Synthetic viruses and their provision

Abstract

The present invention relates to a method for the in vitro amplification of linear viral genomes, in particular bacteriophages, a method for the expression and self-assembly of viruses, in particular bacteriophages, in a cell-free expression system, and the viruses or bacteriophages provided by such a method. A further aspect of the invention relates to synthetic bacteriophages and their uses. According to the invention, the production of synthetic viruses, in particular bacteriophages, can be performed entirely in vitro. The process is based on the combination of an in vitro method for propagating nucleic acids, in particular DNA, optionally also modified in the amplified nucleic acids, and an in vitro method for the complete in vitro production of bacteriophages or viruses by cell-free protein expression in cell extracts or lysates.

Description

[Technical field]

[0001] The present invention relates to methods for the in vitro amplification of viral nucleic acids, particularly bacteriophage genomes, methods for the expression of nucleic acids and self-assembly of viruses, particularly bacteriophages, in cell-free expression systems, and viruses or bacteriophages provided by such methods. Further aspects of the invention relate to synthetic bacteriophages and uses thereof. [Background technology]

[0002] Bacteriophages (hereinafter also referred to as "phages") are specifically viruses that infect a host bacterium and grow at the expense of this host. They are composed of proteins that encapsulate a DNA or RNA genome. Bacteriophages replicate within the host bacterium by injecting their viral genetic material into the host cell, effectively taking over cellular functions for the production of progeny bacteriophage, causing rupture of the cell wall and subsequent bacterial cell death.

[0003] The biotechnological field of applications of bacteriophages is very broad, ranging from evolution-based selection methods such as the evolutionary improvement of the activity of enzymes and other proteins, to phage display that can generate and optimize biopharmaceutical components such as therapeutic antibodies, to the application of bacteriophages themselves as alternatives to antibiotics.

[0004] The latter use is based on the natural properties of bacteriophages, specifically affecting bacteria, especially pathogenic bacteria, causing them to die, ie, lyse.

[0005] Such approaches, which have been applied for a longer time against microbial infections, are of increasing interest due to the strong increase in the number and spread of multidrug-resistant bacterial strains worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) bacteria, for example, are an increasingly common form of infection, usually acquired by transmission within hospitals. MRSA infections are very difficult to treat using conventional antibiotics. The development of new antibiotics is significantly slower than this development.

[0006] Bacteriophages in the food industry already play an important role and are applied in this sector, for example to detect and destroy Listeria monocytogenes in food.

[0007] However, the development of phage-based therapeutics and diagnostics is hindered by problems in the production and / or genetic modification of bacteriophages, the complexity of which is primarily due to the difficulty of altering the bacteriophage genome.

[0008] Furthermore, the development and production of phage-based therapies and diagnostics is still hindered by the difficulty of easy and safe production methods for bacteriophages. Up to now, bacteriophages are generally produced by in vivo culture using suitable bacteria. Depending on the field of application, the bacteria used may be pathogens. This requires compliance with the appropriate safety regulations for each bacterium and the possibility of culturing them. Moreover, the purification of bacteriophages produced by such in vivo culture has turned out to be difficult and complicated.

[0009] Two major aspects of the production of these bacteriophages, namely the cumbersome modification and the easy and safe provision, are both simultaneously addressed by the methods and synthetic bacteriophages of the present invention. Summary of the Invention

[0010] According to the present invention, the production of synthetic viruses, in particular bacteriophages, can be carried out completely in vitro. The process is based on the combination of an in vitro method for propagating nucleic acids, in particular DNA, where the amplified nucleic acid can also be optionally modified, and an in vitro method for the complete in vitro production of bacteriophages or viruses by cell-free protein expression in cell extracts or lysates. Such an inventive completely in vitro method may be divided into two sub-embodiments or methods.

