacterium acnes phylotype or strain may be advantageous depending on the desired specific immune response. Accordingly, in one particular embodiment, the Propionibacterium acnes is derived from a phylotype selected from the group consisting of phylotypes IA-2 p+, IB-1, and IC. In another embodiment, the Propionibacterium acnes is derived from a phylotype selected from the group consisting of phylotypes IA-1, IA-2 p-, and IB-2. In yet another embodiment, the Propionibacterium acnes is derived from a phylotype selected from the group consisting of phylotypes IB-3, II, and III.

[0160] Furthermore, previous studies have shown that in Staphylococcus epidermidis, it is possible to induce different T cell responses using different strains within the same species by manipulating the strains (Chen et al. (2019) "Decoding commensal-host communication through genetic engineering of Staphylococcus epidermidis" bioRxiv https: / / doi.org / 10.1101 / 664656 (Non-patent Literature 7)).

[0161] Therefore, in a particular embodiment, the Propionibacterium acnes strain is a strain that induces a predetermined T-cell response, or a strain that has been engineered to induce it. In a particular embodiment, especially when the Propionibacterium acnes cells are intended to be used in the prevention and / or treatment of cancer, the Propionibacterium acnes strain is a strain that induces elevated levels of IFN-γ and / or IL-17, or a strain that has been engineered to induce it. In a particular embodiment, especially when the Propionibacterium acnes cells are intended to be used in the prevention and / or treatment of infection, the Propionibacterium acnes strain is a strain that induces elevated levels of IFN-γ and / or IL-17, or a strain that has been engineered to induce it. In a particular embodiment, especially when the Propionibacterium acnes cells are intended to be used in the prevention and / or treatment of autoimmune diseases, the Propionibacterium acnes strain is a strain that induces elevated levels of IL-10, or a strain that has been engineered to induce it. In a particular embodiment, if the acne bacteria cells are intended to be used in the prevention and / or treatment of allergies, such as asthma, the acne bacteria are a strain that induces elevated levels of IFN-g and / or IL-10, or a strain that has been manipulated to induce them. In a particular embodiment, if the acne bacteria cells are intended to be used in the prevention and / or treatment of graft rejection, the acne bacteria are a strain that induces elevated levels of IL-10, or a strain that has been manipulated to induce them.

[0162] To regulate the expression of the target molecule and the interaction between *Propionibacterium acnes* and the host, *Propionibacterium acnes* can be generated containing the recombinant self-replicating DNA vector of the present invention (or a plasmid, in particular, a conjugating plasmid as defined above). The target molecule can be carried on the self-replicating DNA vector (or plasmid, in particular a conjugating plasmid) in *Propionibacterium acnes*, or can be inserted into the chromosome of *Propionibacterium acnes* via the action of the self-replicating DNA vector (or plasmid, in particular a conjugating plasmid as defined above).

[0163] In one embodiment, a DNA vector is used for manipulating the chromosomes of Propionibacterium acnes.

[0164] In one embodiment, a DNA vector is used for manipulating the *Propionibacterium acnes* plasmid.

[0165] In one embodiment, a DNA vector is used for manipulating acne phages.

[0166] In one embodiment, a DNA vector (or plasmid, in particular a conjugation plasmid as defined above) is used to regulate the expression of a target molecule and the interaction between *Propionibacterium acnes* and the host. In one embodiment, a DNA vector (or plasmid, in particular a conjugation plasmid as defined above) is used to express a transgene in *Propionibacterium acnes*. The transgene can be cloned into a recombinant autonomous replicating DNA vector (or plasmid, in particular a conjugation plasmid) under the control of a predetermined promoter (constitutive or inductive) followed by a predetermined terminator. The transmission of this vector into *Propionibacterium acnes* enables the expression of the transgene. The transgene may be, for example, a CRISPR-Cas system or may encode a human protein, such as an interleukin. In one embodiment, the DNA vector (or plasmid, in particular a conjugation plasmid) encodes multiple transgenes under the control of a single promoter or under the control of different promoters. The promoter may be endogenous or exogenous, inductive or constitutive.

[0167] In one embodiment, a DNA vector (or plasmid, particularly a conjugated plasmid) is used to modify the Propionibacterium acnes genome. In one embodiment, the transfer of the vector (or plasmid, particularly a conjugated plasmid) into Propionibacterium acnes enables the expression of a CRISPR / Cas system that cleaves the Propionibacterium acnes genome (plasmid or chromosome) at a specific site, resulting in modification of the Propionibacterium acnes genome. In one embodiment, the vector (or plasmid, particularly a conjugated plasmid) further includes the gene of interest and homology to the cleavage site, facilitating the integration of the gene of interest into the Propionibacterium acnes genome.

[0168] Delivery of DNA vectors to acne bacteria strains In one embodiment, delivery of any DNA vector of the present invention into acne-producing cells is performed by contacting the DNA vector of the present invention with acne bacteria.

[0169] In one embodiment, an arbitrary DNA vector of the present invention is delivered into acne-producing cells by transfection into the acne-producing cells (e.g., electroporation), and the DNA vector is stably replicated within the acne-producing cells. In one embodiment, the transfected DNA vector is purified from dam(-) E. coli cells, for example ET12567, and electroporated into acne-producing cells that have been brought to a competent state at 24°C.

[0170] In one embodiment, delivery of any DNA vector of the present invention into acne-producing cells is performed by transfection (e.g., electroporation) into acne-producing protoplasts. In one embodiment, acne-producing protoplasts are generated by resuspension in a hypotonic medium following treatment with mutanoridine or lysozyme, mutanoridine-lysozyme, or mutanoridine-lysozyme bead disruption.

[0171] In one embodiment, an arbitrary DNA vector of the present invention is delivered into acne-producing cells by mixing the DNA vector with acne-producing protoplasts. In another embodiment, glass beads are added together with the DNA vector, and the beads are crushed to introduce the DNA into the acne-producing protoplasts.

[0172] In one embodiment, delivery of the DNA vector of the present invention into acne bacteria is by transduction. In one embodiment, the DNA vector comprises a packaging signal of an acne bacteriophage selected from the group consisting of phage PAC7 (typically the sequence of SEQ ID NO: 68); PAC1 (typically the sequence of SEQ ID NO: 69); PAC9 (typically the sequence of SEQ ID NO: 70); PAC2 (typically the sequence of SEQ ID NO: 71); PAC10 (typically the sequence of SEQ ID NO: 72); PAC22 (typically the sequence of SEQ ID NO: 73); PAC13 (typically the sequence of SEQ ID NO: 74), and PAC263 (typically the sequence of SEQ ID NO: 75); and is packaged within a protein expressed from the genome of an acne bacteriophage selected from the group consisting of phage PAC7 (typically the sequence of SEQ ID NO: 68); PAC1 (typically the sequence of SEQ ID NO: 69); PAC9 (typically the sequence of SEQ ID NO: 70); PAC2 (typically the sequence of SEQ ID NO: 71); PAC10 (typically the sequence of SEQ ID NO: 72); PAC22 (typically the sequence of SEQ ID NO: 73); PAC13 (typically the sequence of SEQ ID NO: 74), and PAC263 (typically the sequence of SEQ ID NO: 75), enabling transduction of the DNA vector into acne bacteria.

[0173] In one embodiment, delivery of any DNA vector of the present invention into acne-producing cells is by conjugation. In one embodiment, the DNA vector includes a transmission origin. In one embodiment, a donor bacterium, such as E. coli, is used to efficiently deliver the DNA vector into acne-producing recipient cells that stably replicate the DNA vector. In one embodiment, the DNA vector is oriT_pMRC01 (typically sequence of SEQ ID NO: 1); oriT_RSF1010 (typically sequence of SEQ ID NO: 2); oriT_pRS01 (typically sequence of SEQ ID NO: 3); oriT_pMV158 (typically sequence of SEQ ID NO: 4); oriT_pTF1 (typically sequence of SEQ ID NO: 5); oriT_pSC101 (typically sequence of SEQ ID NO: 6); oriT_pBTK445 (typically sequence of SEQ ID NO: 7); oriT_pBBR1 (typically sequence of SEQ ID NO: 8); oriT_R721 (typically sequence of SEQ ID NO: 9); oriT_pRmeGR4a (typically sequence of SEQ ID NO: 10); oriT_ColE1 (typically sequence of SEQ ID NO: 11); oriT_pTiC58 (typically sequence of SEQ ID NO: 12); oriT_pMdT1 (typically sequence of SEQ ID NO: 13); oriT_R1 (typically sequence of SEQ ID NO: 14); oriT_Tn5520 (typically sequence of SEQ ID NO: 15); o riT_QKH54 (typically the sequence with sequence number 16); oriT_R64 (typically the sequence with sequence number 17); oriT_R751 (typically the sequence with sequence number 18); oriT_RP4 (typically the sequence with sequence number 19); oriT_pKL1 (typically the sequence with sequence number 20); oriT_RK2 (typically the sequence with sequence number 21); oriT_R1162 (typically the sequence with sequence number 22); oriT_Tn4555 (typically the sequence with sequence number 23); oriT_pHT (typically the sequence with sequence number 24); oriT_Tn4399 (typically the sequence with sequence number 25); oriT_Tn916 (typically the sequence with sequence number 26); oriT_pST12 (typically the sequence with sequence number 27); oriT_pCU1 (typically the sequence with sequence number 28); oriT_pSU233 (typically the sequence with sequence number 29); oriT_F (typically the sequence with sequence number 30); oriT_pMAB01 (typically the sequence with sequence number 31);The transmission origin is selected from the group consisting of oriT_R388 (typically the sequence with sequence number 32); oriT_pS7a (typically the sequence with sequence number 33); oriT_pS7b (typically the sequence with sequence number 34); oriT_R702 (typically the sequence with sequence number 35); oriT_pMUR274 (typically the sequence with sequence number 36); oriT_R100 (typically the sequence with sequence number 37); oriT_pVCR94deltaX (typically the sequence with sequence number 38); oriT_R46 (typically the sequence with sequence number 39); oriT_pGO1 (typically the sequence with sequence number 40); and oriT_pIP501 (typically the sequence with sequence number 41). In one embodiment, the donor bacteria, for example, Escherichia coli, are pMRC01;RSF1010;pRS01;pMV158;pTF1;pSC101;pBTK445;pBBR1;R721;pRmeGR4a;ColE1;pTiC58;pMdT1;R1;Tn5520;QKH54;R64;R751;RP4;pKL1;RK2;R1162;Tn4555;pHT;Tn4399;Tn916;pST A conjugation plasmid, conjugation transposon, or integration and conjugation element (ICE) selected from the group consisting of 12;pCU1;pSU233;F;pMAB01;R388;pS7a;pS7b;R702;pMUR274;R100;pVCR94deltaX;R46;pGO1, and pIP501 is used to efficiently deliver a DNA vector into Propionibacterium acnes recipient cells. In one embodiment, the DNA vector is pMRC01;RSF1010;pRS01;pMV158;pTF1;pSC101;pBTK445;pBBR1;R721;pRmeGR4a;ColE1;pTiC58;pMdT1;R1;Tn5520;QKH54;R64;R751;RP4;pKL1;RK2;R1162;Tn4555;pHT;Tn4399; It contains a conjugation plasmid, conjugation transposon, or origin and associated relaxase of integration and conjugation elements (ICE), selected from the group consisting of Tn916;pST12;pCU1;pSU233;F;pMAB01;R388;pS7a;pS7b;R702;pMUR274;R100;pVCR94deltaX;R46;pGO1, and pIP501.

[0174] In one preferred embodiment, the DNA vector includes the transmission origin oriT_pMRC01 (typically the sequence of SEQ ID NO: 1). In one preferred embodiment, the DNA vector includes the transmission origin oriT_RSF1010 (typically the sequence of SEQ ID NO: 2). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pRS01 (typically the sequence of SEQ ID NO: 3). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pMV158 (typically the sequence of SEQ ID NO: 4). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pTF1 (typically the sequence of SEQ ID NO: 5). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pSC101 (typically the sequence of SEQ ID NO: 6). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pBTK445 (typically the sequence of SEQ ID NO: 7). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pBBR1 (typically the sequence of SEQ ID NO: 8). In one preferred embodiment, the DNA vector includes the transmission origin oriT_R721 (typically the sequence of SEQ ID NO: 9). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pRmeGR4a (typically the sequence of SEQ ID NO: 10). In one preferred embodiment, the DNA vector includes the transmission origin oriT_ColE1 (typically the sequence of SEQ ID NO: 11). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pTiC58 (typically the sequence of SEQ ID NO: 12). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pMdT1 (typically the sequence of SEQ ID NO: 13). In one preferred embodiment, the DNA vector includes the transmission origin oriT_R1 (typically the sequence of SEQ ID NO: 14). In one preferred embodiment, the DNA vector includes the transmission origin oriT_Tn5520 (typically the sequence of SEQ ID NO: 15). In one preferred embodiment, the DNA vector includes the transmission origin oriT_QKH54 (typically the sequence of SEQ ID NO: 16). In one preferred embodiment, the DNA vector includes a transfer origin oriT_R64 (typically the sequence of SEQ ID NO: 17). In another preferred embodiment, the DNA vector includes a transfer origin oriT_R751 (typically the sequence of SEQ ID NO: 18).In one preferred embodiment, the DNA vector includes the transmission origin oriT_RP4 (typically the sequence of SEQ ID NO: 19). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pKL1 (typically the sequence of SEQ ID NO: 20). In one preferred embodiment, the DNA vector includes the transmission origin oriT_RK2 (typically the sequence of SEQ ID NO: 21). In one preferred embodiment, the DNA vector includes the transmission origin oriT_R1162 (typically the sequence of SEQ ID NO: 22). In one preferred embodiment, the DNA vector includes the transmission origin oriT_Tn4555 (typically the sequence of SEQ ID NO: 23). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pHT (typically the sequence of SEQ ID NO: 24). In one preferred embodiment, the DNA vector includes the transmission origin oriT_Tn4399 (typically the sequence of SEQ ID NO: 25). In one preferred embodiment, the DNA vector includes the transmission origin oriT_Tn916 (typically the sequence of SEQ ID NO: 26). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pST12 (typically the sequence of SEQ ID NO: 27). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pCU1 (typically the sequence of SEQ ID NO: 28). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pSU233 (typically the sequence of SEQ ID NO: 29). In one preferred embodiment, the DNA vector includes the transmission origin oriT_F (typically the sequence of SEQ ID NO: 30). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pMAB01 (typically the sequence of SEQ ID NO: 31). In one preferred embodiment, the DNA vector includes the transmission origin oriT_R388 (typically the sequence of SEQ ID NO: 32). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pS7a (typically the sequence of SEQ ID NO: 33). In one preferred embodiment, the DNA vector includes the transmission origin oriT_pS7b (typically the sequence of SEQ ID NO: 34). In one preferred embodiment, the DNA vector includes the transmission origin oriT_R702 (typically the sequence of SEQ ID NO: 35). In another preferred embodiment, the DNA vector includes the transmission origin oriT_pMUR274 (typically the sequence of SEQ ID NO: 36).In a preferred embodiment, the DNA vector comprises the transfer origin oriT_R100 (typically the sequence of SEQ ID NO: 37). In a preferred embodiment, the DNA vector comprises the transfer origin oriT_pVCR94deltaX (typically the sequence of SEQ ID NO: 38). In a preferred embodiment, the DNA vector comprises the transfer origin oriT_R46 (typically the sequence of SEQ ID NO: 39). In a preferred embodiment, the DNA vector comprises the transfer origin oriT_pGO1 (typically the sequence of SEQ ID NO: 40). In a preferred embodiment, the DNA vector comprises the transfer origin oriT_pIP501 (typically the sequence of SEQ ID NO: 41).

[0175] In one embodiment, the donor bacterium is selected from the group consisting of Escherichia coli, Pseudomonas aeruginosa, Lactococcus lactis, Lactobacillus casei, Lactobacillus fermentum, Lactobacillus rhamnosus, Propionibacterium freudenreichii, Lactobacillus brevis, Staphylococcus epidermidis, Staphylococcus aureus, Cutibacterium granulosum, Cutibacterium humerusii, Enterococcus faecalis, and Bacillus subtilis, which carry a conjugative plasmid, a conjugative transposon, or an integrative and conjugative element (ICE).

[0176] In one embodiment, donor bacteria containing a mobile DNA vector and a conjugation mechanism (ICE, plasmid, conjugation transposon), such as E. coli, are grown at high density to perform conjugation. The donor cells are pelletized by centrifugation and washed to remove antibiotics added during growth to maintain the mobile DNA vector and conjugation DNA vector. The donor cells are then mixed in the presence of Propionibacterium acnes cells. The donor cell-Propionibacterium acnes mixture is spotted onto a Brucella agar plate and conjugated under anaerobic conditions at 37°C. After conjugation, the cells are harvested from the conjugation plate and resuspended in BHI broth. - A compound that kills donor cells but does not kill acne bacteria, or - Antibiotics that select mobile DNA vectors Plate the mixture onto Brucella agar plate supplemented with the mixture.

[0177] After several days of incubation, acne bacteria colonies are streaked onto Brucella agar plates supplemented with appropriate selections, and the presence of conjugated plasmids is confirmed by specific PCR. The identity of the acne bacteria, as well as the absence of donor cells, are also confirmed by PCR analysis.

[0178] In one embodiment, conjugation is performed according to the following protocol: 2 mL of overnight culture of E. coli donor cells containing a mobile DNA vector and conjugation mechanism (ICE, plasmid, conjugation transposon) is grown in LB broth and pelletized at 6,000 × g for 1 minute. The supernatant is discarded, the pellet is washed with 500 μL of pre-sterilized LB medium, and centrifuged again under the same conditions. The pellet is then resuspended in 200 μL of 10 × concentrated C. acnes receptor BHI culture, which is growing exponentially (OD600 = 0.5). The E. coli-Propionibacterium acnes mixture is spotted onto Brucella agar plates (50 μL / spot) and conjugated for 24 hours under anaerobic conditions at 37°C. Subsequently, cells are harvested from the conjugation plates, resuspended in 300 μL of BHI broth, and plated onto Brucella agar plate supplemented with 50 μg / mL polymyxin B and 5 μg / mL erythromycin or 3.5 μg / mL chloramphenicol, depending on the selection marker present in the mobile DNA vector. After 7 days, Propionibacterium acnes cells grown in the presence of selection are streaked onto Brucella agar plates supplemented with the appropriate selection, and the presence of the conjugated plasmid is confirmed by specific PCR. The characteristics of Propionibacterium acnes and the absence of E. coli donor strains are also confirmed by PCR analysis.

[0179] Method for modifying endogenous *Propionibacterium acnes* plasmids Naturally occurring *Propionibacterium acnes* plasmids have been described. 21、22 Some of these can be transmitted from one type of acne bacteria to another through conjugation. 20 The ability to modify such plasmids is of interest for studying their effects in acne vulgaris, particularly their pro-inflammatory role, or for using these plasmids for further genetic manipulation of Propionibacterium acnes. The inventors have developed a method for modifying Propionibacterium acnes plasmids.

[0180] In one embodiment, the method involves, in the first step, - The selection marker for acne bacteria as defined above, - The replication origin for acne bacteria as defined above, - The phage packaging signal defined above, and - A template suitable for homologous recombination with endogenous plasmids of Propionibacterium acnes. The process includes introducing a replication vector containing into the acne bacteria.

[0181] In one embodiment, the method involves, in the first step, - The selection marker for acne bacteria as defined above, - The replication origin for acne bacteria as defined above, - The phage packaging signal defined above, - CRISPR-Cas system, and - A template suitable for homologous recombination within endogenous plasmids of Propionibacterium acnes. The process includes introducing a replication vector containing into the acne bacteria.

[0182] Introduction can be achieved by electroporation, electroporation of protoplasts, conjugation, chemical transformation, or transduction. The recombinant *Propionibacterium acnes* is then grown, preferably in the presence of an antibiotic.

[0183] Next, the recombinant organism is infected with a typical *Propionibacterium acnes* phage to produce phage-derived particles that carry the DNA vector.

[0184] Next, phage-derived particles are mixed with acne-bearing cell receivers, typically containing an endogenous plasmid, such as pIMPLE-HL096PA1. Then, acne-bearing cell transdermal cells are typically selected on an appropriate antibiotic.

[0185] In the second step, to increase the likelihood of a homologous recombination event, the *Propionibacterium acnes* transdextrins are grown at high density in the presence of antibiotic A. Homologous recombination typically results in the introduction of a selection marker, conferring resistance to antibiotic B. In a dense culture, *Propionibacterium acnes* strains carrying both wild-type endogenous plasmids and recombinant endogenous plasmids carrying resistance markers are typically present. The dense culture is then preferably washed and placed in the presence of receiver *Propionibacterium acnes* strains that are typically resistant to a third antibiotic C. Selection of conjugated cells by antibiotics C and B typically results in the selection of receiver cells carrying recombinant plasmids.

[0186] Other modifications enabled by the method of the present invention include the insertion of E. coli replicons and E. coli resistance markers on plasmids, enabling the extraction of plasmids from Propionibacterium acnes, as well as their transformation and replication in E. coli.

[0187] Furthermore, a plasmid carrying template DNA suitable for homologous recombination is preferably used to enable the expression of genes that increase the recombination rate.

[0188] A mold suitable for homologous recombination typically contains homology arms upstream and downstream of the recombination point. These homology arms are preferably 50bp, 100bp, 500bp, and 1000bp or longer.

[0189] Genome manipulation of Propionibacterium acnes, and manipulated Propionibacterium acnes strains The present invention includes a method for manipulating the genome of Propionibacterium acnes, and a manipulated Propionibacterium acnes strain manipulated by either of the methods of the present invention. A "manipulated strain" is a strain obtained by either of the methods of the present invention, containing either naturally occurring or unnatural mutations. For example, a manipulated Propionibacterium acnes strain may contain either the vector or DNA of the present invention.

[0190] The present invention includes a method for delivering a target DNA into an acne strain by conjugation. The present invention further includes a method for delivering a target DNA into an acne strain via phage-derived particles. The present invention includes a method for manipulating the acne bacterial chromosome using a replicative vector method and a non-replicative vector method.

[0191] In one embodiment, the delivery of a DNA vector into an acne bacterium is by transduction. In one embodiment, the DNA vector includes a phage packaging signal (cos) derived from an acne bacteriophage. In one embodiment, phage-derived particles containing the DNA vector can be generated, enabling the DNA vector to be transduced into acne bacterial cells.

[0192] In one embodiment, the present invention includes a replicative vector method and a non-replicative vector method using a vector comprising at least one recombination template having one or two homology arms.

[0193] To manipulate the acne bacterial genome, the inventors developed methods using replicative vectors and non-replicative vectors.

[0194] Non-replicative vector method In one embodiment, the non-replicative vector method comprises at least - the phage packaging signal as defined above; - the selection marker for acne bacteria as defined above; - a recombination template having one or two homology arms; - a replication origin that enables replication only within Cutibacterium acnes-producing cells; and - optionally, a counterselection marker, such as SacB using a vector comprising.

[0195] The non-replicating vector method uses a vector that carries an acne bacterium replicon that replicates only within acne bacterium-producing cells but not within other acne bacterium cells. Therefore, such a vector can replicate within acne bacterium-producing cells, and upon contact with a phage genome, can be packaged within a phage capsid, yielding phage-derived particles, which can then be transduced into acne bacterium receiver cells, where they do not replicate.