[0011] Thus, a first aspect of the invention preferably relates to a method for the in vitro amplification of linear, viral or bacteriophage nucleic acid, in particular a bacteriophage genome, comprising the steps of: (a) providing a viral or bacteriophage nucleic acid template to be amplified; (b) amplifying said viral or bacteriophage nucleic acid template using the polymerase chain reaction to amplify different nucleic acid segments, wherein a primer used at the 5' end of at least one of said viral or bacteriophage nucleic acid templates optionally introduces a nucleic acid exonuclease inhibitor modification, in particular a phosphorothioate bond, between the first nucleotide at the 5' end of at least one of said corresponding nucleic acid segments; (c) recession by a 5'-3' exonuclease, in particular T5 exonuclease, to provide a sticky-ended nucleic acid segment; (d) annealing the sticky-end nucleic acid segments; (e) ligating the annealed nucleic acid segments to provide an amplified linear viral or bacteriophage nucleic acid; A preferred embodiment of the present invention is the stepwise / sequential assembly of viral or bacteriophage nucleic acids, particularly phage genomes.

[0012] According to such sequential assembly of a phage genome, different sections of the phage genome may be provided by parallel amplification / assembly reactions comprising steps (a)-(e), and the products of such parallel reactions are combined and assembled to provide a complete, optionally modified, phage genome.

[0013] According to a preferred embodiment, one or more nucleic acids, in particular genome sections, are assembled in a first assembly cycle (Gibson assembly) comprising steps (a) to (e) (where genome modifications are introduced, if necessary). The provided nucleic acids may be amplified by PCR in said steps. The nucleic acids, in particular phage genome sections, are then assembled in a second assembly cycle step (Gibson assembly) to form the desired complete phage genome (optionally modified) which can be used in a cell-free reaction.

[0014] In such an embodiment, in particular a method for providing a modified viral or bacteriophage genome, comprises the steps of: (a) providing a viral or bacteriophage nucleic acid template to be amplified, in particular a portion of a bacteriophage genome; (b) amplifying said viral or bacteriophage nucleic acid template using the polymerase chain reaction to amplify different nucleic acid segments, wherein a primer used at the 5' end of at least one of said viral or bacteriophage nucleic acid templates optionally introduces a nucleic acid exonuclease inhibitor modification, in particular a phosphorothioate bond, between the first nucleotide at the 5' end of at least one of said corresponding nucleic acid segments; (c) invaginating the nucleic acid segment with a 5'-3' exonuclease, in particular T5 exonuclease, to provide a sticky-ended nucleic acid segment; (d) annealing the sticky-end nucleic acid segments; (e) ligating the annealed nucleic acid segments to provide an amplified linear viral or bacteriophage nucleic acid, thereby providing a first set of linear viral or bacteriophage nucleic acid, in particular a first set of linear viral or bacteriophage nucleic acid sections; Optionally, a step (f) of further amplifying the first set of linear viral or bacteriophage nucleic acids, in particular the first set of linear viral or bacteriophage nucleic acid sections, (f) combining the first set of linear viral or bacteriophage nucleic acids with at least a second set of linear viral or bacteriophage nucleic acids, in particular combining the first set of linear viral or bacteriophage nucleic acid sections with at least a second set of linear viral or bacteriophage nucleic acid sections; (h) performing Gibson assembly on the combined first set of linear viral or bacteriophage nucleic acids and at least a second set of linear viral or bacteriophage nucleic acids, in particular the first set of linear viral or bacteriophage nucleic acid sections and at least a second set of linear viral or bacteriophage nucleic acid sections, in particular repeating steps (a) to (e) to provide a bacteriophage genome; Includes.

[0015] According to a preferred embodiment, the combined first set of linear viral or bacteriophage nucleic acids and at least a second set of linear viral or bacteriophage nucleic acids, in particular the first set of linear viral or bacteriophage nucleic acid sections and the at least a second set of linear viral or bacteriophage nucleic acid sections, together form, i.e. encode, a bacteriophage genome. The linear viral nucleic acids, in particular the linear viral or bacteriophage nucleic acid sections, in particular the first and at least a second set of bacteriophage nucleic acid sections, can be derived from different bacteriophages and / or comprise synthetic nucleic acid sequences, thereby providing a modified bacteriophage genome consisting of genes of different wild-type bacteriophages and / or synthetic sequences, e.g. a T7 phage with a T3 tail fiber.