[0196] This method includes the step of introducing a plasmid containing a template suitable for homologous DNA recombination within the genome into acne-producing cells. The template may contain one (Figure 4A) or two (Figure 4B) homologous regions that induce homologous recombination.

[0197] In one embodiment, the method includes acne-producing cells carrying a plasmid containing a template suitable for homologous DNA recombination within chromosomes, where homologous DNA is not present within the chromosomes of the acne-producing cells; a phage packaging signal (cos) derived from acne phage; a selection marker for acne as defined above; and a replication origin for acne-producing cells that does not replicate within acne-producing receiver cells. The template may contain one (Figure 4A) or two (Figure 4B) homologous regions that result in homologous recombination. The producing cells are typically infected with acne phage, resulting in the production of phage-derived particles containing a DNA vector with homology arms. The phage-derived particles are preferably mixed with acne-producing receiver cells (e.g., ATCC11828). Transducers can be selected on an antibiotic plate, streaked on the antibiotic plate, and screened for plasmid integration by PCR. Since the plasmid is non-replicating in acne, only recombinant cells that stably maintain the antibiotic marker can grow on the antibiotic plate.

[0198] When two homology arms are present on the template DNA, a first recombination event (also called a crossover) typically results in complete plasmid integration. This is typically followed by a second recombination event, which involves the removal of the plasmid backbone and results in chromosomal modification or reconstruction of the wild-type locus.

[0199] In one embodiment, *Propionibacterium acnes*-producing cells carry a vector containing a *Propionibacterium acnes* selection marker, e.g., ermE(pEB_HR02), flanked by a left homology arm (LHA) and a right homology arm (RHA). The two homology arms typically do not match the *Propionibacterium acnes*-producing cell chromosome. In one embodiment, the vector further contains a phage packaging signal (cos) derived from a *Propionibacterium acnes* phage, a selection marker for *Propionibacterium acnes*, and an origin of replication for *Propionibacterium acnes*-producing cells that does not replicate in *Propionibacterium acnes* receiver cells. In one embodiment, the DNA vector further contains a *Propionibacterium acnes* counter-selection marker, e.g., sacB, on the plasmid backbone, enabling selection of a second recombination event. *Propionibacterium acnes*-producing cells carrying pEB_HR02 are typically infected with a phage, resulting in the production of phage-derived particles containing pEB_HR02. These phage-derived particles are typically placed in the presence of *Propionibacterium acnes* receiver cells (e.g., ATCC11828). Transduction cells are typically selected on plates supplemented with an antibiotic (e.g., erythromycin), streaked on plates supplemented with the same antibiotic, and plasmid integration is confirmed by PCR. Since the plasmid is not replicated within the *Propionibacterium acnes* receiver cell, *Propionibacterium acnes* clones that can grow in the presence of the antibiotic (e.g., erythromycin) have undergone a single homologous recombination event resulting in the integration of the complete plasmid. For the final recombination locus selection, cells are typically exposed to counterselection (e.g., sucrose) and the antibiotic (e.g., erythromycin), which leads to cell death due to sacB activity (the complete plasmid remains integrated into the chromosome). Surviving cells are typically screened for the presence of a successful final recombination locus by PCR. In one embodiment, the DNA vector contains only one homology arm (pEB_HR01). In one embodiment, both pEB_HR01 and pEB_HR02 phage-derived particles are applied to the skin without antibiotic selection.

[0200] In one embodiment, *Propionibacterium acnes*-producing cells carry a plasmid (vector) containing a left homology arm (LHA) and a right homology arm (RHA) adjacent to the *Propionibacterium acnes* selection marker ErmE (pEB_HR02). The vector preferably further contains an *E. coli* origin of replication, an *E. coli* selection marker, oriT and relaxase derived from the conjugation plasmid, and a *Propionibacterium acnes* counter-selection marker, such as sacB. pEB_HR02 can be transformed into *E. coli* donor cells (e.g., Ec0s2862). Typically, the transformants are selected, grown, and mixed with *Propionibacterium acnes* receiver cells (e.g., ATCC11828). The completed conjugations are typically selected on a plate supplemented with an antibiotic (e.g., erythromycin), streaked on a plate supplemented with an antibiotic (e.g., erythromycin), and plasmid integration is confirmed by PCR. Because plasmids are non-replicating within *Propionibacterium acnes* receiver cells, *Propionibacterium acnes* clones that can grow in the presence of antibiotics (e.g., erythromycin) undergo a single homologous recombination event resulting in the integration of the complete plasmid. The selection of the final recombinant locus typically involves exposing cells to counter-selection (e.g., sucrose) and antibiotics (e.g., erythromycin), which leads to cell death due to sacB activity (the complete plasmid remains integrated into the chromosome). Surviving cells are typically screened for the presence of a successful final recombinant locus by PCR.

[0201] CRISPR-Cas System Selection Vector Method This invention includes a replication vector containing a replication origin for Propionibacterium acnes.

[0202] In one embodiment, the replicated CRISPR-Cas selection vector method is at least - The phage packaging signal (cos) derived from the acne bacterium phage as defined above; - The selection marker for acne bacteria as defined above; - A replication origin for acne bacteria; - Recombination mold having two homology arms; and - CRISPR-Cas system for expression within Propionibacterium acnes Use a vector that includes this.

[0203] In one embodiment, the replicated CRISPR-Cas selection vector method is at least - The selection marker for E. coli as defined above; - Replication origin for E. coli; - The selection marker for acne bacteria as defined above; - Recombination mold with two homology arms; - A replication origin for acne bacteria; and - The CRISPR-Cas system expressed within *Propionibacterium acnes* Use a vector that includes this.

[0204] Therefore, such vectors are replicable in E. coli and also in Propionibacterium acnes. The vectors further carry a CRISPR-Cas system capable of inducing double-strand breaks at the wild-type locus where recombination is desired, leading to the death of Propionibacterium acnes receiver cells.

[0205] In one embodiment, the method involves using *Propionibacterium acnes*-producing cells, such as strain ATCC6919, that carry a replicating CRISPR-Cas system selection vector containing a template suitable for homologous DNA recombination within a chromosome and a phage packaging signal (cos) derived from *Propionibacterium acnes* phage. In one embodiment, the template contains two homologous regions (Figure 5) to induce homologous recombination. The producing cells preferably do not contain the wild-type locus targeted by the CRISPR-Cas system. The *Propionibacterium acnes*-producing cells are typically infected with *Propionibacterium acnes* phage, resulting in the production of phage-derived particles that carry the DNA vector. The phage-derived particles are typically brought into contact with *Propionibacterium acnes* receiver cells. After transduction into the *Propionibacterium acnes* receiver cells, the cells recombined with the DNA template vector are not targeted by the CRISPR-Cas system, for example, because they no longer possess the relevant PAM sequence. Placing on antibiotic-containing media, such as erythromycin plates, typically ensures that viable cells are transduced and still carry DNA vectors (e.g., plasmids) expressing the CRISPR-Cas system. Single colonies are typically streaked on antibiotic-containing media, such as erythromycin plates, and recombinant loci are typically confirmed by PCR and sequencing.

[0206] In one embodiment, a plasmid curing step is performed to remove the plasmid.

[0207] In one embodiment, the acne-producing cells contain the DNA target of the CRISPR-Cas system, but the CRISPR-Cas system is not expressed in the acne-producing cells but is expressed in the acne-receiving cells. More preferably, the CRISPR-Cas system is suppressed in the acne-producing cells but not in the acne-receiving cells.

[0208] Such methods can be used for scarless editing, such as substitution, deletion, or insertion, because it eliminates the need to introduce selection markers for recombinants, as selection is achieved by CRISPR-Cas sterilization.

[0209] Self-targeted replication vector method In one embodiment, the present invention includes a self-targeted replication vector method. In one embodiment, the present invention brings about linearization of the recombinant template, which has been shown to increase recombination efficiency. 9 This includes the use of a CRISPR-Cas system to plan the cleavage of a DNA vector (e.g., plasmid) at one or more target sequences. To enable the cloning of self-targeting vectors, an inducible CRISPR-Cas system can be used, for example, by using an inducible promoter upstream of the gene encoding the Cas nuclease. By combining this inducible promoter with a riboswitch, even tighter inhibition of CRISPR-Cas system expression can be ensured. Another strategy for generating a self-targeting CRISPR-Cas system relies on a promoter that is repressed in *Propionibacterium acnes*-producing cells but not in *Propionibacterium acnes*-receiving cells. In this way, the CRISPR-Cas system becomes active only once introduced into *Propionibacterium acnes*-receiving cells.

[0210] For example, gene substitution can be performed using such strategies, either by using an antibiotic marker adjacent to the homology arm (Figure 6A), or by performing scarless recombination using a CRISPR-Cas system with bacterial-killing capabilities when targeting the acne bacilli chromosome (Figure 6B).

[0211] After introducing and selecting a DNA vector (e.g., plasmid), homologous events typically occur, leading to the removal of the PAM sequence.

[0212] Furthermore, DNA vectors (e.g., plasmids) that carry template DNA suitable for homologous recombination typically enable the expression of genes that increase the recombination rate.

[0213] In one embodiment, the DNA vector comprises a template suitable for homologous recombination and a CRISPR-Cas system that targets the DNA vector itself outside the template region, wherein the RNA guide (crRNA or sgRNA) derived from the CRISPR-Cas system does not perfectly match the DNA target.

[0214] In one embodiment, the present invention is at least - The phage packaging signal (cos) derived from the acne bacterium phage as defined above; - The selection marker for acne bacteria as defined above; - The replication origin for *Propionibacterium acnes* as defined above; and - CRISPR-Cas system for expression within Propionibacterium acnes This includes a vector replication method that uses vectors containing the vector.

[0215] In one embodiment, the vector carries a CRISPR-Cas system capable of inducing double-strand breaks that result in the death of most Propionibacterium acnes receiver cells, except for Propionibacterium acnes receiver cells that no longer carry the CRISPR-Cas system target sequence due to spontaneous mutation or recombination.

[0216] Genome manipulation of *Propionibacterium acnes* phage The present invention includes methods for manipulating the genome of *Propionibacterium acnes* phage, and manipulated *Propionibacterium acnes* phages manipulated by any of the methods of the present invention. “Manipulated or recombinant phage” is a phage obtained by any of the methods of the present invention, containing either naturally occurring or non-naturally occurring mutations. For example, a manipulated *Propionibacterium acnes* phage genome carries one or more nucleotide substitutions, deletions, or insertions. In other embodiments, a *Propionibacterium acnes* phage genome carries one or more transgenes that are expressed immediately upon phage infection.

[0217] In one embodiment, the acne bacterium phage contains one or more genetic modifications within the acne bacterium phage genome.

[0218] In one embodiment, the modification of the acne bacterium phage genome is the insertion, deletion, or substitution of one or more nucleotides within the acne bacterium phage genome.

[0219] In one embodiment, the modification of the acne bacterium phage genome is silent.

[0220] In one embodiment, the modification of the acne bacterium phage genome is not silent.

[0221] In one embodiment, modification of the acne bacterium phage genome does not impair acne bacterium phage production.

[0222] In one embodiment, modification of the *Propionibacterium acnes* phage genome alters the phage host region.

[0223] In one embodiment, the modification of the *Propionibacterium acnes* phage genome results in inhibition of its packaging into the phage capsid without interfering with the production of the capsid itself. More preferably, the genome modification is a modification of the phage packaging sequence. Even more preferably, the modification is a deletion of the phage packaging sequence.

[0224] In one embodiment, the deletion phage packaging sequence is a cos site from a Propionibacterium acnes phage whose sequence is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequence of SEQ ID NO: 66.

[0225] In one embodiment, the deletion phage packaging sequence is a cos site from a Propionibacterium acnes phage whose sequence is at least 80%, 83%, 85%, 87%, 90%, 93%, 95%, 96%, 97%, 98%, 99%, or 100% identical to one or more sequences selected from the group consisting of sequences SEQ ID NO: 76, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.

[0226] In one embodiment, the modified acne bacterium phage is located within the coding sequence.

[0227] In one embodiment, the acne bacterium phage modification is located within a regulatory element, such as a promoter, transcription terminator, origin of replication, RBS, or riboswitch.

[0228] In one embodiment, the modified *Propionibacterium acnes* phage is located within the non-coding sequence and the non-regulatory sequence.

[0229] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome.

[0230] In one embodiment, the modification of the acne bacterium phage involves the insertion of one or more transgenes into the acne bacterium phage genome, and the insertion does not remove any nucleotides that are originally present in the phage genome.

[0231] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome within an intergene region.

[0232] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, the insertion of which replaces one or more nucleotides.

[0233] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, the transgenes replacing one or more genes within a transcription unit containing one or more genes without perturbing the upstream and downstream of the genes.

[0234] In one embodiment, the modification of the acne bacterium phage involves the insertion of one or more transgenes into the acne bacterium phage genome, the transgenes replacing the foline gene.

[0235] In one embodiment, the modification of the acne bacterium phage involves the insertion of one or more transgenes into the acne bacterium phage genome, the transgenes replacing the endolysin gene.

[0236] In one embodiment, the modification of the acne bacterium phage involves the insertion of one or more transgenes into the acne bacterium phage genome, the transgenes replacing the forin gene and the endolysin gene.

[0237] In one embodiment, the modification of the acne bacterium phage involves the insertion of one or more transgenes into the acne bacterium phage genome, and the forin gene and endolysin gene are pre-modified to be inactive.

[0238] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, wherein the transgenes are the selection markers in acne bacterium as defined above.

[0239] In one embodiment, the modification of the acne bacterium phage involves the insertion of a replication origin that enables replication within the acne bacterium.

[0240] In one embodiment, the modification of the acne bacterium phage is below: - The replication origin that enables replication within the acne bacteria as defined above; and - Selective markers in Propionibacterium acnes, as defined above. This is an insertion.

[0241] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, each transgene containing a promoter, one or more coding sequences, and a transcription terminator.

[0242] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, each transgene containing an inducible promoter, one or more coding sequences and their ribosome binding sites, and a transcriptional terminator.

[0243] In one embodiment, the modification of the acne bacterium phage is the insertion of one or more transgenes into the acne bacterium phage genome, each transgene containing a promoter, one or more coding sequences and their ribosome binding sites, and a transcriptional terminator, wherein the promoter is active in a specific acne bacterium strain but inactive or has low activity in other acne bacterium strains.

[0244] In order to manipulate the *Propionibacterium acnes* phage genome, the inventors of this invention have, - Using the introduction of recombinant phage genomes into rebooting bacteria, such as, but not limited to, *Propionibacterium acnes*, which enable in vitro or in vivo DNA assembly of recombinant phage genomes and the production of recombinant phages; and / or - Selection of recombinant phages using a CRISPR-Cas system selection vector for recombination and replication within *Propionibacterium acnes*. We developed a method for generating recombinant acne phage.

[0245] Recombinant acne bacterium phage manipulation by genome assembly and rebooting method The present invention includes a method for producing a manipulated phage, which involves generating a phage containing the assembled modified phage genome, following DNA assembly for generating a modified phage genome.

[0246] In one embodiment, DNA fragments are assembled into the recombinant acne bacterium phage genome and introduced into the rebooting bacteria in order to produce recombinant acne bacterium phages.

[0247] In one embodiment, DNA fragments are assembled into the recombinant acne bacterium phage genome and transformed into rebooting bacteria in order to produce recombinant acne bacterium phages.

[0248] In one embodiment, the rebooting bacteria are from the family Propionibacteriaceae. Preferably, the rebooting bacteria are Propionibacterium freudenreichii, and more preferably, Propionibacterium acnes.

[0249] In one embodiment, to produce recombinant acne bacterium phage, DNA fragments are assembled into the recombinant acne bacterium phage genome within the vector and introduced into the rebooting bacteria.

[0250] In one embodiment, DNA fragments are assembled into the recombinant acne bacterium phage genome inside a vector, and the vector carrying the recombinant acne bacterium phage genome is introduced into a bacterium that conjugates to the acne bacterium producing recombinant phages, such as E. coli, and then amplified on the acne bacterium.

[0251] In one embodiment, DNA fragments are assembled into the recombinant acne bacterium phage genome inside a vector, and the vector carrying the recombinant acne bacterium phage genome is transformed into bacteria that conjugate to the acne bacterium producing recombinant phages, such as E. coli, and then amplified on the acne bacterium.

[0252] In one embodiment, below: - The replication origin that enables replication within E. coli, as defined above; - The selection marker for E. coli as defined above; - The oriT from the conjugated plasmid as defined above, - In some cases, relaxase from the conjugation plasmid; and - Depending on the circumstances, the replication origin that enables replication within the acne bacteria as defined above, DNA fragments are assembled into the recombinant acne bacterium phage genome within the vector containing the recombinant acne bacterium phage genome, and then transformed into E. coli carrying the conjugation vector. Subsequently, the vector containing the recombinant acne bacterium phage genome is typically conjugated into the acne bacterium.

[0253] In one embodiment, the vector into which DNA fragments are assembled into the recombinant acne bacterium phage genome is a bacterial artificial chromosome (BAC).

[0254] In one embodiment, the following techniques: - Gibson Assembly, - PCR assembly, - Golden Gate assemblies and derivatives (MOCLO, GoldenBraid, etc.), - GeneArt® Seamless Assembly, - SLIC assembly, - CPEC assembly, - SLiCE assembly One or a combination of these methods is used to assemble DNA fragments in vitro into the recombinant *Propionibacterium acnes* phage genome.

[0255] In one embodiment, the following method: - PCR using the acne bacterium phage genome, - Digestion of the acne bacterium phage genome, - DNA synthesis (chemical or enzymatic), - Oligo Assembly DNA fragments are generated by one or a combination of these.

[0256] In one embodiment, transformation-associated recombination (TAR) in yeast is used to assemble DNA fragments in vivo into the recombinant acne bacterium phage genome.

[0257] In one embodiment, DNA fragments are assembled in vitro or in vivo into a recombinant phage genome within a cloning vector, such as a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a P1 phage-derived artificial chromosome, a plasmid, or any combination thereof. The recombinant phage genome is then typically extracted from the cloning vector, optionally circularized, and introduced into a rebooting bacterial strain.

[0258] In one embodiment, recombinant acne bacterium phage genome is introduced into acne bacters by transformation. More preferably, recombinant acne bacterium phage genome is introduced into acne bacters by electroporation.

[0259] In one embodiment, a recombinant acne bacterium phage genome is introduced into L-type acne bacterium or acne bacterium protoplasts.

[0260] Recombinant manipulation of acne bacterium phages The present invention includes a method for producing a manipulated phage, comprising the steps of introducing a modification into the acne bacterium phage genome using recombination between a DNA template and the acne bacterium phage genome, and generating a phage containing the modified phage genome.

[0261] In one embodiment, an acne bacterium phage genome is introduced into an acne bacterium strain containing a DNA template, and a recombination event between the phage genome and the DNA template results in a modification of a portion of the phage genome, which is then packaged into an acne bacterium phage.

[0262] In one embodiment, below: - The selection marker for acne bacteria as defined above; - The replication origin for acne bacteria as defined above, - Recombination molds having one or two homology arms, and - Depending on the case, a recombination mechanism After introducing the *Propionibacterium acnes* phage genome into *Propionibacterium acnes* cells containing a replication vector, manipulated *Propionibacterium acnes* phages are produced.

[0263] In one embodiment, below: - Selective markers for donor bacteria, such as E. coli, as defined above. - The donor bacteria, for example, the replication origin for E. coli, as defined above, - The selection marker for acne bacteria as defined above, - The oriT from the conjugated plasmid as defined above; - Depending on the case, relaxase from the conjugation plasmid, - The replication origin for acne bacteria as defined above, - Recombination mold having one or two homology arms, - Depending on the case, a recombination mechanism After introducing the *Propionibacterium acnes* phage genome into *Propionibacterium acnes* containing a replication vector, manipulated *Propionibacterium acnes* phages are produced.

[0264] In one embodiment, the acne bacterium phage genome is introduced into the acne bacterium by electroporation.

[0265] In one embodiment, the acne bacterium phage genome is introduced into the acne bacterium via phage infection.

[0266] In one embodiment, the DNA template suitable for homologous recombination is linear double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) introduced by electroporation.

[0267] In one embodiment, an oligonucleotide is a DNA template suitable for homologous recombination.

[0268] In one embodiment, both the phage genome and the DNA template are transformed into *Propionibacterium acnes*. Preferably, the transformation method is electroporation.

[0269] CRISPR-Cas system selection method for recombinant phages The present invention includes a method for selecting a CRISPR-Cas system for recombinant phages. The present invention includes *Propionibacterium acnes* cells that carry a CRISPR-Cas system expressed within *Propionibacterium acnes* and engineered to target wild-type phage genomes or pre-modified phage genomes but not newly generated recombinant *Propionibacterium acnes* phages.

[0270] In one embodiment, the operational CRISPR system is an endogenous CRISPR-Cas system.

[0271] In another embodiment, the CRISPR-Cas system is an external CRISPR-Cas system.

[0272] In one embodiment, the CRISPR-Cas system is an exogenous CRISPR-Cas system incorporated onto the chromosome of Propionibacterium acnes.

[0273] In one embodiment, the CRISPR-Cas system is an exogenous CRISPR-Cas system on a replication vector.

[0274] In one embodiment, the present invention comprises at least the following: - The selection marker for acne bacteria as defined above, - The replication origin for *Propionibacterium acnes* as defined above; and - An exogenous CRISPR-Cas system that is expressed within *Propionibacterium acnes* and targets wild-type or pre-modified phage genomes, but does not target newly generated recombinant *Propionibacterium acnes* phage genomes. Includes a replica CRISPR-Cas system selection vector.

[0275] In one embodiment, the present invention comprises at least the following: - Selective markers for donor bacteria, such as E. coli, as defined above. - The donor bacteria, for example, the replication origin for E. coli, as defined above, - The selection marker for acne bacteria as defined above, - The oriT from the conjugated plasmid as defined above; - Depending on the case, relaxase from the conjugation plasmid, - The replication origin for *Propionibacterium acnes* as defined above; and - An exogenous CRISPR-Cas system that is expressed within *Propionibacterium acnes* and targets wild-type or pre-modified phage genomes, but does not target newly generated recombinant *Propionibacterium acnes* phage genomes. Includes a replica CRISPR-Cas system selection vector.

[0276] In one embodiment, the method is as follows: - Selective markers for donor bacteria, such as E. coli, as defined above. - The donor bacteria, for example, the replication origin for E. coli, as defined above, - The selection marker for acne bacteria as defined above, - The oriT from the conjugated plasmid as defined above, - Depending on the case, relaxase from the conjugation plasmid, - The replication origin for *Propionibacterium acnes* as defined above; and - An exogenous CRISPR-Cas system that is expressed within *Propionibacterium acnes* and targets wild-type or pre-modified phage genomes, but does not target newly generated recombinant *Propionibacterium acnes* phage genomes. The process includes the step of conjugating a replicated CRISPR-Cas system selection vector containing into a strain of Propionibacterium acnes, such as strain ATCC6919.