[0016] Bacteriophages according to the invention are viruses that infect and replicate in host bacteria and / or archaea. Bacteriophages contain proteins that encapsulate a DNA or RNA genome. They replicate in the host bacteria after injection of the genome into the cytoplasm. The terms "bacteriophage" and "phage" may be applied interchangeably herein. As used herein, the term "genome" is understood by those skilled in the art. It refers to all the genetic information of a virus or bacteriophage and consists of a DNA or RNA nucleotide sequence. It includes protein-coding genes and, optionally, non-coding genes and other functional regions, such as regulatory sequences, i.e. non-coding nucleotide sequences. The genome may be modified, in particular genes and / or parts of genes may be knocked out and / or deleted and / or non-wild-type genes may be added, compared to the corresponding wild-type genome.

[0017] According to a preferred embodiment of the invention, the viral nucleic acid to be amplified, i.e. the template, may be a filamentous bacteriophage nucleic acid, in particular a bacteriophage genome. The genome to be amplified according to the invention is preferably a linear genome. The amplified bacteriophage genome may be modified.

[0018] The nucleic acid template to be amplified may be selected from the group consisting of DNA, RNA, in particular double-stranded DNA or double-stranded RNA, and variants thereof. In particular, "variants" include conservatively modified nucleic acids (e.g., degenerate codon substitutions) and complementary sequences.

[0019] Phages are classified by the International Committee on Taxonomy of Viruses based on morphology and nucleic acid, including dsDNA phages, ssDNA phages, dsRNA phages, and ssRNA phages. The present invention is not limited to a class of phages. However, dsDNA phages are particularly preferred. Preferred dsDNA phages include Ackermanviridae, Autographiviridae, Chaseviridae, Demerecviridae, Drexlerviridae, Herelleviridae, Myoviridae, Podoviridae, Siphoviridae, Tectiviridae, Corticoviridae, Plasmaviridae, Liposthrixviridae, Rudiviridae, Fuselloviridae, Halspiviridae, Guttaviridae, Bycaudaviridae, Thaspiviridae, and Autolykiviridae. According to the present invention, bacteriophages are a preferred embodiment of the virus.

[0020] According to step (a), a viral or bacteriophage nucleic acid template to be amplified is provided. Methods for providing such a nucleic acid template or for providing and / or isolating the corresponding nucleic acid are well established in the art and will be familiar to the skilled artisan.

[0021] Amplifying a nucleic acid template by polymerase chain reaction (PCR) according to step (b) is a commonly established technique.

[0022] Each primer needs to be provided according to the template. Primer design procedures are well known to those skilled in the art. Various biotech companies provide tailor-made primers for every molecular biology application and / or template-specific requirement. In principle, primers can be designed so that the template nucleic acid, in particular the genome to be amplified, is amplified completely and / or the individual nucleic acid segments provided are of substantially equal nucleic acid length. Of course, the individual nucleic acid segments provided may differ in nucleic acid length, in particular when introducing site-specific mutations such as insertions, deletions and / or point mutations.

[0023] Preferably, the size of such a nucleic acid segment is in the range of about 5 kbp to about 30 kbp, preferably about 5 kbp to about 25 kbp, about 5 kbp to about 15 kbp, and particularly preferably about 10 kbp. As understood herein, "segment" and "section" describe a nucleic acid present at a certain step of the method of the invention. Typically, such a "segment" and / or "section" refers to a portion of an optionally modified viral or bacteriophage genome and / or gene.

[0024] The primers used at least at the 5' end of the template nucleic acid introduce a nucleic acid exonuclease inhibitor modification, particularly a backbone modification, and preferably introduce a corresponding modification at the 5' end of one of the templates.

[0025] According to the present invention, DNA exonuclease inhibitory modifications, especially backbone modifications, are modifications that lead to nuclease resistance, especially resistance to 5' exonucleases. In general, such modifications can be distinguished between backbone, terminal and internal base modifications. Only a small selection of backbone modifications is available, with phosphorothioate bonds (PTO bonds), the introduction of locked nucleic acids (LNA) and / or aliphatic spacers being examples of backbone modifications. Terminal modifications at the 5' end, most commonly fluorescent dyes or linker groups, and / or internal base modifications at 2'O exemplify terminal and internal base modifications, especially in RNA and / or mixed oligonucleotides. The use of backbone modifications is preferred.