[0277] After conjugation into *Propionibacterium acnes*, conjugated cells are typically selected on an appropriate antibiotic. The presence of the replicating CRISPR-Cas system selection vector within *Propionibacterium acnes* is optionally confirmed by PCR. Single colonies are typically grown in dense culture in the presence of an appropriate antibiotic and, preferably, mixed with a *Propionibacterium acnes* phage suspension containing a mixture of wild-type phages or pre-produced recombinant phages and newly produced recombinant phages. This suspension is typically then poured onto the upper agar layer. Optionally, the first amplification of recombinant *Propionibacterium acnes* phages may be performed on *Propionibacterium acnes* carrying the replicating CRISPR-Cas system selection vector. A phage suspension is typically obtained and can be mixed with a new culture of *Propionibacterium acnes* carrying the replicating CRISPR-Cas system selection vector for use on the upper agar layer.

[0278] Newly generated recombinant phages typically bind, inject their recombinant phage genome, replicate, and produce new recombinant particles, leading to plaque formation, whereas wild-type or pre-generated recombinant phages inject phage genomes that are recognized and cleaved by the CRISPR-Cas system, resulting in a failure of the phage replication cycle and thus no plaque formation. Typically, a single plaque is picked and isolated to confirm that it is recombinant and carries the expected genetic recombination. Finally, the isolated plaque is typically re-amplified in a *Propionibacterium acnes* indicator strain or in *Propionibacterium acnes* carrying a replication CRISPR-Cas system selection vector, preferably to store a pure phage suspension.

[0279] In one embodiment, recombinant DNA phages are not targeted by the CRISPR-Cas system because, for example, they do not have the relevant PAM sequence.

[0280] Method for producing recombinant phages within Propionibacterium acnes This invention includes a method for producing phage-derived particles within *Propionibacterium acnes*. To date, all *Propionibacterium acnes* phages described are genetically highly conserved and cannot stably replicate in plasmid form or be integrated into the host's chromosomes.1 As a result, acne phages are difficult to manipulate because they cause the death of the cells they infect. To address this problem, the inventors have developed a two-step method for manipulating acne phages.

[0281] In the first step, the E. coli donor strain that carries the conjugation vector is below: - The selection marker for E. coli as defined above; - The replication origin for E. coli as defined above, - The selection marker for acne bacteria as defined above, - Relaxase and oriT from conjugation plasmids, and - Recombination mold with one or two homology arms Transform with a mobile replication vector containing (e.g., pEB-PRECOMB).

[0282] Conjugation occurs between E. coli donor cells (E. coli pEB-PRECOMB) and Propionibacterium acnes receiver cells (e.g., Propionibacterium acnes ATCC6919). The Propionibacterium acnes conjugation cells (PEB-PRECOMB) are typically selected and grown on an appropriate antibiotic and infected with Propionibacterium acnes phage (e.g., PAC7). After phage infection, a recombination event occurs, resulting in a phage lysate containing wild-type Propionibacterium acnes phage (e.g., wt PAC7) or parent Propionibacterium acnes phage (i.e., the phage from which the desired recombinant phage originates) and recombinant phage (e.g., mt PAC7) (Figure 7A).

[0283] In the second step, the following: - The selection marker for E. coli as defined above; - The replication origin for E. coli as defined above, - The selection marker for acne bacteria as defined above, and - The relaxase and oriT from the junction vector as defined above, and - A CRISPR-Cas system expressed in Propionibacterium acnes that targets wild-type phages (e.g., PAC7). Use a replicate plasmid containing (e.g., pEB-PSCREEN).

[0284] In one embodiment, the replication plasmid is transformed into an E. coli donor strain carrying the conjugation plasmid (for example, resulting in the production of the E. coli donor strain pEB-PSCREEN). Conjugation occurs between the E. coli donor strain pEB-PSCREEN and Propionibacterium acnes receiver cells (e.g., ATCC6919). Conjugated Propionibacterium acnes pEB-PSCREEN cells are typically selected on an antibiotic plate, grown in high-density culture, and mixed with a phage lysate containing both wild-type phage (e.g., wt PAC7) and mutant phage (e.g., mt PAC7) (Figure 7B). The wild-type phage genome is typically cleaved by the CRISPR-Cas nuclease and therefore cannot replicate to form plaques, while the recombinant mt phage genome is not recognized by the CRISPR-Cas system, fails to replicate, and does not form plaques. Single plaques are typically isolated and screened by PCR for sequence verification. After confirmation by sequencing, recombinant phages are typically amplified on wild-type Propionibacterium acnes or on Propionibacterium acnes strains containing CRISPR-Cas system vectors that target wild-type phages.

[0285] Recombination events can result in substitutions, deletions, or insertions that lead to the removal of any portion of the PAM sequence or any sequence essential for CRISPR-Cas targeting. Insertions may result in the introduction of a fluorescent or enzyme reporter that aids in the isolation of recombinant plaques.

[0286] A mold suitable for homologous recombination typically contains homology arms upstream and downstream of the recombination point. These homology arms typically have sizes of at least 50 bp, 100 bp, 500 bp, 1000 bp, or at least 5000 bp.

[0287] Examples of applications for manipulating acne phages are as follows: - Expression of therapeutic proteins produced during phage infection and released when cells are lysed. - Expression of therapeutic proteins produced, secreted, or transported to the surface during phage infection. - Specific proteins on the phage capsid, e.g., antigen display, - Modification of the phage host range, and - Acquisition of strictly lytic phage variants This includes, but is not limited to, those items.

[0288] Furthermore, vectors (e.g., plasmids) that carry template DNA suitable for homologous recombination typically enable the expression of genes that increase the recombination rate.

[0289] Furthermore, vectors (e.g., plasmids) carrying template DNA suitable for homologous recombination typically carry inducible endonucleases, such as CRISPR-Cas, and lead to linearization of the vector during phage infection. This linearization of the template vector typically increases the recombination rate.

[0290] Protein expression by manipulated Propionibacterium acnes phages This invention involves the expression of a protein by a manipulated *Propionibacterium acnes* phage. By incorporating an expression cassette into the phage, the protein can be expressed by infected *Propionibacterium acnes*. The promoter within the expression cassette may be inductive or constitutive, enabling inductive or constitutive expression of the protein by the manipulated *Propionibacterium acnes* phage.

[0291] In one embodiment, a phage is used to regulate the expression of a target molecule and / or the interaction between *Propionibacterium acnes* and the host. In one embodiment, a phage genome is used to express a transgene in *Propionibacterium acnes*. The transgene can be cloned within a recombinant autonomously replicating phage vector under the control of a predetermined promoter (constitutive or inductive) followed by a predetermined terminator. Injection of this phage genome into *Propionibacterium acnes* enables the expression of the transgene. The transgene may be, for example, a CRISPR / Cas system, a base-edited or prime-edited expression cassette, or it may encode a human protein, such as an interleukin, or it may encode an antigen as defined below, such as a tumor antigen, viral antigen, bacterial antigen, fungal antigen, autoantigen, allergen, or graft-specific antigen.

[0292] Protein expression by manipulated Propionibacterium acnes strains The present invention includes the expression of proteins by a manipulated strain of Propionibacterium acnes. By incorporating an expression cassette into a DNA vector, the protein can be expressed by a transduced Propionibacterium acnes. The promoter within the expression cassette may be inductive or constitutive, enabling inductive or constitutive expression of the protein by the manipulated strain of Propionibacterium acnes. The expression of multiple proteins may be carried out as a single transcription unit (operon) or as separate transcription units. In a particular embodiment, the protein is an antigen as defined below, for example, a tumor antigen, viral antigen, bacterial antigen, fungal antigen, autoantigen, allergen, or graft-specific antigen.

[0293] Propionibacterium acnes phage The present invention includes acne bacilli phages and related manipulative phages, methods for producing these phages, and methods for using these phages to transduce acne bacilli.

[0294] In one particular embodiment, the manipulated acne bacterium phage of the present invention is unable to self-replicate.

[0295] In connection with the present invention, the terms “self-propagation” and “self-replication” are used without distinction and refer to the ability to have offspring, in particular the ability to produce new phages.

[0296] In this specification, “a phage that cannot self-replicate” means that at least one, more, or all of the functional genes (also referred to herein as “essential genes”) necessary for producing the phage are absent in the manipulated phage (and the phage genome contained in the manipulated phage).

[0297] In a preferred embodiment, at least one, more, or all of the functional genes necessary to produce the manipulated phage are present in the producing cell, preferably in a plasmid, phagemid, chromosome, or helper phage present in the producing cell, enabling the production of the manipulated phage within the producing cell. In such an embodiment, the manipulated acne bacilli phage that cannot self-replicate is further called a conditionally replicating acne bacilli phage.

[0298] In connection with the present invention, the functional gene necessary for producing the manipulated phage may be absent due to (i) the absence of the corresponding gene, or (ii) the presence of a non-functional form of the corresponding gene.

[0299] In one embodiment, the gene sequence necessary for producing the manipulated phage is absent in the manipulated phage. In a preferred embodiment, the gene sequence necessary for producing the manipulated phage is replaced with a target nucleic acid sequence.

[0300] Instead, the gene necessary for producing the manipulated phage exists in the manipulated phage in a non-functional form, for example, a mutant non-functional form or a non-expressible form, for example, accompanied by a deleted or mutated non-functional regulator. In a preferred embodiment, the gene necessary for producing the manipulated phage exists in the manipulated phage in a mutant form that makes it non-functional in receiver cells but remains functional in producing cells.

[0301] In connection with the present invention, the genes necessary for producing the manipulated phage include any coding nucleic acid or non-coding nucleic acid required for the production of the manipulated phage.

[0302] Examples of genes necessary to produce the aforementioned manipulated phages include genes encoding phage structural proteins; phage genes involved in the regulation of gene expression; phage genes involved in the regulation of transcription and / or translation; phage genes involved in phage DNA replication; phage genes involved in the production of phage proteins; phage genes involved in phage protein folding; phage genes involved in phage DNA packaging; and phage genes encoding proteins involved in bacterial cell lysis.

[0303] In one embodiment, the manipulated Propionibacterium acnes phage includes the essential gene defined above, and its expression is controlled by the Propionibacterium acnes phage promoter. The essential gene may be expressed constitutively or under an inductive system, such as an inductive promoter or a riboswitch. When the essential gene is under an inductive system, the manipulated Propionibacterium acnes phage is also considered a conditionally replicating phage.

[0304] In one embodiment, the conditionally replicated phage does not kill, or is unable to kill, unmanipulated acne-producing strains, and in particular, does not kill, or is unable to kill, unmanipulated acne-producing strains that do not contain the essential gene that is not present in the conditionally replicated phage.

[0305] In one embodiment, a conditionally replicated phage carries the CRISPR-Cas system.

[0306] In one embodiment, the conditionally replicating phage contains the transgene.

[0307] Phage-derived particles in acne bacteria The present invention includes phage-derived particles containing any DNA vector of the present invention, and methods for producing such phage-derived particles.

[0308] In one embodiment, a strain of acne bacteria carrying an arbitrary DNA vector of the present invention is brought into contact with an acne bacteria phage, resulting in the introduction of the phage genome into the acne bacteria strain, the expression of phage proteins necessary for phage capsid assembly, and the packaging of the DNA vector inside the phage capsid.

[0309] In one embodiment, a strain of acne bacteria carrying an arbitrary DNA vector including the above-defined selection marker for acne bacteria, the above-defined acne bacteria phage packaging signal (cos site), and the above-defined origin of replication for acne bacteria is brought into contact with an acne bacteria phage, resulting in the introduction of the phage genome into the acne bacteria strain, the expression of phage proteins necessary for phage capsid assembly, and the packaging of the DNA vector into the phage capsid.

[0310] In one embodiment, the phage genome is a wild-type phage genome.

[0311] In one embodiment, the acne bacterium phage is PAC7 (typically the sequence of sequence number 68).

[0312] In one embodiment, the phage genome is a manipulated phage genome.

[0313] Phage-derived particles can be purified by methods known in the art. The present invention comprises purified phage-derived particles containing the DNA vector of the present invention. In one embodiment, the purified phage-derived particles are present in an isolated composition or pharmaceutical composition. The composition contains at least 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pieces, 10 10 pieces, 10 11 pieces, 10 12pieces, 10 13 pieces, 10 14 It may contain more than one purified phage-derived particle.

[0314] Sequence-specific killing of acne bacteria by phage-derived particles In one embodiment, the present invention includes the specific killing of Propionibacterium acnes by phage-derived particles carrying a CRISPR-Cas system.

[0315] Phage-derived particles carrying vectors (e.g., plasmids) encoding the CRISPR-Cas system have recently been used to perform sequence-specific bacterial killing in situ. 10、11 The present inventors have developed a method for producing such phage-derived particles that target Propionibacterium acnes, and this method is included in the present invention.

[0316] In the above method, to produce a high-titer phage suspension, a *Propionibacterium acnes* strain containing the DNA vector of the present invention is brought into contact with a *Propionibacterium acnes* phage, such as PAC7 (typically the sequence of SEQ ID NO: 68).

[0317] In one embodiment, *Propionibacterium acnes* - The selection marker for acne bacteria as defined above, - The acne bacterium phage packaging signal (cos site) defined above, - The replication origin for acne bacteria as defined above, and - CRISPR-Cas system (p target) that targets chromosomal gene loci of specific acne bacteria receiver cells Includes a DNA vector containing [specific components].

[0318] A high-titer acne bacilli phage suspension is typically added to acne bacteria. The suspension typically contains a mixture of wild-type phages and phage-derived particles carrying plasmids. Acne cells carrying the locus targeted by the p-targeted CRISPR-Cas system are typically brought into contact with phage-derived particles containing the p-target. This can be done in vivo or in vitro. Sequence-specific killing is typically observed in lysates containing phage-derived particles with the p-target.

[0319] In one embodiment, phage-derived particles containing a p-targeting vector (e.g., plasmid) are not mixed with the phage, enabling sequence-specific killing of cells carrying DNA targeted by the CRISPR-Cas system.

[0320] Propionibacterium acnes plasmid curing Naturally occurring *Propionibacterium acnes* plasmids have been described, some of which exhibit a pro-inflammatory phenotype. 15、23 and acne vulgaris 16~18 This was related to the fact that such plasmids can be cured, which is of interest for studying their effects in acne vulgaris, particularly their pro-inflammatory role. The inventors have developed a method for curing *Propionibacterium acnes* plasmids.

[0321] In the first step, - The acne bacterium phage packaging signal defined above, - Depending on the case, the selection marker for acne bacteria as defined above, - The origin of replication, as defined above, that enables replication only within acne-producing cells; and - A CRISPR-Cas system that targets a gene sequence, preferably located in a conserved region, e.g., the origin of replication or a locus associated with acne vulgaris, of an endogenous plasmid to be cured in target acne bacterium receiver cells. Acne bacteria-producing cells that carry DNA vectors containing the DNA vector are infected by acne bacteria phages, leading to the production of phage-derived particles that carry the DNA vector.

[0322] Particles derived from *Propionibacterium acnes* phage are brought into contact with *Propionibacterium acnes* receiver cells carrying an endogenous plasmid to be cured, such as pIMPLE-HL096PA1. This can be done in vivo or in vitro.

[0323] In some embodiments, *Propionibacterium acnes* transdermal grafts may be selected on appropriate antibiotics. Single colonies are typically streaked on antibiotic-containing media, and the presence of plasmids is typically screened by PCR. Single colonies without positive PCR for plasmid pIMPLE-HL096PA1 are obtained, and then the vector (e.g., plasmid) containing the CRISPR-Cas system is cured and typically stored as a cryostock.

[0324] Treatment method This invention includes methods for treating disorders or diseases related to acne bacteria.

[0325] The present invention relates to the use of the above-defined manipulated acne bacteria strains, the above-defined phage-derived particles, and the above-defined manipulated phages and / or bacteria that produce them, for the treatment and / or prevention of a wide range of skin diseases and skin disorders.

[0326] The present invention includes a method for treating reduced sebum production, follicular hyperkeratinization, skin bacterial colonization, and inflammation using the manipulated acne bacteria defined above, the phage-derived particles defined above, and the manipulated phages defined above, and / or bacteria that produce them.

[0327] The present invention includes the use of the manipulated acne bacteria strains defined above in cosmetics and other compositions.

[0328] In one embodiment, the present invention includes the expression of therapeutic molecules by manipulated Propionibacterium acnes.

[0329] In one embodiment, the present invention includes the expression of non-therapeutic molecules by manipulated Propionibacterium acnes.

[0330] Cutibacterium acnes is one of the most common and abundant bacteria on human skin, and can be found both on the skin surface (stratum corneum) and in hair follicles. 12 Within the hair follicle, *Propionibacterium acnes* is in direct contact with a considerable variety of living cells, such as keratinocytes, stem cells, sebaceous gland cells, and immune cells. This is not the case on the stratum corneum, where it is primarily in contact with dead keratinocytes. 13 Therefore, it seems interesting to use Propionibacterium acnes as a bacterial chassis for the in situ production and delivery of therapeutic molecules inside and outside the hair follicle.

[0331] The phage-derived particles defined above, and the manipulated phages defined above, and / or bacteria that produce them, can be delivered to the skin of a target by transdermal administration or other appropriate method of administration.

[0332] The subjects of the present invention are animals, preferably mammals, and more preferably humans. However, the term “subject” may further refer to non-human animals in need of treatment, in particular mammals, such as dogs, cats, horses, cattle, pigs, sheep, donkeys, rabbits, ferrets, gerbils, hamsters, chinchillas, rats, mice, guinea pigs, and non-human primates, or non-mammals, such as poultry.

[0333] The human subjects according to the present invention may be fetuses, newborns, children, infants, adolescents, or adults of any age.

[0334] Preferably, the treatment is administered regularly, preferably daily to monthly, more preferably daily to every two weeks, more preferably daily to weekly, and most preferably daily. In a particular embodiment, the treatment is administered multiple times a day, preferably two or three times a day, and more preferably three times a day.

[0335] The treatment period according to the present invention, using the manipulated acne bacteria as defined above, the phage-derived particles as defined above, the manipulated phages as defined above, and / or bacteria that produce them (e.g., Escherichia coli or acne bacteria), is preferably between 1 and 20 weeks, more preferably between 1 and 10 weeks, more preferably between 1 and 4 weeks, and more preferably between 1 and 2 weeks. In a particular embodiment, the treatment period is about 1 week. Alternatively, the treatment may be continued for as long as the infection, disorder, and / or disease persists.

[0336] The form, route of administration, and dosage of the pharmaceutical or veterinary composition according to the present invention of the manipulated acne bacteria, the phage-derived particles, the manipulated phages, and / or bacteria that produce them (e.g., Escherichia coli or acne bacteria) as defined above can be adjusted by those skilled in the art according to the type and severity of the disease, disorder, and / or infection (e.g., according to the bacterial species involved in the disease, disorder, and / or infection, and the localization of the bacterial species within the patient's or subject's body), and according to the patient's or subject, in particular according to their age, weight, sex, and overall health condition.

[0337] In particular, the dosage of the manipulated Propionibacterium acnes as defined above, the phage-derived particles as defined above, the manipulated phages as defined above, and / or bacteria producing them (e.g., Escherichia coli or Propionibacterium acnes) according to the present invention must be determined by standard procedures well known to those skilled in the art. In order to determine an appropriate dose so that a therapeutically effective amount is administered to the patient or subject, physiological data of the patient or subject (e.g., age, size, and weight), as well as the route of administration, must be taken into consideration.

[0338] For example, the total amount of phage-derived particles and / or manipulated phages as defined above, according to the present invention, for each dose is 10 4 From 10 particles 15 It is contained between individual particles.

[0339] Preferably, the total amount according to the present invention for each dose of the phage-derived particles and / or the manipulated bacteria (e.g., Escherichia coli or Propionibacterium acnes) that produce the manipulated phages defined above, or the manipulated Propionibacterium acnes strain defined above, is 10 4 From individual bacteria to 10 15 It is contained between individual bacteria.

[0340] The present invention includes plasmids for expressing toxins, such as nucleases, more preferably, CRISPR-Cas systems that kill transduced populations of Propionibacterium acnes.

[0341] The present invention comprises a plasmid for expressing a CRISPR-Cas system, which targets a sequence present only in specific strains and not in other strains, enabling strain-specific killing among Propionibacterium acnes populations.

[0342] The present invention includes modifications of the *Propionibacterium acnes* chromosome or endogenous plasmid of *Propionibacterium acnes*. For example, modifications that result in changes in the *Propionibacterium acnes*-host relationship, such as deletions, substitutions, and / or insertions, are possible.

[0343] The present invention includes a vector for the expression of a therapeutic molecule, such as a plasmid, which contains one or more genes involved in the production of the therapeutic molecule.

[0344] If the therapeutic molecule does not freely diffuse from acne cells, for example, in the case of a therapeutic protein, a fusion with a signal peptide that enables secretion or transport onto the cell membrane or cell wall of acne cells is preferably encoded on a vector, such as a plasmid. Examples of secretion systems or signal peptides include TAT, SEC, and the type VII / WXG100 secretion system. In particular, the signal peptides are proteins PPA0532 (typically referred to as Q6AAD1 in the UniprotKB database as of November 4, 2020); PPA0533 (typically referred to as Q6AAD0 in the UniprotKB database as of November 4, 2020); PPA0534 (typically referred to as Q6AAC9 in the UniprotKB database as of November 4, 2020); PPA0598 (typically referred to as Q6AA63 in the UniprotKB database as of November 4, 2020); PPA0644 (typically referred to as Q6AA16 in the UniprotKB database as of November 4, 2020); PPA0687 (typically referred to as Q6A9X2 in the UniprotKB database as of November 4, 2020); PPA0721 (typically referred to as Q6A9 in the UniprotKB database as of November 4, 2020) (Typically referred to as T8); PPA0816 (typically referred to as Q6A9J4 in the UniprotKB database as of November 4, 2020); PPA1310 (typically referred to as Q6A856 in the UniprotKB database as of November 4, 2020); PPA1498 (typically referred to as Q6A7M0 in the UniprotKB database as of November 4, 2020); PPA1662 (typically referred to as Q6A771 in the UniprotKB database as of November 4, 2020); PPA1715 (typically referred to as Q6A720 in the UniprotKB database as of November 4, 2020); PPA1939 (typically referred to as Q6A6F6 in the UniprotKB database as of November 4, 2020); PPA2097 (typically referred to as Q6A608 in the UniprotKB database as of November 4, 2020);PPA2105 (typically referred to as Q6A601 in the UniprotKB database as of November 4, 2020); PPA2106 (typically referred to as Q6A600 in the UniprotKB database as of November 4, 2020); PPA2142 (typically referred to as Q6A5W4 in the UniprotKB database as of November 4, 2020); PPA2164 (typically referred to as Q6A5U3 in the UniprotKB database as of November 4, 2020); PPA2175 (typically It can be extracted from a protein selected from the group consisting of Q6A5T2 (as of November 4, 2020, in the UniprotKB database); PPA2152 (typically referred to as Q6A5V4 in the UniprotKB database as of November 4, 2020); PPA1340 (typically referred to as Q6A826 in the UniprotKB database as of November 4, 2020); and PPA2239 (typically referred to as Q6A5M0 in the UniprotKB database as of November 4, 2020).

[0345] If secretion is undesirable or non-functional, a lysis module may be added to the vector, such as a plasmid, to lyse the cells and release the therapeutic molecule.