[0026] Therefore, a preferred example of the main chain modification according to the present invention is a phosphorothioate bond (PTO bond).

[0027] It is preferred that the first 1 to 15, more preferably 1 to 10, even more preferably 1 to 8, and even more preferably 1 to 5 of the first 5' nucleotides and / or corresponding bonds of each primer are modified. According to another preferred embodiment of the invention, 5 to 20, more preferably 5 to 18, and even more preferably 5 to 15 of the first 20 5' nucleotides and / or corresponding bonds of each primer are modified, or 5 to 15, more preferably 5 to 12 of the first 15 5' nucleotides and / or corresponding bonds of each primer are modified.

[0028] Each oligonucleotide is applied as a primer to insert a DNA exonuclease inhibitor modification to prevent exonuclease degradation at each 5' end. The introduction of such modifications can prevent unwanted circularization of linear DNA fragments, particularly in the ligation step (e).

[0029] Therefore, the phosphodiester bond of the first 5' nucleotide of the primer may be replaced by a phosphorothioate bond (PTO bond). During PCR, they are inserted into the DNA fragment and prevent the added 5' exonucleases, especially T5 exonuclease, from producing overhangs at those positions necessary for ligation. This is a surprising result, since 5' exonucleases are not normally stopped by thiophosphates in the DNA backbone.

[0030] Therefore, a particularly preferred embodiment of DNA exonuclease inhibitory modification, i.e., circular inhibitory backbone modification, is based on the introduction of phosphorothioates, i.e., the introduction of phosphorothioate bonds (PTO bonds). DNA phosphorothioate modification is a modification to the DNA backbone, in which non-bridging oxygen atoms are replaced with sulfur atoms. Each phosphorothioate modified DNA may be provided by using phosphorothioate containing primers in a PCR reaction. Each phosphorothioate containing primer may be provided by using phosphorothioate oligonucleotides in which non-bridging oxygen atoms in the DNA backbone are replaced with sulfur atoms when producing and / or providing each primer.

[0031] It is preferred that the first 1 to 20, more preferably 8 to 20, more preferably 10 to 20, and even more preferably 10 to 15 phosphodiester bonds of the first 5' nucleotide of each primer may be replaced with phosphorothioate bonds. According to another preferred embodiment of the present invention, 5 to 20, more preferably 5 to 18, and more preferably 8 to 15 phosphodiester bonds of the first 20 5' nucleotides of each primer may be replaced, or 8 to 15, more preferably 8 to 12 phosphodiester bonds of the first 15 5' nucleotides of each primer may be replaced.

[0032] The 5'-3' exonuclease of step (c) may be any enzyme that acts by cleaving nucleotides from the 5' end (exo) of the amplified nucleic acid template of step (b). A hydrolysis reaction occurs that breaks the phosphodiester bond at the 5' end. The terms "5'-3' exonuclease" and "5' exonuclease" may be used interchangeably herein.

[0033] The action of the 5' exonuclease provides a 3' sticky-ended nucleic acid segment that exhibits a 3' nucleotide overhang.

[0034] Examples of suitable 5' exonucleases include T7 exonuclease, exonuclease VIII (truncated), lambda exonuclease, T5 exonuclease, and any mixture thereof. A particularly preferred 5' exonuclease is T5 exonuclease.

[0035] When backbone modifications are used in the amplification step (b), the use of T5 exonuclease and / or lambda exonuclease is preferred, and when nucleobase modifications are used, the use of T7 exonuclease is preferred.

[0036] The provided sticky-end nucleic acid segments are annealed in step (d). The annealing conditions, particularly the temperature, depend on the template to be amplified and can be determined by those skilled in the art. The stoichiometric ratio of the annealed sticky-end nucleic acid segments is preferably 1:1 to 1:5, particularly about 1:1.