[0346] In a particular embodiment, the therapeutic molecule may be displayed on the cell membrane or cell wall of Propionibacterium acnes cells. To be displayed, the protein of interest typically requires, for example, an N-terminal secretory signaling peptide as described above, and a C-terminal LPXTG motif that allows Propionibacterium acnes-derived class F saltase (Girolamo, SD et al., Biochem J 476, pp. 665-682 (2019) (Non-Patent Literature 8)) to covalently bond the protein of interest to the cell wall. Furthermore, a PT-rich region may be incorporated upstream of the LPXTG motif. Alternatively, a more classical cell wall sorting sequence (CWSS) may be used, combining the LPxTG motif with subsequent hydrophobic amino acids and a positively charged C-terminus.

[0347] To control the expression of therapeutic molecules, one or more genes may be placed under the control of an inductive system, such as an inductive promoter, riboswitch, RNA-based inductive system, or a combination thereof, either as an operon or as a single isolated gene. To optimize the in situ production of therapeutic molecules, multiple promoters of multiple transcriptional intensities may be tested and combined with different RBS intensities. An RBS library approach may be used to select the best RBS variant for in vitro or in situ expression.

[0348] Examples of therapeutic molecules include, but are not limited to, antibodies, antibody-based drugs, Fc fusion proteins, anticoagulants, blood factors, bone morphogenetic proteins, manipulated protein scaffolds, enzymes, growth factors, hormones, interferons, interleukins, and thrombolytic agents. Other examples include those that non-covalently bind to a target (e.g., monoclonal antibodies), those that affect covalent binding (e.g., enzymes), and those that exert activity without specific interactions (e.g., serum albumin).

[0349] For example, therapeutic molecules (e.g., therapeutic recombinant proteins) used to treat cancer, immunodeficiency, infection, and / or other diseases are also intended herein. Manipulated proteins, including bispecific mAbs, multispecific fusion proteins, and proteins having optimized pharmacokinetics, are also intended by this disclosure.

[0350] In some embodiments, the therapeutic protein is etanercept, bevacizumab, rituximab, adalimumab, infliximab, trastuzumab, insulin glargine, epoetin alfa, pegfilgrastim, ranibizumab, darbepoetin alfa, interferon beta-la, interferon beta-la, insulin aspart, Rhu insulin, octocog alfa, insulin lispro, cetuximab, peginterferon alfa-2a, interferon beta-lb, eptacog alfa, insulin aspart, onabotulinum toxin A, epoetin beta, Rec antihemophilia factor, filgrastim, insulin detemir, natalizumab, insulin (humulin), or palivizumab.

[0351] Examples of antibodies, antibody fragments, and / or Fc fusion proteins that may be expressed in connection with this disclosure include, but are not limited to, avagovomab, absiximab, actoxumab, adalimumab, adecatumumab, aferimomab, aftuzumab, alacizumab pegol, ALD, alemtuzumab, alirocumab, altumomab pentetate, amatsuximab, and anatumomab mafenatox. mafenatox), aniflorumab, anrukinzumab, apolizumab, alsitumomab, aselizumab, atinumab, atorizumab (or tocilizumab), atorolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, beralizumab, vertilimumab, besilesomab, bevacizumab, bezlotoxumab, bicilomab, bimagrumab, bibatuzumab meltansine mertansine), blinatumomab, brosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumakisomab, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetantetraxetan), conatumumab, concizumab, crenezumab, dasetuzumab, dacrizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox aritox), drozitumab, duligotumab, dupilumab, dusigitumab, ecromeximab, eculizumab, edovacomab, edrecolomab, efalizumab, efungumab, eldelumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab situxetan cituxetan), epratuzumab, erlizumab, ertumaxomab, etalacizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, falletuzumab, facinumab, FBTA, felvizumab, fezakinumab, ficl atuzumab, figtumumab, flambotumab, fortrizumab, foralumab, foravirumab, fresolimumab, fluranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotinVedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomab / tiuxetan, icurucumab, igobomab, imciromab, imgatuzumab, inclacumab, indatuximab / labtansine ravtansine), infliximab, intetumumab, inolimomab, inotuzumab-ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keriximab, rabetsuzumab, lambrolizumab, lampalizumab, lebrikizumab rikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Ligelizumab, Lintuzumab, Lirilumab, Lodelcizumab, Lorbotuzumab meltansine Mertansine), Lucatumumab, Lumiliximab, Mapatumumab, Margetuximab, Maslimomab, Maplilimumab, Matsuzumab, Mepolizumab, Metelimumab, Miratuzumab, Minretumomab, Mitumomab, Mogamulizumab, Morolimmumab, Motabizumab, Moxetumomab pasudotox, Muromonab-CD3, Nacolomab Tafenatox), Namilumab, Naptumomab Estafenatox, Narnatumab, Natalizumab, Nevacumab, Necitumumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab, Nofetumomab MerpentanMerpentan, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Oportuzumab monatox Monatox), olegobomab, orticumab, oterixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pinatuzumab vedotin Vedotin, Pintumomab, Placulumab, Polatuzumab vedotin, Ponezumab, Priliximab, Pritoxaximab, Pritumumab, PRO, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ramucirumab, Ranibizumab, Laxibakumab Legavirumab, reslizumab, rilotumumab, rituximab, lobatumumab, rolledumab, romosozumab, rontalizumab, lovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetidependetide), secukinumab, cerivanthumab, setoxaximab, sevilumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, ciprizumab, silukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, subizumab, tabarumab, tacutuzumab tetraxetan tetraxetan), tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomab aritox), tenatumomab, teneliximab, teplizumab, teprotumumab, TGN, tisilimmab (or tremelimumab), tildrakizumab, tigatuzumab, TNX-, tocilizumab (or atrizumab), toralizumab, tocitumomab, tovetumab, tralokinumab, trastuzumab, TRBS, tregalizumab, tremelimumab, tucotuzumab cermoloukin celmoleukin), tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vantictumab, vapaliximab, batelizumab, vedolizumab, vertzuzumab, bepalimomab, besencumab, vizilizumab, borosiximab, borsetuzumab mafodotinThis includes mafodotin, botumumab, zaltumumab, zanolimmab, zatuximab, ziralimumab, and zolimomab aritox.

[0352] Other examples of Fc fusion proteins that may be expressed in connection with this disclosure include, but are not limited to, etanercept, alefacept, abatacept, lilonacept, romiplostim, belatacept, and aflibercept.

[0353] Examples of anticoagulants and / or blood factors that may be expressed in connection with this disclosure include, but are not limited to, protein C, protein S, and antithrombin, factors I through VIII, prothrombinase, prothrombin, thrombin, von Willebrand factor (vWF), fibrinogen, fibrin, and fibrinopeptides.

[0354] Examples of bone morphogenetic proteins (BMPs) that may be expressed in connection with this disclosure include, but are not limited to, BMP1-BMP7, BMP8a, BMP8b, BMP10, and BMP15.

[0355] Examples of enzymes that may be expressed in connection with this disclosure include, but are not limited to, any enzyme to which an enzyme nomenclature (EC number) (e.g., EC1 to EC6) has been assigned by the International Union of Biochemistry and Molecular Biology (IUBMB) (Webb, Edwin C. Enzyme nomenclature 1992: recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology on the nomenclature and classification of enzymes. San Diego: Academic Press, published for the International Union of Biochemistry and Molecular Biology. ISBN 0-12-227164-5 (1992) (Non-Patent Literature 9) is incorporated herein by reference). Other examples include styrene monooxygenase (StyAB), toluene dioxygenase (TODC1C2AB), luciferase, and lactase. In some embodiments, the enzyme is toluene dioxygenase. In some embodiments, the enzyme is styrene monooxygenase.

[0356] Examples of growth factors that may be expressed in connection with this disclosure include, but are not limited to, adrenomedullin (AM), angiopoietin (Ang), autologous motility factors, bone morphogenetic proteins (BMPs), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth and differentiation factor 9 (GDF9), hepatocyte growth factor (HGF), and hepatocyte growth factor. It contains mammary gland growth factor (HDGF), insulin-like growth factor (IGF), migration stimulant, myostatin (GDF-8), nerve growth factor (NGF), and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-P), tumor necrosis factor alpha (TNF-), vascular endothelial growth factor (VEGF), placental growth factor (P1GF), fetal bovine somatotropin (FBS), and IL-1 to IL-7.

[0357] Examples of peptide hormones that may be expressed in connection with this disclosure include, but are not limited to, amylin (or islet amyloid polypeptide), anti-Müllerian hormone (or Müllerian inhibitor or hormone), adiponectin, adrenocorticotropic hormone (or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atriopeptin), brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin hormone-releasing hormone, enkephalin, endothelin, erythropoietin, and follicle-stimulating hormone. This includes lumon, galanin, gastrin, ghrelin, glucagon, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (or somatomedin), leptin, lipotropin, luteinizing hormone, melanocyte-stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, prolactin, prolactin-releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (or thyrotropin), and thyrotropin-releasing hormone.

[0358] Examples of interferons (IFNs) that may be expressed in connection with this disclosure include, but are not limited to, IFN-α, IFN-β, IFN-ω, and IFN-γ.

[0359] Examples of interleukins that may be expressed in connection with this disclosure include, but are not limited to, interleukins 1 to 17. In some embodiments, the interleukin is interleukin-4, interleukin-6, interleukin-10, interleukin-11, or interleukin-13.

[0360] Other examples of therapeutic proteins that may be expressed in connection with this disclosure include, but are not limited to, insulin (glycemic regulator), pramulintide acetate (glucose management), growth hormone GH (growth inhibitor), pegvisomant (growth hormone receptor antagonist), mecasermin (IGFl, growth inhibitor), factor VIII (coagulation factor), factor IX (coagulation factor, protein C concentrate (anticoagulation)), al-proteinase inhibitor (antitrypsin inhibitor), erythropoietin (stimulates erythrogenesis), filgrastim (granulocyte colony-stimulating factor, G-CSF; stimulates neutrophil proliferation), and salglamostim. 36、37(Granulocyte-macrophage colony-stimulating factor, GM-CSF), Oprelbequin (Interleukin II, IL-11), Human follicle-stimulating hormone (FSH), Human chorionic gonadotropin (HCG), Lutropin-a (Human luteinizing hormone), Interleukin-2 (IL-2), Interleukin-1 receptor agonist, Denileukin difutitox (a fusion of IL-2 and diphtheria toxin), Interferon alphacon-1 (Consensus Interferon), Interferon-2a (IFNa2a), Interferon-2b (IFNa2b), Interferon-n3 (IFNan3), Interferon-pia (rIFN-β), Interferon-β lb(rIFN-β), interferon-ylb(IFNy, salmon calcitonin (a linear polypeptide hormone of 32 amino acids)), teriparatide (part of human parathyroid hormone, residues 1-34), exenatide (incretin mimetic with effects similar to glucagon-like peptide 1), octreotide (octapeptide that mimics natural somatostatin), dibotermin-a (recombinant human osteomorphic protein 2), recombinant human osteomorphic protein 7, histrelin acetate (gonadotropin-releasing hormone, GnRH), parifermin (keratinocyte growth factor, KGF), becaprelmin (platelet-derived growth factor, PDGF), nesiritide (recombinant human type B natriuretic peptide), repildin (recombinant variant of hirudin, other variants are bivalirudine), anakinra (interleukin-1 (IL-1) receptor antagonist), enfuvirtide (HIV-1 gp41-derived peptide), β-glucocerebrosidase (hydrolyzes glucose and ceramide), alglucosidase-a (decomposes glycogen), laronidase (digests glycosaminoglycans in lysosomes), idursulfase (cleaves O-sulfates and prevents GAG accumulation), galsulfase (cleaves terminal sulfates from GAGs), agalsidase-β (human α-galactosidase A, hydrolyzes sphingoglycolipids), lactase (digests lactose), pancreatic enzymes (lipase, amylase, protease;(Digestion of food), adenosine deaminase (metabolizes adenosine), tissue plasminogen activator (tPA, serine protease involved in the breakdown of blood clots), Vila factor (serine protease, causes blood coagulation), drolecogin-a (serine protease, human activated protein C), trypsin (serine protease, hydrolyzes proteins), botulinum toxin type A (protease, inactivates SNAP-25 involved in synaptic vesicle fusion), botulinum toxin type B (protease that inactivates SNAP-25 involved in synaptic vesicle fusion), collagenase (endopeptidase, digests natural collagen), star deoxyribonuclease I (endonuclease, DNase) This includes I (DNA cleavage), hyaluronidase (hydrolyzes hyaluronan), papain (cysteine ​​protease, hydrolyzes proteins), L-asparaginase (catalyzes the conversion of L-asparagine to aspartic acid and ammonia), rasburicase (uric acid oxidase, catalyzes the conversion of uric acid to allantoin), streptokinase (anistreplase, anisoylated plasminogen streptokinase activator complex (APSAC)), and antithrombin III (serine protease inhibitor).

[0361] Other examples of therapeutic proteins that may be expressed in connection with this disclosure include the antigens defined below.

[0362] The present invention further includes a modified Propionibacterium acnes comprising a vector (e.g., plasmid) for the expression of an antigen, such as a tumor antigen, viral antigen, bacterial antigen, fungal antigen, autoantigen, allergen, or graft-specific antigen.

[0363] As used herein, “antigen” refers to a molecule containing one or more epitopes (e.g., linear, structural, or both) that induce an immunological response. The antigen may be of any type. In particular, an antigen may be a protein, polypeptide, or peptide, a carbohydrate, a lipid, a nucleic acid, such as DNA or RNA. In a particular embodiment, the antigen is a protein, polypeptide, or peptide. “Protein” as intended herein is understood to include proteins, polypeptides, and peptides. Furthermore, for the purposes of the present invention, “antigen” includes proteins that have undergone modifications to their native sequence, such as deletions, additions, and substitutions (generally conserved), as long as the protein maintains its ability to induce an immunological response. These modifications may be planned by site-directed mutagenesis or may be accidental, for example, due to mutations in the host producing the antigen.

[0364] In one particular embodiment, the antigen induces activation or elevation of an immune response, particularly an immune response specific to the antigen. In an alternative embodiment, the antigen results in tolerance or suppression of an immune response, particularly an immune response to the antigen.

[0365] In one particular embodiment, the antigen reduces the target inflammatory response.

[0366] In one particular embodiment, the antigen is a tumor antigen.

[0367] In this specification, “tumor antigen” means an antigenic substance produced in tumor cells. Tumor antigens may be, for example, peptide-containing tumor antigens, such as polypeptide tumor antigens or glycoprotein tumor antigens. Tumor antigens may further be, for example, sugar-containing tumor antigens, such as glycolipid tumor antigens or ganglioside tumor antigens.

[0368] Tumor antigens include, but are not limited to, (a) polypeptide-containing tumor antigens comprising polypeptides (e.g., those with a length of approximately 8 to 20 amino acids, but lengths outside this range are also common), lipopolypeptides, and glycoproteins, and (b) sugar-containing tumor antigens comprising polysaccharides, mucins, gangliosides, glycolipids, and glycoproteins. Furthermore, tumor antigens may be (a) full-length molecules associated with cancer cells, (b) homologs and modified forms of the same molecule (including molecules with partial deletions, additions, and / or substitutions), and (c) fragments of the same molecule. Tumor antigens include, for example, class I-limited antigens recognized by CD8+ lymphocytes, or class II-limited antigens recognized by CD4+ lymphocytes.

[0369] Numerous tumor antigens are known in the field, including (a) cancer testicular antigens, e.g., NY-ESO-1, SSX2, SCP1, and polypeptides of the RAGE, BAGE, GAGE, MAGE family, e.g., GAGE-1, GAGE-2, MAGE-1, MAGE-2, MAGE-3, MAGE-4, MAGE-5, MAGE-6, and MAGE-12 (e.g., usable to target melanoma, lung tumors, head and neck tumors, NSCLC, mammary gland tumors, gastrointestinal tumors, and bladder tumors), and (b) variant antigens, e.g., p53 (various solid Tumors (e.g., associated with colorectal cancer, lung cancer, and head and neck cancer), p21 / Ras (e.g., associated with melanoma, pancreatic cancer, and colorectal cancer), CD4 (e.g., associated with melanoma), MUM1 (e.g., associated with melanoma), caspase-8 (e.g., associated with head and neck cancer), CIA0205 (e.g., associated with bladder cancer), HLA-A2-R1701, betacatenin (e.g., associated with melanoma), TCR (e.g., associated with T-cell non-Hodgkin lymphoma), BCR-abl (e.g., associated with chronic myeloid leukemia), triose phosphate isomerase, IA 0205, CDC-27, and LDLR-FUT, (c) Overexpressed antigens, e.g., galectin 4 (e.g., associated with colorectal cancer), galectin 9 (e.g., associated with Hodgkin's disease), proteinase 3 (e.g., associated with chronic myeloid leukemia), WT1 (e.g., associated with various leukemias), carbonic anhydrase (e.g., associated with renal cancer), aldolase A (e.g., associated with lung cancer), PRAME (e.g., associated with melanoma), HER-2 / neu (e.g., breast cancer, (d) Shared antigens, e.g., melanoma-melanocyte differentiation antigen, e.g., MART-1 / Me(a) gplOO, MC1R, melanocyte-stimulating hormone receptor, tyrosinase, tyrosinase-related protein-1 / TRP1, and tyrosinase-related protein-2 / TRP2 (e.g., associated with melanoma), (e) prostate-related antigens, e.g., PAP, PSA, PSMA, PSH-P1, PSM-P1, PSM-P2 (e.g., associated with prostate cancer), (f) immunoglobulin idiotypes (e.g., associated with myeloma and B-cell lymphoma), and (g) other tumor antigens, e.g., (i) glycoproteins, e.g., sialyl Tn and sialyl Lex (e.g., associated with breast cancer and uterine cancer). The antigens include (ii) various mucins (associated with colorectal cancer), glycoproteins that can bind to carrier proteins (e.g., MUC-1 can bind to LH); (ii) lipopeptides (e.g., MUC-1 bound to the lipid portion); (iii) polysaccharides that can bind to carrier proteins (e.g., KLH) (e.g., Globo-H synthetic hexasaccharide); and (iv) polypeptide-containing antigens and polysaccharide-containing antigens, including gangliosides that can also bind to carrier proteins (e.g., KLH), such as GM2, GM12, GD2, GD3 (e.g., associated with brain cancer, lung cancer, melanoma).

[0370] Other tumor antigens include pi 5, Hom / Mel-40, H-Ras, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein-Barr virus antigen, EBNA, human papillomavirus (HPV) antigens including E6 and E7, hepatitis B and C virus antigens, human T-cell lymphotropic virus antigen, TSP-180, pl85erbB2, pl80erbB-3, c-met, mn-23H l, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, p 16, TAGE, PSCA, CT7, 43-9F, 5T4, 791 Tgp72, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA Includes 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein / cyclophyllin C-related protein), TAAL6, TAG72, TLP, TPS, etc.

[0371] In another embodiment, the antigen is a viral antigen.

[0372] In this specification, “viral antigen” means a protein encoded by the viral genome.

[0373] In certain embodiments, the viral antigen preferably comprises an epitope that is exposed to its surface for at least one stage of the life cycle of the virus. The viral antigen is preferably conserved across multiple serotypes or isolates. Viral antigens suitable for use in connection with this disclosure include, but are not limited to, antigens derived from one or more of the viruses described below, as well as examples of specific antigens identified below.

[0374] Orthomyxovirus: Viral antigens include, but are not limited to, those derived from orthomyxoviruses, such as influenza A, B, and C. In certain embodiments, the orthomyxovirus antigen is selected from one or more viral proteins comprising one or more hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), substrate protein (M1), membrane protein (M2), and transcriptase components (PB1, PB2, and PA). In certain embodiments, the viral antigen comprises HA and NA. In certain embodiments, the influenza antigen is derived from an interpandemic (annual) influenza strain, and in other embodiments, the influenza antigen is derived from a strain with the potential to cause a global pandemic (i.e., an influenza strain with novel hemagglutinin compared to the currently circulating strain, or an influenza strain that is pathogenic in avian subjects and has the potential to horizontally transmit to human populations, or an influenza strain that is pathogenic to humans).

[0375] Paramyxoviridae viruses: Viral antigens include, but are not limited to, those derived from paramyxoviridae viruses, such as pneumovirus (RSV), paramyxovirus (PIV), metapneumovirus, and morbillivirus (measles).

[0376] Pneumovirus: Viral antigens include, but are not limited to, those derived from pneumoviruses, such as respiratory syncytial virus (RSV), bovine respiratory syncytial virus, mouse pneumonia virus, and turkey rhinotracheitis virus. Preferably, the pneumovirus is RSV. In certain embodiments, the pneumovirus antigen is selected from one or more proteins, including the following: surface protein, fusion protein (F), glycoprotein (G), and small hydrophobic protein (SH), substrate proteins M and M2, nucleocapsid proteins N, P and L, and non-structural proteins NS1 and NS2. In other embodiments, the pneumovirus antigen includes F, G, and M.

[0377] Paramyxovirus: Viral antigens include, but are not limited to, those derived from paramyxoviruses, such as parainfluenza viruses types 1-4 (PIV), mumps, Sendai virus, Simian virus 5, bovine parainfluenza virus, Nipah virus, Henipa virus, and Newcastle disease virus. In certain embodiments, the paramyxovirus is PIV or mumps. In certain embodiments, the paramyxovirus antigen is selected from one or more of the following proteins: hemagglutinin-neuraminidase (HN), fusion proteins F1 and F2, nucleoprotein (NP), phosphoprotein (P), large protein (L), and substrate protein (M). In other embodiments, the paramyxovirus protein includes HN, F1, and F2. In other embodiments, the paramyxovirus is a nipah virus or henipavirus, and the antigen is selected from one or more of the following proteins: fusion (F) protein, glycoprotein (G), substrate (M) protein, nucleocapsid (N) protein, large (L) protein, and phosphoprotein (P).

[0378] Poxviridae: Viral antigens include, but are not limited to, those derived from orthopoxviruses, such as those from *Varicum poxum* (including, but not limited to, *Varicum poxum* var. *vegetativeum*) and *Varicum poxum* var. *vegetativeum* (including, but not limited to, *Varicum poxum* var. *vegetative

[0379] Metapneumovirus: Viral antigens include, but are not limited to, metapneumoviruses, such as human metapneumovirus (hMPV) and trimeta-pneumovirus (aMPV). In certain embodiments, the metapneumovirus antigen is selected from one or more proteins, including the following: surface protein, fusion protein (F), glycoprotein (G), and small hydrophobic protein (SH), substrate proteins M and M2, and nucleocapsid proteins N, P, and L. In other embodiments, the metapneumovirus antigen includes F, G, and M.

[0380] Morbillivirus: Viral antigens include, but are not limited to, those derived from morbilliviruses, such as measles. In certain embodiments, the morbillivirus antigen is selected from one or more of the following proteins: hemagglutinin (H), glycoprotein (G), fusion factor (F), large protein (L), nucleoprotein (NP), polymerase phosphoprotein (P), and substrate (M).

[0381] Picornavirus: Viral antigens include, but are not limited to, those derived from picornaviruses, such as enteroviruses, rhinoviruses, heparnaviruses, cardioviruses, and aftviruses. In certain embodiments, the antigen is derived from an enterovirus, and in other embodiments, the enterovirus is a poliovirus. In yet another embodiment, the antigen is derived from a rhinovirus.