[0037] After annealing according to step (d), a polymerase, in particular a DNA polymerase such as Taq polymerase, may fill in nucleotide gaps between the annealed nucleic acid segments with free nucleotides prior to ligation according to step (e).

[0038] The annealed sticky-ended nucleic acid segments are ligated according to step (e) to provide an amplified linear nucleic acid, particularly a bacteriophage genome, in which a ligase enzyme is utilized to link the 3' hydroxyl terminus of one nucleic acid segment to the 5' phosphate terminus of another nucleic acid segment by forming a phosphodiester bond.

[0039] According to a preferred embodiment, steps (a)-(e) can be carried out in a single tube reaction without isolation and / or purification of any intermediates, in particular without the use of a one-step masterbatch of the enzyme.

[0040] The above method comprising steps (a) to (e) is based on the so-called "Gibson assembly", an in vitro enzymatic reaction known to link two or more DNA fragments with overlapping sequences at their ends. The basis of the Gibson assembly method is the production of DNA fragments by PCR, where the primers are selected such that the DNA fragments have overlapping sequences. The fragments are then joined by the combining activity of three enzymes. An exonuclease, preferably T5 exonuclease, produces a 3' overhang that hybridizes with the complementary strand of the complementary sequence of the adjacent strand. A DNA polymerase fills the generated gap and a ligase covalently joins the DNA. The method can be used for site-directed mutagenesis to introduce site-specific mutations such as insertions, deletions and / or point mutations.

[0041] However, there is a problem with the known Gibson assembly method. Many phage genomes have identical sequences at both ends of the genome that are joined by Gibson assembly, producing circular genomes and making in vitro propagation of linear DNA impossible. To prevent circularization of the ends of the genome, which often contain overlapping sequences, primers at the ends of the genome are modified.

[0042] This problem is particularly solved by the present invention by applying to at least one 5' end of a viral or bacteriophage nucleic acid template a primer into which an exonuclease degradation inhibiting modification is inserted.

[0043] When a nucleic acid template, particularly a linear genome, is modified, the template may be previously methylated in a separate step to distinguish it from any new products, i.e., the amplified modified nucleic acid segments. In step (d), after amplifying the template DNA via PCR, the methylated template DNA is removed by a methylation-sensitive restriction enzyme. The respective enzymes are known to those skilled in the art and include, for example, Dpnl.

[0044] By this method, any false positive viruses due to the modification, in particular false positive phages due to the modification, can be reduced to a minimum that would be generated based on unmodified DNA. The method can be applied to any suitable modification, such as deletions, insertions and / or mutations, in particular point mutations.

[0045] Thus, according to a preferred embodiment, the method of the invention comprises a step of methylating the nucleic acid template prior to the amplification step (b) and / or a step of digesting the methylated nucleic acid template after the amplification step (b).

[0046] A second aspect of the present invention is (a) providing a viral or bacteriophage nucleic acid, in particular a bacteriophage genome; (b) providing a cell-free expression system and optionally adding at least one protein and / or a nucleic acid encoding a protein that enhances or improves the reaction, preferably selected from the group consisting of cofactors, chaperones, polymerases, transcription regulators and / or any mixtures thereof; (c) combining said viral or bacteriophage nucleic acid with said cell-free expression system; (d) incubating the combined bacteriophage nucleic acid and cell-free expression system under conditions suitable for expression and self-assembly of the bacteriophage; The present invention relates to a method for expressing and self-assembling a virus or bacteriophage in a cell-free expression system, comprising:

[0047] According to particularly preferred embodiments, viral or bacteriophage nucleic acids or genomes are provided by the methods described herein.

[0048] As understood herein, a "cell-free expression system" includes the complete transcription and translation machinery for the transcription and expression of a viral or bacteriophage nucleic acid, particularly a bacteriophage genome. The term "cell-free" is understood by those of skill in the art and refers to "substantially free."

[0049] A preferred example of a cell-free expression system is a cell lysate. By using such a cell lysate, several proteins or metabolites can be synthesized simultaneously.