[0382] Enterovirus: Viral antigens include, but are not limited to, those derived from enteroviruses, such as poliovirus types 1, 2, or 3, coxsackievirus A types 1-22 and 24, coxsackievirus B types 1-6, echovirus (ECHO virus) types 1-9, 11-27 and 29-34, and enteroviruses 68-71. In certain embodiments, the antigen is derived from an enterovirus, and in other embodiments, the enterovirus is poliovirus. In certain embodiments, the enterovirus antigen is selected from one or more of the following capsid proteins VP0, VP1, VP2, VP3, and VP4.

[0383] Bunyavirus: Viral antigens include, but are not limited to, those derived from orthobunyaviruses, e.g., California encephalitis virus, phleboviruses, e.g., Rift Valley fever virus, or nairoviruses, e.g., Crimean-Congo hemorrhagic fever virus. Rhinovirus: Viral antigens include, but are not limited to, those derived from rhinoviruses. In certain embodiments, the rhinovirus antigen is selected from one or more of the following capsid proteins: VP0, VP1, VP2, VP2, and VP4.

[0384] Heparnavirus: Viral antigens include, but are not limited to, those derived from heparnaviruses, such as, for example, hepatitis A virus (HAV).

[0385] Togavirus: The viral antigen includes, but is not limited to, those derived from togaviruses, such as rubivirus, alphavirus, or arterivirus. In certain embodiments, the antigen is derived from rubivirus, for example, rubella virus. In certain embodiments, the togavirus antigen is selected from E1, E2, E3, C, NSP-1, NSPO-2, NSP-3, or NSP-4. In certain embodiments, the togavirus antigen is selected from E1, E2, or E3.

[0386] Flavivirus: Viral antigens include, but are not limited to, those derived from flaviviruses, such as tick-borne encephalitis (TBE) virus, dengue (type 1, 2, 3, or 4) virus, yellow fever virus, Japanese encephalitis virus, Kasanul Forest disease virus, West Nile encephalitis virus, St. Louis encephalitis virus, Russian spring-summer encephalitis virus, and Poissant encephalitis virus. In certain embodiments, the flavivirus antigen is selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. In certain embodiments, the flavivirus antigen is selected from PrM, M, and E.

[0387] Pestivirus: Viral antigens include, but are not limited to, those derived from pestiviruses, such as bovine viral diarrhea (BVDV), swine cholera (CSFV), or border disease (BDV).

[0388] Hepadnavirus: Viral antigens include, but are not limited to, those derived from hepadnavirus, such as hepatitis B virus. In certain embodiments, the hepadnavirus antigen is selected from surface antigens (L, M, and S) and core antigens (HBc, HBe).

[0389] Hepatitis C virus: Viral antigens include, but are not limited to, those derived from hepatitis C virus (HCV). In certain embodiments, the HCV antigen is selected from one or more peptides derived from E1, E2, E1 / E2, NS345 polyprotein, NS345 core polyprotein, core, and / or non-structural region. In certain embodiments, the hepatitis C virus antigen includes one or more of the following: HCV E1 and / or E2 proteins, E1 / E2 heterodimer complexes, core proteins, and non-structural proteins that retain immunogenicity but may be modified to remove enzymatic activity, or fragments of these antigens.

[0390] Rhabdovirus: Viral antigens include, but are not limited to, those derived from rhabdoviruses, such as lyssavirus (rabies virus) and becyclovirus (VSV). Rhabdovirus antigens may be selected from glycoproteins (G), nucleoproteins (N), large proteins (L), and non-structural proteins (NS).

[0391] Caliciviridae: Viral antigens include, but are not limited to, those derived from the Caliciviridae family, such as Norwalk virus, and Norwalk-like viruses, such as Hawaiian virus and Snow Mountain virus.

[0392] Coronavirus: Viral antigens include, but are not limited to, those derived from coronavirus, SARS, human respiratory coronavirus, avian infectious bronchitis (IBV), mouse hepatitis virus (MHV), and porcine infectious gastroenteritis virus (TGEV). In certain embodiments, the coronavirus antigen is selected from the spike (S), envelope (E), substrate (M), nucleocapsid (N), and hemagglutinin-esterase glycoprotein (HE). In certain embodiments, the coronavirus antigen is derived from the SARS virus. In certain embodiments, the coronavirus is derived from the SARS virus antigen described in International Publication No. 04 / 92360 (Patent Document 1).

[0393] Retrovirus: Viral antigens include, but are not limited to, those derived from retroviruses, such as oncoviruses, lentiviruses, or spumaviruses. In certain embodiments, the oncovirus antigen is derived from HTLV-1, HTLV-2, or HTLV-5. In certain embodiments, the lentivirus antigen is derived from HIV-1 or HIV-2. In certain embodiments, the antigen is derived from an HIV-1 subtype (or clade), including, but is not limited to, HIV-1 subtypes (or clades) A, B, C, D, F, G, H, J, K, and O. In other embodiments, the antigen is derived from a recombinant HIV-1 epidemic strain (CRF), including, but is not limited to, A / B, A / E, A / G, A / G / I, etc. In certain embodiments, the retrovirus antigen is selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr. In certain embodiments, the HIV antigen is selected from gag (p24 gag and p55 gag), env (gp160 and gp41), pol, tat, nef, rev vpu, and miniprotein (preferably p55 gag and gp140v deletion type). In certain embodiments, the HIV virus antigen is derived from one or more of the following strains: HIVIIIb, HIVSF2, HIVLAV, HIVLAI, HIVMN, HIV-1CM235, HIV-1US4, HIV-1SF162, HIV-1TV1, and HIV-1MJ4. In certain embodiments, the antigen is derived from endogenous human retrovirus and includes, but is not limited to, HERV-K ("old" HERV-K and "new" HERV-K).

[0394] Reovirus: Viral antigens include, but are not limited to, those derived from reoviruses, such as orthoreovirus, rotavirus, orbivirus, or cortivirus. In certain embodiments, the reovirus antigen is selected from structural proteins λ1, λ2, λ3, μ1, μ2, σ1, σ2, or σ3, or non-structural proteins σNS, μNS, or ols. In certain embodiments, the reovirus antigen is derived from rotavirus. In certain embodiments, the rotavirus antigen is selected from VP1, VP2, VP3, VP4 (or cleavage products VP5 and VP8), NSP1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5. In certain embodiments, the rotavirus antigen includes VP4 (or cleavage products VP5 and VP8) and VP7.

[0395] Parvovirus: Viral antigens include, but are not limited to, those derived from parvoviruses, such as parvovirus B19. In certain embodiments, the parvovirus antigen is selected from VP-1, VP-2, VP-3, NS-1, and NS-2. In certain embodiments, the parvovirus antigen is the capsid protein VP1 or VP-2.

[0396] Hepatitis delta virus (HDV): Viral antigens include, but are not limited to, those derived from HDV, particularly the δ-antigen from HDV.

[0397] Hepatitis E virus (HEV): Viral antigens include, but are not limited to, those derived from HEV.

[0398] Hepatitis G virus (HGV): Viral antigens include, but are not limited to, those derived from HGV.

[0399] Human herpesviruses: Viral antigens include, but are not limited to, those derived from human herpesviruses, such as, for example, herpes simplex virus (HSV), varicella-zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), human herpesvirus 6 (HHV6), human herpesvirus 7 (HHV7), and human herpesvirus 8 (HHV8). In certain embodiments, the human herpesvirus antigen is selected from immediate early protein (a), early protein (β), and late protein (γ). In certain embodiments, the HSV antigen is derived from HSV-1 or HSV-2 strain. In certain embodiments, the HSV antigen is selected from glycoproteins gB, gC, gD, and gH, fusion protein (gB), or immune escape protein (gC, gE, or gl). In certain embodiments, the VZV antigen is selected from core, nucleocapsid, tegument, or envelope protein. Live attenuated VZV vaccines are commercially available. In certain embodiments, the EBV antigen is selected from initial antigen (EA) protein, viral capsid antigen (VCA), and membrane antigen (MA) glycoproteins. In certain embodiments, the CMV antigen is selected from capsid protein, envelope glycoprotein (e.g., gB and gH), and tegument protein.In other embodiments, the CMV antigen is the following proteins: pp65, IE1, gB, gD, gH, gL, gM, gN, gO, UL128, UL129, gUL130, UL150, UL131, UL33, UL78, US27, US28, RL5A, RL6, RL10, RL11, RL12, RL13, UL1, UL2, UL4, UL5, UL6, UL7, UL8, UL9, UL10, UL11, UL14, UL15A, UL16, UL17, UL18, UL22A, UL38, UL40, UL41A, UL42, UL116, UL119, One or more of the following may be selected: UL120, UL121, UL124, UL132, UL147A, UL148, UL142, UL144, UL141, UL140, UL135, UL136, UL138, UL139, UL133, UL135, UL148A, UL148B, UL148C, UL148D, US2, US3, US6, US7, US8, US9, US10, US11, US12, US13, US14, US15, US16, US17, US18, US19, US20, US21, US29, US30, and US34A. The CMV antigen may also be a fusion of one or more CMV proteins, for example, pp65 / IE1 (Reap et al., Vaccine (2007) 25: pp. 7441-7449 (Non-Patent Literature 10)).

[0400] Papovavirus: Antigens include, but are not limited to, those derived from papovaviruses, such as papillomaviruses and polyomaviruses. In certain embodiments, papillomaviruses include HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63, and 65. In certain embodiments, the HPV antigen is derived from serotype 6, 11, 16, or 18. In certain embodiments, the HPV antigen is selected from capsid proteins (L1) and (L2), or E1-E7, or fusions thereof. In certain embodiments, the HPV antigen is formulated into virus-like particles (VLPs). In certain embodiments, polyomavirus viruses include BK viruses and JK viruses. In certain embodiments, the polyomavirus antigen is selected from VP1, VP2, or VP3.

[0401] Adenovirus: Antigens include those derived from adenovirus. In certain embodiments, the adenovirus antigen is derived from adenovirus serotype 36 (Ad-36). In certain embodiments, the antigen is derived from a protein or peptide sequence, or a fragment thereof, that encodes the Ad-36 coat protein (International Publication No. 2007 / 120362 (Patent Document 2)).

[0402] In other embodiments, the antigen is a bacterial antigen.

[0403] Examples of bacterial antigens suitable for use in connection with the present invention include, but are not limited to, proteins, polysaccharides, and lipopolysaccharides derived from bacteria. In certain embodiments, the bacterial antigen comprises an epitope that is exposed to the surface of the bacterium for at least one stage of its life cycle. The bacterial antigen is preferably conserved across multiple serotypes. In certain embodiments, the bacterial antigen comprises an antigen derived from one or more of the bacteria described below, as well as examples of specific antigens identified below.

[0404] Neisseria meningitidis: Meningococcal (N. meningitidis) antigens include, but are not limited to, proteins and sugars (including polysaccharides or lipooligosaccharides) derived from meningococcal serotypes, e.g., A, C, W135, Y, X, or B. Useful combinations of meningococcal protein antigens include one, two, or three of NHBA, fHbp, and / or NadA immunogens.

[0405] Streptococcus pneumoniae: Streptococcus pneumoniae antigens include, but are not limited to, sugars (including polysaccharides or oligosaccharides) and / or proteins from Streptococcus pneumoniae. In certain embodiments, the sugar antigen is selected from one or more of the following serotypes of Streptococcus pneumoniae: 1, 2, 3, 4, 5, 6A, 6B, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F, and / or 33F. Vaccines or immunogenic compositions may contain multiple serotypes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or more serotypes. Heptavalent, nonavalent, decavalent, elevenvalent, and thirteenvalent conjugate combinations are already known in the art, as are 23valent unconjugate combinations. For example, a decavalent combination may contain sugars from serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F. An elevenvalent combination may further contain sugars from serotype 3. A 12-valent combination may be formed by adding serotypes 6A and 19A; 6A and 22F; 19A and 22F; 6A and 15B; 19A and 15B; r22F and 15B to a 10-valent mixture; a 13-valent combination may be formed by adding serotypes 19A and 22F; 8 and 12F; 8 and 15B; 8 and 19A; 8 and 22F; 12F and 15B; 12F and 19A; 12F and 22F; 15B and 19A; 15B and 22F, etc. to an 11-valent mixture.In certain embodiments, the protein antigen is described in International Publication No. 98 / 18931 (Patent Document 3), 98 / 18930 (Patent Document 4), U.S. Patent No. 6,699,703 (Patent Document 5), U.S. Patent No. 6,800,744 (Patent Document 6), International Publication No. 97 / 43303 (Patent Document 7), 97 / 37026 (Patent Document 8), 02 / 079241 (Patent Document 9), 02 / 34773 (Patent Document 10), 00 / 06737 (Patent Document 11), 00 / 06738 (Patent Document 12), 00 / 58475 (Patent Document 13), and 2003 / 08. No. 2183 (Patent Document 14), No. 00 / 37105 (Patent Document 15), No. 02 / 22167 (Patent Document 16), No. 02 / 22168 No. (Patent Document 17), No. 2003 / 104272 (Patent Document 18), No. 02 / 08426 (Patent Document 19), No. 01 / 12219 ( Patent Document 20), No. 99 / 53940 (Patent Document 21), No. 01 / 81380 (Patent Document 22), No. 2004 / 092209 (Patent Document 23), No. 00 / 76540 (Patent Document 24), No. 2007 / 116322 (Patent Document 25), LeMieux et al., Infect. Imm. (2006) 74:2453-2456 (Non-Patent Document 11), Hoskins et al., J. Bacteriol. (2001) 183:5709-5717 (Non-Patent Document 12), Adamou et al., Infect. Infect. Dis. (2000) 182:1694-1701 (Non-Patent Document 14), Talkington et al., Microb. Pathog. (1996) 21(1): 17-22 (Non-Patent Document 15), Bethe et al., FEMS Microbiol. Lett. (2001) 205(1): pp. 99-104 (Non-Patent Document 16), Brown et al., Infect. Proteins can be selected from those identified in Immun. (2001) 69: pp. 6702-6706 (Non-Patent Literature 17), Whalen et al., FEMS Immunol. Med. Microbiol. (2005) 43: pp. 73-80 (Non-Patent Literature 18), Jomaa et al., Vaccine (2006) 24(24): pp. 5133-5139 (Non-Patent Literature 19).In other embodiments, the Streptococcus pneumoniae protein may be selected from the polyhistidine triad family (PhtX), the choline-binding protein family (CbpX), CbpX truncate, the LytX family, LytX truncate, CbpX truncate-LytX truncate chimeric protein, pneumolysin (Ply), PspA, PsaA, Spl28, SpIOl, Spl30, Spl25, Spl33, and the pneumococcal pneumoniae subunit.

[0406] Streptococcus pyogenes (Group A Streptococcus): Group A Streptococcus antigens include, but are not limited to, proteins identified in International Publication No. 02 / 34771 (Patent Document 26) or No. 2005 / 032582 (Patent Document 27) (including GAS40), fusions of GAS M protein fragments (including those described in International Publication No. 02 / 094851 (Patent Document 28), and Dale, Vaccine (1999) 17: pp. 193-200 (Non-Patent Document 20) and Dale, Vaccine 14(10): pp. 944-948 (Non-Patent Document 21)), fibronectin-binding protein (Sfbl), streptococcal heme-related protein (Shp), and streptolynzin S (SagA).

[0407] Moraxella catarrhalis: Moraxella antigens include, but are not limited to, the antigens identified in International Publication No. 02 / 18595 (Patent Document 29) and No. 99 / 58562 (Patent Document 30), outer membrane protein antigen (HMW-OMP), C-antigen, and / or LPS.

[0408] Bordetella pertussis: The pertussis antigen includes, but is not limited to, a combination of pertussis toxoid (PT) and filamentous hemagglutinin (FHA) from Bordetella pertussis, and possibly further with partactin and / or agglutinogens 2 and 3.

[0409] Burkholderia: Burkholderia antigens include, but are not limited to, *Burkholderia mallei*, *Burkholderia pseudomallei*, and *Burkholderia cepacia*.

[0410] Staphylococcus aureus: Antigens include, but are not limited to, polysaccharides and / or proteins from Staphylococcus aureus. Staphylococcus aureus polysaccharides include, but are not limited to, type 5 and type 8 capsular polysaccharides (CP5 and CP8) optionally bound to non-toxic recombinant Pseudomonas aeruginosa exotoxin A, e.g., StaphVAX®, type 336 polysaccharide (336PS), and polysaccharide intercellular adhesion (PIA, also known as PNGA). Staphylococcus aureus proteins include, but are not limited to, antigens derived from surface proteins, invasins (leucocidine, kinase, hyaluronidase), surface factors that inhibit phagocytic engulfment (capsule, protein A), carotenoids, catalase production, protein A, coagulase, coagulation factors, and / or membrane-damaging toxins (hemolysin, leukotoxin, leucocidine) that lyse eukaryotic cell membranes (optionally detoxified). In certain embodiments, the Staphylococcus aureus antigen is described in International Publication No. 02 / 094868 (Patent Document 31), No. 2008 / 019162 (Patent Document 32), No. 02 / 059148 (Patent Document 33), No. 02 / 102829 (Patent Document 34), No. 03 / 011899 (Patent Document 35), No. 2005 / 079315 (Patent Document 36), No. 02 / 077183 (Patent Document 37), No. 99 / 27109 (Patent Document 38), and No. 01 / 70955. The proteins can be selected from those identified in Patent Document 39, Patent Document 40 (Patent Document 00 / 12689), Patent Document 41 (Patent Document 00 / 12131), Patent Document 42 (Patent Document 2006 / 032475), Patent Document 43 (Patent Document 2006 / 032472), Patent Document 44 (Patent Document 2006 / 032500), Patent Document 45 (Patent Document 2007 / 113222), Patent Document 46 (Patent Document 2007 / 113223), and Patent Document 47 (Patent Document 2007 / 113224). In other embodiments, the Staphylococcus aureus antigen may be selected from IsdA, IsdB, IsdC, SdrC, SdrD, SdrE, ClfA, ClfB, SasF, SasD, SasH(AdsA), Spa, EsaC, EsxA, EsxB, Emp, HlaH35L, CP5, CP8, PNGA, and 336PS.

[0411] Staphylococcus epidermidis: Staphylococcus epidermidis antigens include, but are not limited to, slime-associated antigens (SAAs).

[0412] Clostridium tetani (tetanus): Tetanus antigens include, but are not limited to, tetanus toxoid (TT).

[0413] Clostridium perfringens: Antigens include, but are not limited to, epsilon toxin from Clostridium perfringens.

[0414] Clostridium botulinum (botulism): Botulism antigens include, but are not limited to, those derived from Clostridium botulinum.

[0415] Corynebacterium diphtheriae (diphtheria): The diphtheria antigen preferably includes, but is not limited to, detoxified diphtheria toxin, such as CRM197. In certain embodiments, a diphtheria toxoid is used as a carrier protein.

[0416] Haemophilus influenzae B (Hib): Hib antigens include, but are not limited to, Hib sugar antigens. Hib antigens can be bound to.

[0417] Pseudomonas antigens include, but are not limited to, endotoxin A, Wzz protein, Pseudomonas LPS, LPS isolated from PAO1 (O5 serotype), and / or outer membrane proteins including outer membrane protein F (OprF).

[0418] Brucella: Bacterial antigens derived from Brucella include, but are not limited to, Bovine abortifacient (B. abortus), Canine abortifacient (B. canis), Goat abortifacient (B. melitensis), Siberian abortifacient (B. neotomae), Sheep abortifacient (B. ovis), Pig abortifacient (B. suis), and Brucella pinnipediae.

[0419] Francisella: Bacterial antigens derived from Francisella include, but are not limited to, F. novicida, F. philomiragia, and F. tularensis.

[0420] Streptococcus agalactiae (Group B Streptococcus): Group B Streptococcus antigens include, but are not limited to, protein antigens or sugar antigens identified in International Publication No. 02 / 34771 (Patent Document 26), No. 03 / 093306 (Patent Document 48), No. 04 / 041157 (Patent Document 49), and No. 2005 / 002619 (Patent Document 50) (including proteins GBS80, GBS104, GBS276, and GBS322, and sugar antigens derived from serotypes Ia, Ib, Ia / c, II, III, IV, V, VI, VII, and VIII).

[0421] Neiserria gonorrhoeae: The gonorrhea antigen consists of Por (or porin) protein, e.g., PorB (see Zhu et al., Vaccine (2004) 22: pp. 660-669 (Non-Patent Literature 22)), transferrin-binding protein, e.g., TbpA and TbpB (see Price et al., Infection and Immunity (2004) 71(1): pp. 277-283 (Non-Patent Literature 23)), opacity protein (e.g., Opa), reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see Plante et al., J Infectious See Disease (2000) 182: pp. 848-855 (Non-Patent Document 24), and also see, for example, International Publication No. 99 / 24578 (Patent Document 51), No. 99 / 36544 (Patent Document 52), No. 99 / 57280 (Patent Document 53), and No. 02 / 079243 (Patent Document 54).

[0422] Chlamydia trachomatis: Chlamydia trachomatis antigens include, but are not limited to, antigens derived from serotypes A, B, Ba, and C (the agent of trachoma, a cause of blindness), serotypes L1, L2, and L3 (associated with lymphogranulomatosis of the inguinal region), and serotypes D-K. In certain embodiments, the Chlamydia trachomatis antigen includes, but is not limited to, the antigens identified in International Publication No. 00 / 37494 (Patent Document 55), No. 03 / 049762 (Patent Document 56), No. 03 / 068811 (Patent Document 57), or No. 05 / 002619 (Patent Document 58), and includes PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7 / L12 (CT316), OmcA (CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761).

[0423] Treponema pallidum (syphilis): Syphilitic antigens include, but are not limited to, the TmpA antigen.

[0424] Haemophilus ducreyi (cause of chancroid): The Ducrey antigen includes, but is not limited to, the outer membrane protein (DsrA).

[0425] Enterococcus faecalis or Enterococcus faecium: Antigens include, but are not limited to, trisaccharide repeats or other antigens derived from enterococci.

[0426] Helicobacter pylori: H. pylori antigens include, but are not limited to, CagA, VacA, NAP, HopX, HopY, and / or urease antigens.

[0427] Staphylococcus saprophyticus: The antigen contains, but is not limited to, 160 kDa hemagglutinin, a saprophytic staphylococcus antigen.

[0428] Yersinia enterocolitica: Antigens include, but are not limited to, LPS.

[0429] E. coli: E. coli antigens may be derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), extraintestinal pathogenic E. coli (ExPEC), and / or enterohemorrhagic E. coli (EHEC). ExPEC antigens include, but are not limited to, accessory fixation factors (orf3526), ​​orf353, bacterial Ig-like domain (group 1) protein (orf405), orfl364, NodT family outer membrane factor lipoprotein efflux transporter (orfl767), gspK (orf3515), gspJ (orf3516), tonB-dependent siderophora receptor (orf3597), ciliated protein (orf3613), upec-948, upec-1232, A-chain precursor of type 1 ciliated protein (upec-1875), yap H homolog (upec-2820), and hemolysin A (recp-3768).

[0430] Bacillus anthracis (anthrax): The anthrax antigen includes, but is not limited to, component A (lethal factor (LF) and edema factor (EF)), both of which may share a common component B known as the protective antigen (PA). In certain embodiments, the anthrax (B. anthracis) antigen may be detoxified.

[0431] Yersinia pestis (plague): Plague antigens include, but are not limited to, F1 capsular antigen, LPS, and Yersinia pestis V antigen.