[0050] A "cell lysate" according to the present invention refers to a fluid containing cellular components derived from cells after lysis. Lysis methods are known to those skilled in the art and include, for example, disrupting the membrane of a cell by viruses, enzymes, or osmotic mechanisms that compromise its integrity. Such a cell lysate is substantially free of intact cells, i.e., acellular. The terms "cell lysate" and "cell extract" can be used interchangeably herein.

[0051] After purification, this lysate is free of host DNA and allows the expression of the desired protein by exogenous addition of DNA, in particular an amplified bacteriophage genome, as described herein.The cell lysate used is therefore preferably free of nucleic acids, in particular DNA and / or membranes, from the cells from which it is derived.

[0052] The cell lysate used according to the invention may be derived from a microorganism, in particular a pathogenic bacterium, such as E. coli, yeast, insect, mammal and / or plant, in particular, for example, wheat or rice, or may even be artificial. "Microorganism" refers to bacteria or archaea. Preferably, the microorganism is a bacterium.

[0053] Several cell-free expression systems are known to those skilled in the art and may be applied according to the present invention. For example, the "PURE" system consists of a few isolated proteins (Shimizu et al.), whereas unprocessed cell lysates (crude extracts) of bacteria such as E. coli contain almost all intracellular proteins, even those not necessary for expression.

[0054] The use of E. coli lysates, in particular crude E. coli lysates, is a further preferred embodiment. A preferred example of a crude E. coli lysate is an E. coli S30 cell extract produced by a method based on the protocol described in E. Falgenhauer et al. (Falgenhauer, S. von Schoenberg, C. Meng, A. Mueckl, K. Vogele, Q. Emslander, C. Ludwig, FC Simmel, ChemBioChem 2021, 22, 2805).

[0055] To enhance or improve the reaction, external factors may be added, such as energy carriers, cofactors, amino acids, polymerases, transcription factors, chaperones, etc. Such factors may be added as proteins and / or nucleic acids encoding the respective proteins.

[0056] If the cell-free lysate used is derived from a cell or microorganism different from the natural host of the virus or bacteriophage, at least one bacteriophage host-specific expression factor and / or a nucleic acid encoding at least one bacteriophage host-specific expression factor, preferably a transcription factor, can be added if necessary.

[0057] According to one embodiment of the present invention, at least one nucleotide sequence, preferably encoding at least one bacteriophage host-specific expression factor or any other protein selected from the group consisting of cofactors, chaperones, polymerases, transcription regulators, is cloned into the cell line used to provide the cell lysate. The nucleotide sequence is expressed in the cell before lysis. Thus, after lysis, the cell lysate already contains all the nucleic acids and / or proteins necessary for the amplification of the virus or bacteriophage, in particular at least one host-specific expression factor or any other protein selected from the group consisting of cofactors, chaperones, polymerases, transcription regulators and / or any mixture thereof.

[0058] The method can also be used to produce modified bacteriophages, in which at least one protein of interest has been modified or added. The modified or added protein of the bacteriophage can be provided by adding the protein to a cell-free expression system and / or by adding a nucleic acid encoding the protein. The protein itself can be the naturally occurring amino acid sequence or can be artificially modified for optimization.

[0059] According to a preferred embodiment, the modified bacteriophage of the present invention comprises: (i) providing a modified bacteriophage genome; Step (ii) of adding to the bacteriophage a modified or unmodified protein to be assembled, the protein being different from the corresponding protein of the bacteriophage; adding to the bacteriophage a nucleic acid encoding a modified or unmodified protein to be assembled, the protein being different from the corresponding protein of the bacteriophage; (v) inhibiting expression of a protein of the bacteriophage by the nucleic acid molecule; and / or It may be produced by applying at least one step selected from the group consisting of any combination of steps (i) to (v) and step (vi).

[0060] Cell-free synthesis of proteins, i.e., outside living cells, has several advantages over cellular expression, all of which are more true when a protein is expressed that is toxic to bacteria or when unnatural amino acids are inserted into the protein.

[0061] Also, production of bacteriophages in cell-free lysates may be carried out without any bacteriophage host bacteria, allowing production of bacteriophages locally, for example in laboratories with no biosafety level, while reducing the amount of toxic by-products produced by the method according to the invention, due to the absence of lysed pathogenic host bacteria.

[0062] Another advantage is that the in vitro method is based on cell-free lysates and pure solutions of viruses or bacteriophages can be produced without any proviruses or prophages, since only the genome of the added virus or bacteriophage is added. Cell-free expression combined with in vitro propagation of nucleic acids, especially genomes, represents a broadly applicable method to accelerate the application of bacteriophages in research and development and biomedical applications.

[0063] According to a preferred embodiment, expression and self-assembly during incubation step (d) can be monitored.

[0064] Thus, a further aspect of the present invention relates to an all in vitro method for amplifying and providing synthetic viruses, in particular bacteriophages, i.e. viruses and / or bacteriophages produced entirely in vitro. Such an all in vitro method relates to the combination of an in vitro amplification method with an expression and self-assembly method, as described herein.

[0065] Thus, the completely in vitro method includes method (I) and method (II), The method (I) for the in vitro amplification of filamentous viral or bacteriophage nucleic acids, in particular bacteriophage genomes, comprises the steps of: (a) providing a viral or bacteriophage nucleic acid template to be amplified; (b) amplifying said viral or bacteriophage nucleic acid template using the polymerase chain reaction to amplify different nucleic acid segments, wherein a primer used at the 5' end of at least one of the viral or bacteriophage nucleic acid templates introduces a nucleic acid cycling inhibitory modification, in particular a phosphorothioate bond, between the first nucleotide at the 5' end of at least one of the corresponding nucleic acid segments; (c) invaginating with an exonuclease, in particular T5 exonuclease, to provide a sticky-ended nucleic acid segment; (d) annealing the attached nucleic acid segments; (e) ligating the annealed nucleic acid segments to provide an amplified linear viral or bacteriophage nucleic acid; Including, The method (II) for expression of the provided amplified linear viral or bacteriophage nucleic acid and self-assembly of the expressed viral or bacteriophage proteins in a cell-free expression system comprises: (a') providing a viral or bacteriophage nucleic acid, in particular a bacteriophage genome; (b') providing a cell-free expression system and optionally adding at least one protein and / or a nucleic acid encoding a protein that enhances or improves the reaction, preferably selected from the group consisting of cofactors, chaperones, polymerases, transcription regulators and / or any mixtures thereof; (c') combining the viral or bacteriophage nucleic acid with a cell-free expression system; (d') incubating the combined bacteriophage nucleic acid and cell-free expression system under conditions suitable for expression and self-assembly of the bacteriophage; Includes.

[0066] Such methods can be used to provide wild-type viruses and / or modified phages. According to the present invention, a "modified phage" can be modified at the genome level and / or at the proteome level.

[0067] The present invention shows for the first time that synthetic phages can be produced entirely in vitro, accelerating and enhancing the production as well as modification of viruses and / or bacteriophages. The method of the present invention is the first known "completely in vitro method" that combines in vitro amplification, particularly using PCR, of filamentous viral or bacteriophage nucleic acid, in vitro expression of this amplified nucleic acid, and in vitro self-assembly of the expressed proteins in a cell-free system to provide the desired virus or bacteriophage. The completely in vitro method of the present invention is cell-free, i.e., does not require the use of cells at any step.

[0068] The concentrations of bacteriophages produced in vitro are so high that the corresponding compositions can be directly applied in phage therapy without further concentration steps, although of course further concentration steps, if necessary, are included within the scope of the present invention, especially after purification.

[0069] A further aspect of the invention relates to a synthetic virus or bacteriophage provided by an in vitro method. The synthetic bacteriophage may have the same lytic activity and / or phenotypic characteristics as the corresponding wild-type bacteriophage. According to a preferred embodiment, such phages have a modified genome and / or proteome.

[0070] A further aspect of the present invention relates to the use of bacteriophages as described herein for use in the pharmaceutical, chemical, biotechnological, agricultural and / or food industries, including but not limited to the bacterial field, for example as vaccines against infections and tumors, and as delivery vehicles for any kind of pharmaceutical agent, especially anti-cancer agents, to target cells, especially human target cells.