[0432] Mycobacterium tuberculosis: Tuberculosis antigens include, but are not limited to, lipoproteins, LPS, BCG antigen, 85B antigen (Ag85B) fusion protein, ESAT-6, Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase-associated antigen, and MPT51 antigen.

[0433] Rickettsia: Antigens include, but are not limited to, outer membrane proteins including outer membrane protein A and / or B (OmpB), LPS, and surface protein antigens (SPAs).

[0434] Listeria monocytogenes: Bacterial antigens include, but are not limited to, those derived from Listeria monocytogenes.

[0435] Chlamydia pneumoniae: Antigens include, but are not limited to, those identified in International Publication No. 02 / 02606 (Patent Document 59).

[0436] Vibrio cholerae: Antigens include, but are not limited to, proteinase antigens, LPS, in particular, lipopolysaccharide of Vibrio cholerae II, O1 Inaba O-specific polysaccharide, Vibrio cholerae O139, IEM108 vaccine antigens, and occlusion toxin (Zot).

[0437] Salmonella typhi (typhoid fever): The antigen includes, but is not limited to, capsular polysaccharides, preferably conjugates (Vi, i.e., vax-TyVi).

[0438] Borrelia burgdorferi (Lyme disease): Antigens include, but are not limited to, lipoproteins (e.g., OspA, OspB, OspC, and OspD), other surface proteins (e.g., OspE-related protein (Erp), decorin-binding protein (e.g., DbpA), and antigenically variable VI proteins (e.g., antigens associated with P39 and P13 (membrane-bound proteins) and VlsE antigen mutant proteins).

[0439] Porphyromonas gingivalis: Antigens include, but are not limited to, the outer membrane protein (OMP) of Porphyromonas gingivalis.

[0440] Klebsiella: The antigen includes, but is not limited to, OMP containing OMP A, or, in some cases, polysaccharides bound to tetanus toxoid.

[0441] Other bacterial antigens used in connection with the present invention include, but are not limited to, any of the aforementioned capsular antigens, polysaccharide antigens, or protein antigens. In certain embodiments, the bacterial antigens used in connection with the present invention are derived from Gram-negative bacteria, while in other embodiments, they are derived from Gram-positive bacteria. In certain embodiments, the bacterial antigens used in connection with the present invention are derived from aerobic bacteria, while in other embodiments, they are derived from anaerobic bacteria.

[0442] In other embodiments, the antigen is a fungal antigen.

[0443] Examples of fungal antigens used in connection with the present invention include, but are not limited to, those derived from one or more of the fungi described below.

[0444] Fungal antigens can originate from dermatophytes, including hairy epidermal fungi (Epidermophyton floccosum), Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, and Trichophyton galine. Trichophyton gallinae), Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleinii, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, T. discoides var. discoides, T. okraceim var. This includes Trichophyton ochraceum, Trichophyton violaceum, and / or Trichophyton faviforme.

[0445] The fungal antigens also include Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowii, Aspergillus flavarus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, and Candida tropicalis. Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida krusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondii, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatichidis dermatitidis), Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniaeIt can originate from pneumoniae, Microsporidia, species of the genus Encephalitozoon, Septata intestinalis, and Enterocytozoon bieneusi, and less commonly from the genera Brachiola, Microsporidium, Nosema, Pleistophora, Trachipleistophora, Vittaforma, Paracoccidioides brasiliensis, Pneumocystis carinii, and Pythium incidiosum. insidiosum), Pityrosporum ovale, budding yeast (Saccharomyces cerevisiae), Saccharomyces boulardii, Saccharomyces pombe, Scedosporium apiospermum, Sporothrix schenckii, Trichosporon beigelii, Toxoplasma gondii, Penicillium marnefeimarneffei, Malassezia species, Fonsecaea species, Wangiella species, Sporothrix species, Basidiobolus species, Conidiobolus species, Rhizopus species, Mucor species, Absidia species, Mortierella species, Cunninghamella species, Saxenea species a) Species of the genera Alternaria, Curvularia, Helminthosporium, Fusarium, Aspergillus, Penicillium, Monilinia, Rhizoctonia, Paecilomyces, Pithomyces, and Cladosporium.

[0446] For example, fungal antigens can trigger an immune response to Candida fungi, such as C. albicans.

[0447] In other embodiments, the antigen is a self-antigen.

[0448] In relation to the present invention, the term "autoantigen" refers to an immunogenic antigen or epitope that is present in the subject from birth and may be involved in the development of an autoimmune disease.

[0449] In some embodiments, the autoantigen is a central nervous system (CNS) antigen. In some embodiments, the autoantigen is a multiple sclerosis-related antigen, a diabetes-related antigen, a rheumatoid arthritis-related antigen, a myocarditis-related autoantigen, or a thyroiditis-related antigen.

[0450] Exemplary autoantigens are disclosed, for example, in U.S. Patent Application Publication No. 2016 / 0022788 (Patent Document 60), which is incorporated herein by reference in its entirety.

[0451] In some embodiments, the autoantigen is a multiple sclerosis-associated antigen. In some embodiments, the autoantigen is an antigenic peptide of myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), alpha-B crystallin, S100 beta, or proteolipid protein (PLP), or is derived from myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin-associated glycoprotein (MAG), alpha-B crystallin, S100 beta, or proteolipid protein (PLP).

[0452] In some embodiments, the autoantigen is a diabetes-related antigen. In some embodiments, the autoantigen is selected from insulin, chromogranin A, glutamate decarboxylase (GAD1;GAD67), glutamate decarboxylase 2 (GAD2;GAD65), and islet-specific glucose-6-phosphatase catalytic subunit-related proteins, and combinations thereof. Antigenic fragments and antigenic derivatives of these antigens are also intended. In some embodiments, the antigen may be proinsulin.

[0453] In some embodiments, the autoantigen is a rheumatoid arthritis-related antigen. In some embodiments, the rheumatoid arthritis-related autoantigen may be the peptide (Q / R)(K / R)RAA. In some embodiments, the arthritis-related autoantigen may be type II collagen or a fragment thereof.

[0454] In some embodiments, the autoantigen is a myocarditis-related autoantigen. In some embodiments, the myocarditis-related autoantigen is myosin, an antigenic fragment, or an antigenic derivative. In some embodiments, the antigen may be a peptide contained in human myosin. In some embodiments, the antigen may be a peptide contained in α-myosin.

[0455] In some embodiments, the autoantigen is a thyroiditis-associated autoantigen. In some embodiments, the autoantigen is selected from thyroid peroxidase (TPO), thyroglobulin, or pendrin.

[0456] In other embodiments, the antigen is an allergen.

[0457] An "allergen" is defined as a substance, usually a protein, that induces the production of IgE antibodies in an individual with a predisposition. Similar definitions are presented in the following references: Clin. Exp. Allergy, No. 26, pp. 494-516 (1996) (Non-Patent Literature 25); Mol. Biol. of Allergy and Immunology, edited by R. Bush, Immunology and Allergy Clinics of North American Series (August 1996) (Non-Patent Literature 26). In a particular embodiment, the antigen is a protein allergen, i.e., any amino acid chain that is likely to induce an allergic response, and includes short-chain peptides, polypeptides, or full proteins of about 6 to 20 amino acids.

[0458] Non-exclusive examples of allergens include pollen allergens (e.g., pollen allergens from trees, herbs, grasses, and Poaceae), insect allergens (e.g., inhalant allergens, salivary allergens, and venomous allergens, e.g., cockroach allergens and midge allergens, bee venom allergens), dust mite allergens, animal hair allergens and dander allergens (e.g., from dogs, cats, horses, rats, mice, etc.), and food allergens.

[0459] For example, protein allergens include: Dermatophagoides (house dust mites); Felis (cats); Ambrosia (ragweed); Lolium (poison rye); Cryptomeria (Japanese cedar); Alternaria (alder); Betula (birch); and Blomia (bamboo). Protein allergens; Quercus genus protein allergens; Olea genus protein allergens; Artemisia genus protein allergens; Plantago genus protein allergens; Parietaria genus protein allergens; Canine genus protein allergens; Blattella genus protein allergens; Apis genus protein allergens; Cupressus genus protein allergens; Protein allergens of the genus Thuya; protein allergens of the genus Chamaecyparis; protein allergens of the genus Periplaneta; protein allergens of the genus Agropyron; protein allergens of the genus Secale; protein allergens of the genus Triticum; protein allergens of the genus Cynorhodon; protein allergens of the genus Juniperus; protein allergens of the genus Dactylis Protein allergens of the genus Festuca; protein allergens of the genus Poa; protein allergens of the genus Avena; protein allergens of the genus Holcus; protein allergens of the genus Anthoxanthum; protein allergens of the genus Arrhenatherum; protein allergens of the genus Agrostis; protein allergens of the genus Phleum; protein allergens of the genus Phalaris;The allergens may be selected from a group consisting of protein allergens of the genus Paspalum and protein allergens of the genus Sorghum.

[0460] Examples of various known protein allergens derived from some of the aforementioned genera include: Betula (verrucosa) Bet v I; Bet v II; Bromia Blo t I; Blo t III; Blo t V; Blo t XII; Rosehip Cyn d I; Dermatophagoides (pteronyssinus) or Dermatophagoides (farinae) Der p I; Der p II; Der p III; Der p VII; Der f I; Der f II; Der f III; Der f VII; Domestic cat (Felis (domesticus)) Fel d I; Ragweed (Ambrosia (artemiisfolia)) Amb a I.1; Amb a I.2; Amb a I.3; Amb a I.4; Amb a II; Ryegrass (Lollium (perenne)) Lo p I; Lot p II; Lol p III; Lot p IV; Lol p IX (Lol p V or Lol p Ib); Japanese cedar (Cryptomeria (japonica)) Cry j I; Cry j II; Japanese dogwood (Canis (familiaris)) Can f I; Can f II; Juniperus (sabinoides) or pencil juniper (Juniperus (virginiana)) Jun s I; Jun v I; Cedarwood Texas (Juniperus (ashei)) Jun a I; Jun a II; Orchard grass (Dactylis (glomerata)) Dac g I; Dac g V; Kentucky bluegrass (Poa (pretensis)) Poa p I; Phl p I; Phl p V; Phl p VI, and sorghum (Halepensis) (halepensis)) Includes Sor h I

[0461] Food allergens can originate from milk, dairy products, eggs, legumes (peanuts and soybeans), nuts, grains (e.g., wheat), crustaceans (e.g., mustard), crustaceans, fish, and mollusks. In particular, food allergens may be ovalbumin or gluten.

[0462] The present invention further comprises a vaccine and / or immunogenic composition and / or immunotherapeutic composition, or the DNA vector, comprising a nucleic acid encoding the antigen as defined above, for example, a tumor antigen, viral antigen, bacterial antigen, fungal antigen, autoantigen, allergen, or graft-specific antigen; and optionally an adjuvant-containing modified Propionibacterium acnes.

[0463] Any conventional, exploratory, synthetic, or biological adjuvants for vaccination may be used, including thermolabile toxins (LT), cholera toxin (CT), cholera toxin B subunit (CTB), polymerized liposomes, mutant toxins, probiotic bacteria, oligonucleotides, RNA, siRNA, DNA, and lipids.

[0464] The present invention further includes a method for preventing and / or treating cancer in a subject requiring prevention and / or treatment of cancer, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the tumor antigen defined above, or manipulated acne bacteria containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the tumor antigen defined above, or manipulated acne bacteria containing the DNA vector, for use in a method for preventing and / or treating cancer in a subject.

[0465] As used herein, the term “cancer” means certain hyperproliferative diseases, including malignant tumors, characterized by unregulated or uncontrolled cell proliferation. Cancers of substantially any tissue are known. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias, or lymphoid neoplasms. Specific examples of such cancers are described below and include squamous cell carcinoma (e.g., epithelial squamous cell cancer), lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, hepatocellular carcinoma, gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, thyroid cancer, liver cancer, and head and neck cancers. The term "cancer" includes primary malignant cells or tumors (for example, those in which the cells have not migrated to a site different from the primary malignant tumor or site of tumor within the subject's body) and secondary malignant cells or tumors (for example, those resulting from metastasis, the migration of malignant cells or tumor cells to a secondary site different from the site of the primary tumor).

[0466] The term "cancer" is included within the broader term "abnormal cell proliferation," which may also be called "excessive cell proliferation" or "cell proliferation disorders." Examples of diseases associated with abnormal cell proliferation include metastatic tumors, malignant tumors, benign tumors, cancer, precancerous conditions, hyperplasia, warts, and polyps, as well as noncancerous conditions such as benign melanoma, benign chondroma, benign prostatic hyperplasia, moles, dysplastic nevi, dysplasia, hyperplasia, and other cell proliferations occurring within the epidermis. The precancerous class includes acquired small or microscopic precancerous conditions, acquired large lesions with nuclear atypia, prodromal lesions occurring with hereditary hyperplastic syndromes that progress to cancer, and acquired diffuse hyperplasia and diffuse metaplasia. Examples of small or microscopic precancerous conditions include HGSIL (high-grade squamous intraepithelial lesion of the cervix), AIN (anal intraepithelial neoplasia), vocal cord dysplasia, abnormal crypts (of the colon), and PIN (prostatic intraepithelial neoplasia). Examples of large acquired lesions with nuclear atypia include tubular adenoma, AILD (angioimmunoblastic lymphadenopathy with heteroproteinosis), atypical meningioma, gastric polyps, large plaque parapsoriasis, myelodysplasia, papillary transitional cell carcinoma, refractory anemia with blast cell proliferation, and Schneiderian papilloma.

[0467] The present invention further includes a method for preventing and / or treating cancer in a subject requiring prevention and / or treatment of a viral infection, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the viral antigen defined above, or manipulated acne bacteria containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the viral antigen defined above, or manipulated acne bacteria containing the DNA vector, for use in a method for preventing and / or treating a viral infection in a subject.

[0468] In the above embodiment, the antigen preferably induces activation or enhancement of an immune response, particularly an immune response specific to the antigen.

[0469] Specific examples of viral infections include, but are not limited to, cytomegalovirus (CMV) pneumonia, enteritis, and retinitis; Epstein-Barr virus (EBV) lymphoproliferative disorder; varicella / herpes zoster (caused by varicella-zoster virus, VZV); HSV-1 and -2 mucositis; HSV-6 encephalitis, BK virus hemorrhagic cystitis; viral influenza; pneumonia caused by synovial respiratory virus (RSV); AIDS (caused by HIV); and hepatitis A, B, or C.Further examples of viral infections include: Retroviridae; Picornaviridae (e.g., poliovirus, hepatitis A virus; enterovirus, anthropogenic coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., gastroenteritis strains); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronavirusidae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., Hantan virus, Bunyaviridae). Yaviruses, phleboviruses, and nairoviruses); Arenaviridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviruses, and rotaviruses); Bimaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papillomaviruses, polyomaviruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella-zoster virus, cytomegalovirus (CMV), herpesviruses); Poxviridae (smallpox virus, vaccinia virus, poxviruses); and Iridoviridae (e.g., African swine cholera virus); and Unclassified viruses (e.g., virulence factors of spongiform encephalopathy (etiological)). This includes infections caused by the Norwalk virus, Norwalk-associated viruses, and astroviruses, as well as factors for hepatitis delta (presumably defective satellites of the hepatitis B virus), non-hepatitis A and non-hepatitis B factors (class 1 = internal transmission; class 2 = parenteral transmission (i.e., hepatitis C)); and Norwalk virus and Norwalk-associated viruses.

[0470] The present invention further includes a method for preventing and / or treating a bacterial infection in a subject requiring prevention and / or treatment of a bacterial infection, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the bacterial antigen defined above, or a manipulated acne bacterium containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the bacterial antigen defined above, or a manipulated acne bacterium containing the DNA vector, for use in a method for preventing and / or treating a bacterial infection in a subject.

[0471] In the above embodiment, the antigen preferably induces activation or enhancement of an immune response, particularly an immune response specific to the antigen.

[0472] Examples of bacterial infections include Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, Mycobacteria species (e.g., Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii). kansasii), M. gordonae, Staphylococcus aureus, Neisseria gonoloe, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactia (Group B Streptococcus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic species), Streptococcus pneumoniae, pathogenic Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium species, Erysipelothrix rugiopathies Infections caused by rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella maltosida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, and Actinomyces israelii include, but are not limited to, infections caused by these organisms.

[0473] The present invention further includes a method for preventing and / or treating a fungal infection in a subject requiring prevention and / or treatment of a fungal infection, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the fungal antigen defined above, or a manipulated acne bacterium containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the fungal antigen defined above, or a manipulated acne bacterium containing the DNA vector, for use in a method for preventing and / or treating a fungal infection in a subject.

[0474] In the above embodiment, the antigen preferably induces activation or enhancement of an immune response, particularly an immune response specific to the antigen.

[0475] Examples of fungal infections include, but are not limited to, aspergillosis; thrush (caused by Candida albicans); cryptococcosis (caused by Cryptococcus); and histoplasmosis. Therefore, examples of fungal infections include, but are not limited to, infections caused by Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides imitis, Blastomyces dermatichidis, Chlamydia trachomatis, or Candida albicans.

[0476] The present invention further includes a method for preventing and / or treating an autoimmune disease in a subject requiring prevention and / or treatment of the autoimmune disease, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the autoantigen defined above, or manipulated acne bacteria containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the autoantigen defined above, or manipulated acne bacteria containing the DNA vector, for use in a method for preventing and / or treating an autoimmune disease in a subject.

[0477] In the above embodiment, the antigen preferably leads to an immune response, particularly tolerance or suppression of the immune response to the antigen.

[0478] Autoimmune diseases include, but are not limited to, multiple sclerosis, rheumatoid arthritis, myasthenia gravis, psoriasis, systemic lupus erythematosus, autoimmune thyroiditis (Hashimoto's thyroiditis), Graves' disease, inflammatory bowel disease, autoimmune uretinitis, myocarditis, polymyositis, and certain types of diabetes, including type 1 diabetes.

[0479] The present invention further includes a method for preventing and / or treating allergies, such as asthma, in subjects requiring prevention and / or treatment of allergies, such as asthma, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the allergen defined above, or a manipulated acne bacterium containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the allergen defined above, or a manipulated acne bacterium containing the DNA vector, for use in a method for preventing and / or treating allergies, such as asthma, in subjects.

[0480] In the above embodiment, the antigen preferably leads to an immune response, particularly tolerance or suppression of the immune response to the antigen.

[0481] In relation to this disclosure, allergy refers to asthma or allergies caused by the allergens defined above.

[0482] The present invention further includes a method for preventing and / or treating graft rejection in a subject requiring prevention and / or treatment of graft rejection, comprising the step of administering to the subject a therapeutically or prophylactically efficient amount of a DNA vector containing a nucleic acid encoding the graft-specific antigen defined above, or manipulated acne bacteria containing the DNA vector. The present invention further relates to a DNA vector containing a nucleic acid encoding the graft-specific antigen defined above, or manipulated acne bacteria containing the DNA vector, for use in a method for preventing and / or treating graft rejection in a subject.

[0483] In the above embodiment, the antigen preferably leads to an immune response, particularly tolerance or suppression of the immune response to the antigen.

[0484] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as that generally understood by one person skilled in the art in which the present invention pertains.

[0485] All publications referenced herein are incorporated herein by reference. This disclosure is understood to supersede any disclosures in the incorporated publications to the extent that they conflict.

[0486] When used in this specification and the accompanying claims, the singular forms "a," "an," and "the" are to be used to indicate that there may be multiple references unless otherwise clearly indicated. For example, a reference to "an antigen" may include a mixture of two or more antigens.

[0487] definition Delivery Vehicle As used herein, the term "delivery vehicle" refers to any means that enables the delivery of a payload into a bacterium.

[0488] The present invention includes, but is not limited to, several types of delivery vehicles, such as bacteriophage scaffolds, viral scaffolds, chemical-based delivery vehicles (e.g., cyclodextrin, calcium phosphate, cationic polymer, cationic liposome), protein-based or peptide-based delivery vehicles, lipid-based delivery vehicles, nanoparticle-based delivery vehicles, non-chemical-based delivery vehicles (e.g., transformation, electroporation, sonoporation, optical transfection), particle-based delivery vehicles (e.g., gene guns, magnetofection, impalefection, particle collision, cell membrane-permeable peptides), or donor bacteria (conjugation).

[0489] Any combination of delivery vehicles is further included in the present invention.

[0490] The delivery vehicle may be derived from bacteriophage-derived scaffolds, natural, evolved, or manipulated capsids.

[0491] In some embodiments, the delivery vehicle is the payload, because bacteria inherently have the ability to take in payloads from the environment on their own.

[0492] 《Joining》 Conjugation is the process by which a donor bacterium actively transfers DNA to a recipient bacterium. DNA transfer involves the recognition of the origin of transfer (oriT) by proteins known as relaxases, which covalently bind to oriT DNA by making nicks (breaks). The relaxase and single-stranded DNA are then typically injected into the recipient cell via the type IV secretory system. During plasmid or ICE (integration and conjugation element) conjugation, relaxase transfer is linked to the rolling-circle replication of the plasmid or ICE. As soon as it enters the recipient cell, the relaxase re-circulates the transferred strand at the oriT. - Smillie et al., Microbiology and Molecular Biology Rev, 2010, pp. 434-452 (Non-Patent Literature 27).

[0493] Examples of conjugation plasmids are F, R388, RP4, RK2, and R6K. Plasmids from the following groups: IncA, IncB / O (Ind O), IncC, IncD, IncE, IncFI, lncF2, IncG, IncHM, lncHI2, Inch, Incl2, IncJ, IncK, IncL / M, IncN, IncP, IncQI, lncQ2, IncR, IncS, IncT, IncU, IncV, IncW, IncXI, lncX2, IncY, IncZ, ColE1, ColE2, ColE3, p15A, pSC101, lncP-2, lncP-5, lncP-7, lncP-8, lncP-9, Ind, Inc4, Inc7, Inc8, Inc9, Inc11, Inc13, Ind4, or Ind8 are frequently conjugated and carry the type IV secretory system.

[0494] A list of type IV secretory systems can be found in public databases, such as AtlasT4SS.

[0495] Conjugation is not limited to plasmids and can also occur from bacterial chromosomes if oriT is present. It can occur spontaneously through recombination of the conjugation plasmid within the chromosome, or artificially through the introduction of oriT at a desired location within the chromosome. A specific class of conjugation elements is known as integration and conjugation elements (ICEs). Integration and conjugation elements are not maintained in a circular plasmid form but are integrated into the host chromosome. Upon transmission, ICEs are excised from the chromosome and then transmitted in a manner similar to that of conjugation plasmids. Immediately upon entering the recipient cell, ICEs are integrated into the recipient's chromosome. Lists of ICE elements can be found in public databases, such as ICEberg.

[0496] An ICE or plasmid containing both the origin gene and the type IV secretory gene is generally called a mobile element, while an ICE or plasmid containing only the oriT gene may be called a mobile plasmid. A mobile element can be transmitted from a donor cell to a recipient cell only if the type IV secretory gene is expressed in trans by either another plasmid or from the host cell's chromosome.

[0497] "payload" As used herein, the term "payload" refers to any nucleic acid sequence, amino acid sequence, or combination thereof (e.g., peptide nucleic acid or peptide-oligonucleotide conjugate, but not limited to) that is delivered into a bacterium by a delivery vehicle.

[0498] The term "payload" may further refer to plasmids, vectors, or cargo.

[0499] The payload may be a phagemide or fasmid obtained from a natural, evolved, or manipulated bacteriophage genome. The payload may further consist only partially of a phagemide or fasmid obtained from a natural, evolved, or manipulated bacteriophage genome.

[0500] In some embodiments, the payload is the delivery vehicle, because bacteria inherently have the ability to take in payloads from the environment on their own.

[0501] 《Nucleic acid》 As used herein, the term “nucleic acid” refers to a sequence of at least two covalently linked nucleotides, which may be single-stranded or double-stranded, or may contain portions of both single-stranded and double-stranded sequences. The nucleic acids of the present invention may be natural, recombinant, or synthetic. Nucleic acids may be in the form of a circular sequence or a linear sequence, or a combination of both. Nucleic acids may be DNA, genomic DNA or cDNA, or RNA, or a combination of both. Nucleic acids may contain any combination of deoxyribonucleotides and ribonucleotides, as well as any combination of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, 5-hydroxymethylcytosine, and isoguanine. Other examples of modified bases that may be used in the present invention are listed in Chemical Reviews 2016, 116(20) pp. 12655-12687 (Non-Patent Literature 28). The term "nucleic acid" further includes, but is not limited to, nucleic acid analogs that may contain other skeletons, including phosphoramides, phosphorothioates, phosphorodithioates, O-methylphosphoroamidite bonds, and / or deoxyribonucleotide nucleic acids and ribonucleotide nucleic acids. Any combination of the aforementioned characteristics of nucleic acids is further included in the present invention.

[0502] "vector" As used herein, the term “vector” refers to any construct of a sequence capable of expressing a polypeptide in a given host cell. When a vector is used, the choice of vector depends, as is well known to those skilled in the art, on the method used to transform the host bacterium. Vectors include, but are not limited to, plasmid vectors and recombinant phage vectors, or any other vector known in the art that is suitable for delivering the polypeptide of the present invention to target bacteria. Those skilled in the art are well aware of the genetic elements that must be present on a vector in order to efficiently transform, select, and grow host cells containing any of the isolated nucleotide or nucleic acid sequences of the present invention.

[0503] 《Phagemid》 As used herein, the terms “phagemide” or “phasmid” are equivalent and refer to a recombinant DNA vector containing at least one sequence of a bacteriophage genome. The phagemides of this disclosure include a phage packaging site and optionally an origin of replication (ori), particularly a bacterial and / or phage origin of replication. In one embodiment, the phagemide of this disclosure does not contain a bacterial origin of replication and therefore cannot replicate on its own when injected into a bacterium. Instead, the phagemide includes a plasmid origin of replication, particularly a bacterial and / or phage origin of replication.

[0504] Packaged phagemids As used herein, the terms “packaged phagemide” or “phage-derived particle” refer to a phagemide capsidized within a bacteriophage scaffold, bacterial virus particle, or capsid. In particular, it refers to a bacteriophage scaffold, bacterial virus particle, or capsid lacking a bacteriophage genome. Packaged phagemide, or phage-derived particle, can be produced by helper phage strategies well known to those skilled in the art. The helper phage contains all the genes encoding the structural and functional proteins essential for capsidizing the phagemide according to the present invention. Packaged phagemide, or phage-derived particle, can also be produced by satellite virus strategies well known to those skilled in the art. A satellite virus is a subviral factor consisting of nucleic acids that, with respect to all morphogenetic functions, depend on co-infection of host cells by a helper virus, but with respect to all its episomal functions (integration and immunology, multicopy plasmid replication), the satellite is completely autonomous from the helper. In one embodiment, the satellite gene may encode a protein that promotes a reduction in the capsid size of a helper phage, and is described in relation to a P4 Sid protein that regulates the P2 capsid size to match its smaller genome.

[0505] "peptide" As used herein, the term "peptide" refers to both a short chain of at least two linked amino acids, and a portion, subset, or fragment thereof that is not expressed independently of the rest of the protein. In some cases, the peptide is a protein. In other cases, the peptide is not a protein, and the peptide refers only to a portion, subset, or fragment of a protein. Preferably, the size of the peptide ranges from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 amino acids to 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 100, 200 amino acids.

[0506] "Manipulative type"

[01] As used herein, the term “manipulated” means that the bacterial cells, phages, phage-derived particles, phagemids, or vectors of the present invention have been modified by molecular biological techniques. As those skilled in the art will understand, the manipulation of bacterial cells, phages, phage-derived particles, phagemids, or vectors implies an intentional action to introduce or modify nucleic acid sequences, and does not encompass the introduction or modification of nucleic acid sequences by the natural evolution of bacterial cells, phages, phage-derived particles, phagemids, or vectors.

[0507] "Percent of Identity"

[02] The percentage of identity used herein is calculated with respect to a polymer (e.g., polynucleotide or polypeptide) with those sequences aligned. The percentage of identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences, and the length of each cap. The comparison of sequences and the determination of the percentage of identity between two sequences can be achieved using mathematical algorithms, as described in the non-limiting examples below.

[03] The percentage of identity between two amino acid sequences may be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17 (1988) (Non-Patent Literature 29)), incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. Furthermore, the percentage of identity between two amino acid sequences may be determined using the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970) (Non-Patent Literature 30)), incorporated into the GAP program in the GCG software package (available from www.gcg.com), using the BLOSUM62 matrix, BLOSUM30 matrix, or PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6. In one specific embodiment, the BLOSUM30 matrix is ​​used with 12 gap-opening penalties and 4 gap-extending penalties.

[0508] CRISPR-Cas system The CRISPR-Cas system refers to DNA encoding two distinct elements: i) an endonuclease, in this case a CRISPR-associated nuclease (Cas or "CRISPR-associated protein"), and ii) a guide RNA. Depending on the type of CRISPR system, the guide RNA may be a chimeric RNA consisting of a combination of CRISPR (crRNA) bacterial RNA and tracrRNA (transactivating RNA CRISPR) (Jinek et al., Science 2012). The guide RNA combines the targeting specificity of the crRNA, which corresponds to the "spacing sequence" used as a guide to the Cas protein, and the conformational properties of the tracrRNA in a single transcript. When the guide RNA and Cas protein are simultaneously expressed in a cell, the target genomic sequence can be permanently interrupted (and, depending on its location, cause loss and / or cell death of the targeted and surrounding sequences), or modified. Modifications may be guided by a repair matrix.

[0509] The CRISPR-Cas system comprises two main classes, depending on the nuclease action mechanism: - Class 1 consists of multi-subunit effector complexes and includes types I, III, and IV. - Class 2 consists of single-unit effector modules, such as Cas9 nucleases, and includes Type II (II-A, II-B, II-C, II-C variant), Type V (VA, VB, VC, VD, VE, V-U1, V-U2, V-U3, V-U4, V-U5), and Type VI (VI-A, VI-B1, VI-B2, VI-C, VI-D).

[0510] The target sequence according to the present invention includes a nucleic acid sequence encoding a Cas protein. Various CRISPR enzymes can be used as the target sequence on the plasmid according to the present invention. In some embodiments, the CRISPR enzyme is a type II, type II-A, or type II-B CRISPR enzyme. In other embodiments, the CRISPR enzyme is a type I or type III CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some other embodiments, the CRISPR enzyme catalyzes RNA cleavage. In one embodiment, the CRISPR enzyme may be bound to a guide RNA or a single guide RNA (sgRNA). In certain embodiments, the guide RNA or sgRNA targets a gene selected from the group consisting of antibiotic resistance genes, pathogenic protein or virulence factor genes, toxin protein or toxin factor genes, bacterial receptor genes, membrane protein genes, structural protein genes, secreted protein genes, genes that express resistance to drugs in general, and genes that cause adverse effects on the host.

[0511] The target sequence may include, alone, a nucleic acid sequence encoding a guide RNA or sgRNA that intrinsically directs the Cas protein to the target bacterium, or a combination of the Cas protein encoded by the payload and / or the RNA guide.

[0512] Non-limiting examples of Cas proteins, either as part of a multi-subunit effector or as a single-unit effector, include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas11(SS), Cas12a(Cpf1), Cas12b(C2c1), Cas12c(C2c3), Cas12d(CasY), Cas12e(CasX), C2c4, C2c8, C2c5, C2c10, C2c9, Cas13a(C2c2), Cas13b(C2c6), Cas13c(C2c7), Cas13d, Csa5, Csc 1, Csc2, Cse1, Cse2, Csy1, Csy2, Csy3, Csf1, Csf2, Csf3, Csf4, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csn2, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10 This includes Csx16, CsaX, Csx13, Csx1, Csx15, SdCpf1, CmtCpf1, TsCpf1, CmaCpf1, PcCpf1, ErCpf1, FbCpf1, UbcCpf1, AsCpf1, LbCpf1, their homologs, their orthologues, their variants, or modified versions thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the protospacer adjacent motif (PAM) site.

[0513] In one particular embodiment, the CRISPR enzyme is any Cas9 protein, e.g., any naturally occurring bacterial Cas9, as well as any variant, homolog, or orthologue thereof.

[0514] "Cas9" refers to the protein Cas9 (also known as Csn1 or Csx12), or its functionality, i.e., a fragment of a protein, peptide, or polypeptide capable of interacting with guide RNA and exhibiting enzymatic activity (nuclease) that causes double-strand breaks in target genome DNA. Therefore, "Cas9" also refers to a modified protein, for example, a modified protein that has been cleaved to remove a protein domain that is not essential for the protein's defined function, particularly a domain not required for interaction with gRNA.

[0515] The sequences encoding Cas9 (whole protein or fragment thereof) used in connection with the present invention can be obtained from any known Cas9 protein (Fonfara et al., 2014; Koonin et al., 2017). Examples of Cas9 proteins useful in the present invention include, but are not limited to, the Cas9 proteins of Streptococcus pyogenes (SpCas9), Streptococcus thermophiles (St1Cas9, St3Cas9), Streptococcus mutans, Staphylococcus aureus (SaCas9), Campylobacter jejuni (CjCas9), Francisella novicida (FnCas9), and Neisseria meningitidis (NmCas9).

[0516] The sequences encoding Cpf1(Cas12a) (whole protein or fragment thereof) used in connection with the present invention can be obtained from any known Cpf1(Cas12a) protein (Koonin et al., 2017). Examples of Cpf1(Cas12a) proteins useful in the present invention include, but are not limited to, Cpf1(Cas12a) proteins from Acidaminococcus species, Lachnospiraceae bacteria, and Francisella novicida.

[0517] The sequences encoding Cas13a (whole protein or fragment thereof) used in connection with the present invention can be obtained from any known Cas13a(C2c2) protein (Abudayyeh et al., 2017). Examples of Cas13a(C2c2) proteins useful in the present invention include, but are not limited to, the Cas13a(C2c2) protein of Leptotrichia wadei (LwaCas13a).

[0518] The sequences encoding Cas13d (whole protein or fragment thereof) used in connection with the present invention can be obtained from any known Cas13d protein (Yan et al., 2018). Examples of Cas13d proteins useful in the present invention include, but are not limited to, Cas13d proteins from the species Eubacterium siraeum and Ruminococcus.

[0519] In certain embodiments, the CRISPR / Cas9 system targets the reduction or inactivation of gene expression of a target nucleic acid sequence selected from the group consisting of antibiotic resistance genes, virulence factor or pathogenic protein genes, toxin factor or toxin protein genes, bacterial receptor expressing genes, membrane proteins, structural proteins, secreted proteins, genes expressing resistance to drugs in general, and genes that cause adverse effects on the host.

[0520] In one embodiment, the CRISPR-Cas system is used to target and inactivate virulence factors. Vulnerability factors can be any substance produced by a pathogen that alters the host-pathogen interaction by increasing the degree of damage inflicted on the host. Vulnerability factors are used by pathogens in many ways, including, for example, to evade the host immune response in cell adhesion or colonization within the host, to facilitate entry into and exit from host cells, to obtain nutrients from the host, or to inhibit other physiological processes in the host. Vulnerability factors may include enzymes, endotoxins, adhesion factors, motility factors, complement evasion factors, scavenging factors, and factors that promote biomembrane formation. For example, such targeted virulence factor genes include, but are not limited to, EHEC-HlyA, Stx1(VT1), Stx2(VT2), Stx2a(VT2a), Stx2b(VT2b), Stx2c(VT2c), Stx2d(VT2d), Stx2e(VT2e), and Stx2f(VT2f), Stx2h(VT2h), stx2k, fimA, fimF, fimH, neuC, kpsE, sfa, foc, iroN, aer, iha, papC, papGI, papGII, papGIII, hlyC, cnf1, hra, sat, ireA, usp Possible target virulence factor genes include ompT, ibeA, malX, fyuA, irp2, traT, afaD, ipaH, eltB, estA, bfpA, eaeA, espA, aaiC, aatA, TEM, CTX, SHV, csgA, csgB, csgC, ​​csgD, csgE, csgF, csgG, csgH, T1SS, T2SS, T3SS, T4SS, T5SS, and T6SS (secretionary system). For example, such targeted virulence factor genes could be Shigella dysenteriae virulence factor genes, for example, stx1 and stx2, but are not limited to these. For example, such targeted virulence factor genes could be Yersinia pestis virulence factor genes, for example, yscF (plasmid-mediated (pCDl) T3SS external needle subunit), but are not limited to these.For example, such targeted virulence gene may be a tularemia virulence gene, such as fslA, for example. For example, such targeted virulence gene may be a Bacillus anthracis virulence gene, such as pag (anthrax toxin, cell-bound protective antigen), for example. For example, such targeted virulence gene may be a Vibrio cholera virulence gene, such as ctxA and ctxB (cholera toxin), tcpA (toxin coregulatory ciliary), and toxT (master pathogenicity regulator), for example. For example, such targeted virulence factor genes may include Pseudomonas aeruginosa virulence factor genes, such as, but are not limited to, pioverdin (e.g., sigma factor pvdS, biosynthetic genes pvdL, pvdI, pvdJ, pvdH, pvdA, pvdF, pvdQ, pvdN, pvdM, pvdO, pvdP, transporter genes pvdE, pvdR, pvdT, opmQ), siderofoapioquelin (e.g., pchD, pchC, pchB, pchA, pchE, pchF, and pchG), and toxins (e.g., exoU, exoS, and exoT). For example, such targeted virulence genes may include Klebsiella pneumoniae virulence genes, e.g., fimA (adhesion, type I ciliary major subunit), and cps (capsular polysaccharide). For example, such targeted virulence genes may include Acinetobacter baumannii virulence genes, e.g., ptk (capsule polymerization), and epsA (assembly). For example, such targeted virulence genes may include Salmonella enterica cifi virulence genes, e.g., MIA (invasion, SPI-1 regulator), ssrB (SPI-2 regulator), and those associated with bile resistance, including efflux pump genes acrA, acrB, and tolC. For example, such targeted virulence factor genes could be Fusobacterium nucleatum virulence factor genes, such as, but not limited to, FadA and TIGIT.For example, such a targeted virulence factor gene could be a Bacteroides fragilis virulence factor gene, such as bft, but is not limited to bft. For example, such targeted virulence factor genes may include the porphyrin gene of Cutibacterium acnes, CAMP factors (CAMP1, CAMP2, CAMP3, CAMP4), hyaluronic acid lyase (HYL-IB / II, HYL-IA), lipase (GehA, GehB), hemolysin, sialidase, endoglycoceramidase, endo-β-N-acetylglucosaminidase, dermatan sulfate adhesin (DsA1, DsA2), proline-threonine repeat (PTR), or any virulence factor located on acne-related genomic loci 1, 2, 3 (plasmids), 4 as described by Tomida et al., such as the tight adhesion locus (tad), streptolyn S-related gene (sag), and non-ribosomal peptide synthase (NRPS).

[0521] In other embodiments, the CRISPR / Cas9 system is used to target and inactivate antibiotic resistance genes, including, but are not limited to, GyrB, ParE, ParY, AAC(1), AAC(2'), AAC(3), AAC(6'), ANT(2"), ANT(3"), ANT(4'), ANT(6), ANT(9), APH(2"), APH(3"), APH(3'), APH(4), APH(6), APH(7"), APH(9), ArmA, RmtA, RmtB, RmtC, Sgm, AER, BLA1, CTX-M, KPC, SHV, TEM, BlaB, CcrA, IMP, NDM, VIM, ACT, AmpC, CMY, LAT, PDC, OXA β-lactamase, mecA, Omp36, OmpF, PIB, bla(blaI, blaR1), and mec(mecI, mecR1) operon, chloramphenicol acetyltransferase (CAT), chloramphenicol phosphotransferase, ethambutol-resistant arabinosyltransferase (EmbB), MupA, MupB, membrane-bound protein MprF, Cfr 23S rRNA methyltransferase, rifampin ADP-ribosyltransferase (Arr), rifampin lycosyltransferase, rifampin monooxygenase, rifampin phosphotransferase, DnaA, RbpA, rifampin-resistant beta subunit of RNA polymerase (RpoB), Erm 23SrRNA methyltransferase, Lsa, MsrA, Vga, VgaB, streptogramin Vgb lyase, Vat acetyltransferase, fluoroquinolone acetyltransferase, fluoroquinolone-resistant DNA topoisomerase, fluoroquinolone-resistant GyrA, GyrB, ParC, quinolone-resistant protein (Qnr), FomA, FomB, FosC, FosA, FosB, FosX, VanA, VanB, VanD, VanR, VanS, lincosamide nucleotidyltransferase (Lin), EreA, EreB, GimA, Mgt, Ole, macrolide phosphotransferase (MPH), Me fA, MefE, Mel, streptoslysine acetyltransferase (sat), Sul1, Sul2, Sul3, sulfonamide resistance FolP, tetracycline inactivating enzyme TetX, TetA, TetB, TetC, Tet30, Tet31, TetM, TetO, TetQ, Tet32, Tet36, MacAB-TolC, MsbA, MsrA, VgaB, EmrD, EmrAB-TolC, NorB, GepA, MepA, AdeABC, AcrD, MexAB-OprM, mtrCDE, EmrE, adeR, acrR, baeSR, mexR, phoPQ, mtrR, or any antibiotic resistance gene listed in the Comprehensive Antibiotic Resistance Database (CARD https: / / card.mcmaster.ca / ).

[0522] In other embodiments, the CRISPR-Cas system is used to target and inactivate bacterial toxin genes. Bacterial toxins may be classified as exotoxins or endotoxins. Exotoxins are produced and actively secreted; endotoxins remain part of the bacterium. Responses to bacterial toxins may include severe inflammation and may lead to sepsis. Such toxins may be, for example, botulinum neurotoxin, tetanus toxin, staphylococcal toxin, diphtheria toxin, anthrax toxin, alpha toxin, pertussis toxin, Shiga toxin, thermostable enterotoxin (Escherichia coli ST), colibactin, BFT (B. fragilis toxin), or any toxin described in Henkel et al. (Toxins from Bacteria in EXS. 2010;100:1-29 (Non-Patent Literature 31)).

[0523] Base editing Base editing (BE) refers to the ability to replace specific nucleotide base pairs on a DNA or RNA molecule with other base pairs. Until recently, the only way to perform specific substitutions on DNA in vivo was by using recombination of template DNA carrying the specific base pair change with the target gene locus. Base editing techniques rely on entirely different strategies. There is no DNA exchange; instead, enzymatic reactions convert nucleotides to other nucleotides, resulting in a mismatch at the dsDNA level, which is then corrected by cellular mechanisms.

[0524] One of the main challenges in base editing is how to restrict the activity of the enzyme performing nucleotide conversion to the target nucleotide, such as a SNP involved in pathogenicity. This spatial constraint has recently been achieved through the reuse of the CRISPR-Cas system for other purposes. In fact, by fusing a catalytically impaired or inactive Cas nuclease with a base-modifying enzyme that is active only on single-stranded DNA, it is possible to achieve highly efficient base editing. This is possible thanks to the ability of CRISPR-Cas to generate a locally occurring ssDNA bubble within the "R loop" when the complex anneals to its DNA target strand via RNA-DNA base pairing.

[0525] So far, seven types of DNA base editors have been described: - Cytosine base editor (CBE) that converts C:G to T:A (Komor, A et al., Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420-44 (2016) (Non-patent document 32)), - Adenine base editor (ABE) that converts A:T to G:C (Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage. Nature 551(7681) pp. 464-471 (2017) (Non-patent document 33)), - C:G to G:C conversion cytosing anine base editor (CGBE), Chen, L et al., Precise and programmable C:G to G:C base editing in genomic DNA. Biorxiv (2020) (Non-patent document 34); Kurt, I et al., CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nature Biotechnology (2020) (Non-patent document 35) - Cytosine adenine base editor (CABE) that converts C:G to A:T, Zhao, D et al., New base editors change C to A in bacteria and C to G in mammalian cells. Nature Biotechnology (2020) (Non-patent document 36) - Adenine cytosine base editor (ACBE) for converting A:T to C:G (Liu, D et al., A:T to C:G base editors and uses thereof. International Patent Application Publication No. 2020181180 (2020) (Patent Document 61)), - Adenine thymine base editor (ATBE) for converting A:T to T:A (Liu, D et al., A:T to C:G base editors and uses thereof. International Patent Application Publication No. 2020181180 (2020) (Patent Document 61)), - Thymine adenine base editor (TABE) for converting T:A to A:T (Liu, D et al., T:A to A:T base editing through adenosine methylation. International Patent Application Publication No. 2020181193 (2020) (Patent Document 62); Liu, D et al., T:A to A:T base editing through thymine alkylation. International Patent Application Publication No. 2020181178 (2020) (Patent Document 63); Liu, D et al., T:A to A:T base editing through adenine excision. International Patent Application Publication No. 2020181195 (2020) (Patent Document 64)).

[0526] Base editors differ in terms of the base-modifying enzyme. CBEs rely on ssDNA cytidine deaminases: APOBEC1, rAPOBEC1, APOBEC1 variants or evolved forms (evoAPOBEC1), and APOBEC homologs (APOBEC3A (eA3A), Anc689), cytidine deaminase 1 (CDA1), evoCDA1, FERNY, evoFERNY. ABEs rely on tandem fusion TadA-TadA * Based on the deoxyadenosine deaminase activity, TadA * This is an evolved form of TadA, an E. coli tRNA adenosine deaminase enzyme, which can convert adenosine on ssDNA to inosine. * This includes TadA-8a-e and TadA-7.10.

[0527] In addition to the base modification enzymes, further modifications were made to the base editor to improve the effectiveness, accuracy, and modularity of the editing process: - Addition of one or two uracil DNA glycosylase inhibitor domains (UGIs) to prevent the base excision repair mechanism that reverses the base edition. - Addition of Mu-GAM, which reduces insertion-deletion rates by inhibiting the non-homologous end joining mechanism (NHEJ) in cells. - Use of nickase-active Cas9 (nCas9 D10A) to promote repair by creating nicks on the unedited strand, thereby promoting the fixation of edited bases. - For example, the use of various Cas proteins (e.g., Cas12a) from different organisms, different PAM motifs or variants with different fidelity, or different families.

[0528] Non-limiting examples of DNA-based editor proteins include BE1, BE2, BE3, BE4, BE4-GAM, HF-BE3, Sniper-BE3, Target-AID, Target-AID-NG, ABE, EE-BE3, YE1-BE3, YE2-BE3, YEE-BE3, BE-PLUS, SaBE3, SaBE4, SaBE4-GAM, Sa(KKH)-BE3, VQR-BE3, VRER-BE3, EQR-BE3, xBE3, Cas12a-BE, Ea3A-BE3, A3A-BE3, TAM, CRISPR-X, ABE7.9, ABE7.10, and ABE7.10. * , xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, ABE8e, SpRY-ABE, SpRY-CBE, SpG-CBE4, SpG-ABE, SpRY-CBE4, SpCas9-NG-ABE, SpCas Contains 9-NG-CBE4, enAsBE1.1, enAsBE1.2, enAsBE1.3, enAsBE1.4, AsBE1.1, AsBE1.4, CRISPR-Abest, CRISPR-Cbest, eA3A-BE3, AncBE4.

[0529] The Citrous Anine Base Editor (CGBE) is as follows: - Cytosine deaminase (rAPOBEC) and base excision repair proteins (e.g., rXRCC1) (Precise and programmable C:G to G:C base editing in genomic DNA. Biorxiv (2020) (Non-patent document 34)), - Rat APOBEC1 variant (R33A) protein and Escherichia coli-derived uracil DNA N-glycosylase (eUNG) (Kurt, I et al., CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nature Biotechnology (2020) (Non-patent document 35)) It consists of nicasse CRISPR fused with [another ingredient]. - Cytosine adenine base editors (CABEs) consist of Cas9 nickase, cytidine deaminase (e.g., AID), and uracil DNA glycosylase (Ung). Zhao, D et al., New base editors change C to A in bacteria and C to G in mammalian cells. Nature Biotechnology (2020) (Non-patent document 36). - ACBE contains a nucleic acid programmed DNA-binding protein and adenine oxidase. Liu, D et al., A:T to C:G base editors and uses thereof. International Patent Application Publication No. 2020181180 (2020) (Patent Document 61). - ATBE consists of Cas9 nickase and one or more adenosine deaminase or oxidase domains. Liu, D et al., A:T to T:A base editing through adenine deamination and oxidation. International Patent Application Publication No. 2020181202 (2020) (Patent Document 65). - TABE consists of Cas9 niccasse and adenosine methyltransferase, thymine alkyltransferase, or adenosine deaminase domains. (Liu, D et al., T:A to A:T base editing through adenosine methylation. International Patent Application Publication No. 2020181193 (2020) (Patent Document 62); Liu, D et al., T:A to A:T base editing through thymine alkylation. International Patent Application Publication No. 2020181178 (2020) (Patent Document 63); Liu, D et al., T:A to A:T base editing through adenine excision. International Patent Application Publication No. 2020181195 (2020) (Patent Document 64)).

[0530] Base editor molecules may further consist of two or more of the aforementioned editor enzymes fused to a Cas protein (for example, a combination of ABE and CBE). These biomolecules are called dual base editors and enable the editing of two different bases (Grunewald, J et al., A dual-deaminase CRISPR base editor enables concurrent adenine and cytosine editing, Nature Biotechnology (2020) (Non-Patent Literature 37); Li, C et al., Targeted, random mutagenesis of plant genes with dual cytosine and adenine base editors, Nature Biotechnology (2020) (Non-Patent Literature 38)).

[0531] In one embodiment, a base editor is used to inactivate gene expression by editing one or more nucleotides involved in transcription or translation. In particular, the base editor targets one or more nucleotides of the promoter, RBS, or start codon.

[0532] In one embodiment, a base editor is used to introduce an immature stop codon.

[0533] In one embodiment, a base editor is used to introduce one or more rare codons.

[0534] In other embodiments, a base editor is used to regulate gene expression by editing one or more nucleotides involved in transcription or translation. In particular, the base editor targets one or more nucleotides of the promoter, RBS, or start codon, resulting in an increase or decrease in gene expression.

[0535] In other embodiments, a base editor is used to reverse mutations that result in inactivation, decreased activity, or increased activity of a gene or pathway.

[0536] In other embodiments, a base editor is used to reverse mutations that result in increased pathogenicity.

[0537] In one embodiment, a base editor is used to modify the regulation of a gene by editing one or more nucleotides involved in gene regulation, such as operator sequences, transcription factor binding sites, riboswitches, RNAse recognition sites, protease cleavage sites, methylation sites, and post-translational modification sites (phosphorylation, glycosylation, acetylation, pupyrulation, etc.).

[0538] RNA-based editing RNA base editing is based on the same principle as DNA base editing. That is, an enzyme that catalyzes the conversion of one RNA base to another needs to be brought near the target base in order to perform the conversion locally. To date, the only enzyme used for RNA editing is adenosine deaminase from the ADAR family, which converts adenosine to inosine in dsRNA structures. Several promising studies have used this specificity for dsRNA to fuse the ADAR deaminase domain (ADARDD) with antisense oligonucleotides to program localized RNA base editing. More recently, the ability of some CRISPR-Cas systems to bind RNA molecules has been repurposed for RNA editing. By using a catalytically dead Cas13b enzyme (dPspCas13b) fused with an overactive variant of the ADAR2 deaminase domain (ADAR2DD-E488Q for REPAIRv1 and ADAR2DD-E488Q-T375G for REPAIRv2), Cox et al. improved specificity and efficiency compared to previous RNA editing strategies.

[0539] Non-exclusive examples of RNA-based editors include REPAIRv1 and REPAIRv2.

[0540] In one embodiment, an RNA base editor is used to inactivate gene expression by editing one or more nucleotides involved in translation. In particular, the base editor targets one or more nucleotides in the 5'UTR, RBS, or start codon.

[0541] In one embodiment, an RNA base editor is used to introduce an immature stop codon.

[0542] In one embodiment, an RNA base editor is used to introduce one or more rare codons.

[0543] In other embodiments, RNA base editors are used to regulate gene expression by editing one or more nucleotides involved in translation. Specifically, base editors target one or more nucleotides in the 5'UTR, RBS, or start codon, resulting in increased or decreased gene expression.

[0544] In other embodiments, an RNA base editor is used to reverse mutations that result in the inactivation or reduced activity of a gene or pathway.

[0545] In other embodiments, a base editor is used to reverse mutations that result in increased pathogenicity.

[0546] Prime Edit The prime editor (PE) described in Anzalone et al. (Anzalone, AV et al., Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, pp. 149-157 (2019) (Non-Patent Literature 39)), which is incorporated by reference herein, consists of Cas9 fused with reverse transcriptase, used in combination with prime editing RNA (pegRNA; guide RNA containing the template region of reverse transcriptase).

[0547] Prime editing enables insertions, deletions (indels), and 12 base-to-base conversions. Prime editing relies on the ability of reverse transcriptase (RT) fused with a Cas nickase variant to convert the RNA sequence provided by prime editing guide RNA (pegRNA) into DNA at the nick site generated by the Cas protein. The DNA flap generated by this process is then either included in or excluded from the target DNA sequence.

[0548] The Prime editing system is: - Cas nickerse variants, such as Cas9-H840A, fused with a reverse transcriptase domain, e.g., M-MLV RT, or its variants (M-MLV RT(D200N), M-MLV RT(D200N / L603W), M-MLV RT(D200N / L603W / T330P / T306K / W313F)). - Prime Editing Guide RNA (pegRNA) Includes.

[0549] To facilitate editing, the prime editing system may ideally include the expression of additional sgRNA that directs Cas nuclease activity to the unedited DNA strand only after the edited strand flap has been resolved, by designing it to anneal with the edited strand but not with the original strand.

[0550] Non-limiting examples of prime editing systems include PE1, PE1-M1, PE1-M2, PE1-M3, PE1-M6, PE1-M15, PE1-M3inv, PE2, PE3, PE3b, "CRISPEY" (Cas9 Retron precISe Parallel Editing via homologY), retron RNA fused with sgRNA and expressed together with Cas9, and retron proteins containing at least reverse transcriptase (Sharon, E. et al., Functional Genetic Variants Revealed by Massively Parallel Precise Genome Editing. Cell 175, pp. 544-557. e16 (2018) (Non-patent Literature 40)), and the SCRIBE strategy: a retron system expressed in combination with a recombinase that promotes single-stranded DNA recombination, also known as single-stranded annealing protein (SSAP) 12.Such recombinases include phage recombinases, such as lambdared, recET, Sak, Sak4, and the recently described SSAP in Wannier et al. (Wannier, TM et al., Improved bacterial recombineering by parallelized protein discovery. Biorxiv 2020.01.14.906594(2020) doi:10.1101 / 2020.01.14.906594. (Non-Patent Literature 41)), the Group II introns described in Karberg et al. (Karberg, M. et al., Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol 19, pp. 1162-1167 (2001) (Non-Patent Literature 42)), targetron systems adapted to many bacterial species, and Simon et al. (Simon, AJ, Ellington, AD & Finkelstein, IJ, Retrons and their applications in genome engineering). This includes, but is not limited to, other retrons based on gene targeting approaches, as described in Nucleic Acids Res 47, pp. 11007-11019 (2019) (Non-Patent Literature 43).

[0551] In one embodiment, a prime editing system is used to inactivate gene expression by substituting, deleting, or inserting one or more nucleotides involved in transcription or translation. Specifically, the prime editing system substitutes, deletes, or inserts one or more nucleotides in the promoter, RBS, or coding sequence.

[0552] In one embodiment, a prime editing system is used to introduce one or more immature stop codons.

[0553] In one embodiment, a prime editing system is used to introduce one or more rare codons.

[0554] In one embodiment, a prime editing system is used to introduce or delete nucleotides and induce a frameshift in the reading frame.

[0555] In other embodiments, a prime editing system is used to regulate gene expression by substituting, deleting, or inserting one or more nucleotides involved in transcription or translation. Specifically, the prime editing system substitutes, deletes, or inserts one or more nucleotides in the promoter, RBS, or start codon, resulting in increased or decreased gene expression.

[0556] In other embodiments, a prime editing system is used to reverse mutations that result in the inactivation or reduced activity of a gene or pathway.

[0557] In other embodiments, a prime editing system is used to reverse mutations that result in increased pathogenicity.

[0558] The present invention includes the following embodiments. 1. Recombinant acne bacterium phage. 2. The recombinant Propionibacterium acnes phage according to Embodiment 1, comprising at least one transgene. 3. The recombinant acne bacterium phage according to Embodiment 2, wherein the introduced gene is the CRISPR-Cas system or a part of the CRISPR-Cas system. 4. The recombinant Propionibacterium acnes phage according to Embodiment 1, wherein the host range is different from that of the corresponding wild-type Propionibacterium acnes phage. 5. Recombinant Propionibacterium acnes phage according to Embodiment 1, comprising a manipulative capsid. 6. Recombinant Propionibacterium acnes phage according to Embodiment 5, wherein the antigen is displayed on the surface of the operational capsid. 7. The following: - DNA template suitable for homologous recombination with the acne bacterium phage genome - A replication origin that enables replication within the acne bacteria; and - In some cases, a first selection marker that allows for the selection of DNA vectors in *Propionibacterium acnes*; Propionibacterium acnes cells carrying recombinant DNA vectors containing [the specified ingredient]. 8. The Propionibacterium acnes cell according to Embodiment 7, wherein the Propionibacterium acnes phage genome is introduced into the cell. 9. The Propionibacterium acnes cell according to Embodiment 7, wherein the Propionibacterium acnes phage genome recombinates with a DNA vector to produce recombinant phages. 10. Below: - A replication origin that enables replication within the acne bacteria; - A CRISPR-Cas system that is expressed within the aforementioned Propionibacterium acnes but does not target the newly generated recombinant Propionibacterium acnes phage genome, and - In some cases, a first selection marker that enables the selection of DNA vectors in Propionibacterium acnes. Propionibacterium acnes cells carrying a DNA vector for the selective production of recombinant Propionibacterium acnes phages, including [specific component]. 11. A method for producing a phage lysate containing wild-type acne bacilli or parent acne bacilli and recombinant acne bacilli, comprising introducing a wild-type acne bacilli phage genome or parent acne bacilli phage genome into acne bacilli cells as described in Embodiment 7. 12. A method for selecting recombinant acne bacilli phage, comprising mixing a phage lysate containing wild-type acne bacilli phage or parent acne bacilli phage and recombinant acne bacilli phage with acne bacilli cells as described in Embodiment 10, thereby resulting in the selective production of recombinant acne bacilli phage. 13. A method for treating a disorder or disease related to Propionibacterium acnes, comprising the step of administering to a subject a recombinant Propionibacterium acnes phage described in any one of Embodiments 1 to 6, or a recombinant Propionibacterium acnes phage obtained by the method described in Embodiment 11 or 12. 14. - A replication origin that enables replication within *Propionibacterium acnes*; - Depending on the circumstances, a selection marker that enables the selection of DNA vectors in Propionibacterium acnes, and - Target gene Recombinant DNA vector containing [the specified ingredient]. 15. The DNA vector according to Embodiment 14, further comprising oriT, which enables conjugation into Propionibacterium acnes; a replication origin that enables replication within the donor organism; and a second selection marker that enables selection within the donor organism. 16. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is R6K (typically the sequence of SEQ ID NO: 42). 17. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is RK2 (typically the sequence of SEQ ID NO: 43). 18. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pBBR1 (typically the sequence of SEQ ID NO: 44). 19. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pRO1600 (typically the sequence of SEQ ID NO: 45). 20. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is RSF1010 (typically the sequence of SEQ ID NO: 46). 21. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pAMβ1 (typically the sequence of SEQ ID NO: 47). 22. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pLME106 (typically the sequence of SEQ ID NO: 48). 23. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pTZC1 (typically the sequence of SEQ ID NO: 49). 24. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pBC1 (typically the sequence of SEQ ID NO: 50). 25. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pEP2 (typically the sequence of SEQ ID NO: 51). 26. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pWVO1 (typically the sequence of SEQ ID NO: 52). 27. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pAP1 (typically the sequence of SEQ ID NO: 53). 28. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pWKS1 (typically the sequence of SEQ ID NO: 54). 29. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pLME108 (typically the sequence of SEQ ID NO: 55). 30. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pLS1 (typically the sequence of SEQ ID NO: 56). 31. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pUB6060 (typically the sequence of SEQ ID NO: 57). 32. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is p545 (typically the sequence of SEQ ID NO: 58). 33. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pJD4 (typically the sequence of SEQ ID NO: 59). 34. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pIJ101 (typically the sequence of SEQ ID NO: 60). 35. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pSN22 (typically the sequence of SEQ ID NO: 61). 36. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pGP01 (typically the sequence of SEQ ID NO: 62). 37. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pIP501 (typically the sequence of SEQ ID NO: 63). 38. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pCU1 (typically the sequence of SEQ ID NO: 64). 39. The DNA vector according to Embodiment 14 or 15, wherein the origin of replication that enables replication within Propionibacterium acnes is pBAV1K-T5 (typically the sequence of SEQ ID NO: 65). 40. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pMRC01 (typically the sequence of Sequence ID No. 1). 41. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_RSF1010 (typically the sequence of Sequence ID No. 2). 42. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pRS01 (typically the sequence of Sequence ID No. 3). 43. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pMV158 (typically the sequence of Sequence ID No. 4). 44. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pTF1 (typically the sequence of Sequence ID No. 5). 45. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pSC101 (typically the sequence of Sequence ID No. 6). 46. ​​A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pBTK445 (typically the sequence of SEQ ID NO: 7). 47. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pBBR1 (typically the sequence of Sequence ID No. 8). 48. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R721 (typically the sequence of Sequence ID No. 9). 49. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pRmeGR4a (typically the sequence of Sequence ID No. 10). 50. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_ColE1 (typically the sequence of SEQ ID NO: 11). 51. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pTiC58 (typically the sequence of SEQ ID NO: 12). 52. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pMdT1 (typically the sequence of Sequence ID No. 13). 53. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R1 (typically the sequence of SEQ ID NO: 14). 54. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_Tn5520 (typically the sequence of SEQ ID NO: 15). 55. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_QKH54 (typically the sequence of SEQ ID NO: 16). 56. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R64 (typically the sequence of SEQ ID NO: 17). 57. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R751 (typically the sequence of Sequence ID No. 18). 58. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_RP4 (typically the sequence of SEQ ID NO: 19). 59. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pKL1 (typically the sequence of Sequence ID No. 20). 60. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_RK2 (typically the sequence of SEQ ID NO: 21). 61. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R1162 (typically the sequence of Sequence ID No. 22). 62. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_Tn4555 (typically the sequence of SEQ ID NO: 23). 63. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pHT (typically the sequence of SEQ ID NO: 24). 64. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_Tn4399 (typically the sequence of SEQ ID NO: 25). 65. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_Tn916 (typically the sequence of SEQ ID NO: 26). 66. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pST12 (typically the sequence of Sequence ID No. 27). 67. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pCU1 (typically the sequence of Sequence ID No. 28). 68. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pSU233 (typically the sequence of Sequence ID No. 29). 69. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_F (typically the sequence of SEQ ID NO: 30). 70. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pMAB01 (typically the sequence of Sequence ID No. 31). 71. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R388 (typically the sequence of Sequence ID No. 32). 72. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pS7a (typically the sequence of Sequence ID No. 33). 73. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pS7b (typically the sequence of Sequence ID No. 34). 74. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R702 (typically the sequence of Sequence ID No. 35). 75. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pMUR274 (typically the sequence of Sequence ID No. 36). 76. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_R100 (typically the sequence of Sequence ID No. 37). 77. A DNA vector according to any one of Embodiments 15 to 39, wherein oriT is oriT_pVCR94deltaX (typi...

Claims

1. A recombinant acne bacterium phage comprising at least one transgene that is foreign to acne bacterium.

2. The recombinant acne bacterium phage according to claim 1, wherein the introduced gene is a CRISPR-Cas system or a part of a CRISPR-Cas system, or a prime or base editing system.

3. The recombinant acne bacterium phage according to claim 1, wherein the introduced gene encodes an antigen, a human protein, or a therapeutic protein.

4. The recombinant acne bacterium phage according to claim 3, wherein the human or therapeutic protein is an interleukin.

5. A recombinant acne bacterium phage according to any one of claims 1 to 4, wherein the host range of the recombinant phage is different from that of the corresponding wild-type acne bacterium phage.

6. Recombinant acne phage according to any one of claims 1 to 5, comprising a manipulative capsid.

7. The recombinant acne bacterium phage according to claim 6, wherein the antigen is displayed on the surface of the manipulated capsid.

8. Propionibacterium acnes cells comprising a recombinant DNA vector, wherein the vector is: - A DNA template suitable for homologous recombination with wild-type or parental *Propionibacterium acnes* phage genome, comprising an upstream homology arm and a downstream homology arm, - At least one transgene that is foreign to *Propionibacterium acnes*, and is sandwiched between the upstream homology arm and the downstream homology arm, - A replication origin that enables replication within the acne bacteria, and - In some cases, a first selection marker that enables the selection of DNA vectors in Propionibacterium acnes. Including acne bacteria cells.

9. A Propionibacterium acnes cell that produces a phage lysate containing a wild-type or parental Propionibacterium acnes phage and a recombinant Propionibacterium acnes phage according to any one of claims 1 to 7, wherein the cell is obtained by contacting the Propionibacterium acnes cell according to claim 8 with a wild-type or parental Propionibacterium acnes phage, and the wild-type or parental Propionibacterium acnes phage genome is recombined with a DNA template for homologous recombination of the DNA vector of the Propionibacterium acnes cell.

10. A method for producing a phage lysate containing a wild-type or parental acne bacterium phage and a recombinant acne bacterium phage according to any one of claims 1 to 7, wherein the method comprises the step of contacting a wild-type or parental acne bacterium phage genome with an acne bacterium cell according to claim 8, wherein the wild-type or parental acne bacterium phage genome is recombined with a DNA template for homologous recombination of the acne bacterium cell's DNA vector.

11. A Propionibacterium acnes cell comprising a DNA vector useful for the selective production of recombinant Propionibacterium acnes phage according to any one of claims 1 to 7, wherein the vector is: - A replication origin that enables replication within the acne bacteria, - A CRISPR-Cas system expressible within the aforementioned Propionibacterium acnes, wherein the system targets the wild-type or parental Propionibacterium acnes phage genome as defined in claim 9 or 10, but does not target the genome of the recombinant Propionibacterium acnes phage described in any one of claims 1 to 6, and - In some cases, a first selection marker that enables the selection of DNA vectors in Propionibacterium acnes. Including acne bacteria cells.

12. A Propionibacterium acnes cell that selectively produces recombinant Propionibacterium acnes phage according to any one of claims 1 to 7, the cell obtained by contacting the Propionibacterium acnes cell according to claim 11 with a phage lysate obtained by the method of claim 10.

13. A method for selectively producing recombinant acne bacilli phage according to any one of claims 1 to 7, comprising the step of contacting a phage lysate obtained by the method of claim 10 with acne bacilli cells according to claim 11.

14. A composition useful for treating acne-related disorders or diseases, comprising acne bacteria cells as described in Claim 12, or recombinant acne bacteria phage as described in any one of Claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in Claim 13.

15. A vaccine and / or immunogenic composition comprising acne bacteria cells as described in Claim 12, or recombinant acne bacteria phage as described in any one of Claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in Claim 13, wherein the introduced gene encodes an antigen.

16. A composition for preventing and / or treating cancer in a subject requiring prevention and / or treatment of cancer, comprising acne bacteria cells as described in claim 12, or recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the introduced gene encodes a tumor antigen.

17. A composition for preventing and / or treating viral infection in a subject requiring prevention and / or treatment of viral infection, comprising acne bacteria cells as described in claim 12, recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the introduced gene encodes a viral antigen.

18. A composition for preventing and / or treating bacterial infection in a subject requiring prevention and / or treatment of bacterial infection, comprising acne bacteria cells as described in claim 12, or recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the transgene encodes a bacterial antigen.

19. A composition for preventing and / or treating fungal infections in subjects requiring prevention and / or treatment of fungal infections, comprising acne bacteria cells as described in claim 12, recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the transgene encodes a fungal antigen.

20. A composition for preventing and / or treating an autoimmune disease in a subject requiring prevention and / or treatment of an autoimmune disease, comprising acne bacteria cells as described in claim 12, or recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the introduced gene encodes an autoantigen.

21. A composition for preventing and / or treating allergies in subjects requiring prevention and / or treatment of allergies, comprising acne bacteria cells as described in claim 12, recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the introduced gene encodes an allergen.

22. A composition for preventing and / or treating graft rejection in subjects requiring prevention and / or treatment of graft rejection, comprising acne bacteria cells as described in claim 12, or recombinant acne bacteria phage as described in any one of claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in claim 13, wherein the introduced gene encodes a graft-specific antigen.

23. A composition for preventing and / or treating a skin disease in a subject requiring prevention and / or treatment of a skin disease, comprising acne bacteria cells as described in Claim 12, or recombinant acne bacteria phage as described in any one of Claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in Claim 13, wherein the introduced gene encodes a human or therapeutic protein.

24. The composition for preventing and / or treating a skin disease according to claim 23, wherein the human or therapeutic protein is an interleukin.

25. A composition for preventing and / or treating an inflammatory disease in a subject requiring prevention and / or treatment of an inflammatory disease, comprising acne bacteria cells as described in Claim 12, or recombinant acne bacteria phage as described in any one of Claims 1 to 7, or recombinant acne bacteria phage obtained by the method described in Claim 13, wherein the transgene encodes a human or therapeutic protein.

26. The composition for preventing and / or treating an inflammatory disease according to claim 25, wherein the human or therapeutic protein is an interleukin.

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