[0071] However, their use in the field of bacteria is preferred. In particular, bacteriophages may be used as a microbial population control alternative to antibiotics. They are much more specific than antibiotics and are typically harmless not only to the host organism but also to other beneficial bacteria, such as the gut microbiota, because they have a high selectivity for the bacterial strains they effectively combat. This significantly reduces the likelihood of opportunistic infections. Advantages include further reduced side effects and reduced risk of bacterial resistance development. Small effective doses can be used, since phages replicate in vivo.

[0072] These unique properties make it extremely promising for antibacterial properties.

[0073] Thus, the synthetic bacteriophages provided herein may be part of a pharmaceutical composition. Such a composition may comprise (i) at least one bacteriophage strain capable of generating a lytic infection, and (ii) a pharma- ceutically acceptable carrier. Of course, such a composition may comprise different bacteriophage strains, i.e., a mixture of phages.

[0074] The pharmaceutical composition may be used in combination with antibiotics for the treatment of bacterial infections and / or antibiotic resistant bacteria. In particular, bacteriophages are more successful than antibiotics in the presence of biofilms that are usually covered by a polysaccharide layer that is impermeable to antibiotics. According to a preferred embodiment, the composition may be freeze-dried.

[0075] "Pharmaceutically acceptable carrier" refers to a pharma- ceutically acceptable filler or diluent used to prepare a pharmaceutical composition for animal or human administration. The pharmaceutical composition may further comprise pharma- ceutically acceptable adjuvants, and optionally other therapeutic agents. Particularly for oral administration, it is preferred to add an antacid, i.e., a substance that neutralizes stomach acidity, to increase the number of phages that find their way through the stomach.

[0076] The dosage and concentration of administration of the pharmaceutical composition will necessarily depend on the therapeutic effect to be achieved (e.g., treatment of IBD) and may vary depending on the particular bacteriophage strain in the composition, the route of administration, and the age and condition of the subject to whom the pharmaceutical composition is administered.

[0077] When the pharmaceutical composition is administered with one or more antibiotics, they may be administered simultaneously, separately or sequentially.

[0078] Pharmaceutical compositions and routes of administration include those suitable for or for administration orally (including buccal, sublingual and intraorbital), rectally, nasally, topically (including transdermal), ophthalmic, vaginal, bronchial, pulmonary or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intraperitoneal, intrapleural, intraductal and intrathecal) or by implant.

[0079] In particular, the lyophilized composition may be administered orally, preferably in tablet form.

[0080] Agriculture includes plant and animal agriculture, where bacteriophages can replace or complement antibiotics and / or pesticides.

[0081] Bacterial pathogens relate to several plant diseases that may also be treated with the synthetic bacteriophages provided herein. Non-limiting examples of bacterial plant diseases that may be treated include leaf blight on onion, common scab or soft rot of potato, bacterial wilt on tobacco, bacterial spot or citrus canker on citrus, fire blight on apple, black spot of cabbage, soft rot of calla, bacterial wilt of tomato, and bacterial spot of peach.

[0082] The use of the synthetic bacteriophages of the present invention in animal agriculture is exemplified by their use against Salmonella, pathogenic E. coli, Clostridium and Campylobacter in the poultry industry, and against Salmonella and pathogenic E. coli in the swine industry.

[0083] Additionally, the phages provided herein can be used to ensure food safety and prevent spoilage bacteria. Since 2006, several bacteriophage products have been approved by the U.S. Food and Drug Administration (FDA) and the United States Department of Agriculture (USDA). For example, LMP-102 (Intralytix) was approved for the treatment of ready-to-eat (RTE) poultry and meat products. In the same year, FDA approved LISTEX to use bacteriophages in cheese to kill Listeria monocytogenes.

[0084] With regard to biotechnology-related uses, the methods described herein and the bacteriophages provided herein can be used, for example, in phage display-related uses.

[0085] The present invention further includes kits comprising a pharmaceutical composition of the present invention and instructions for using the composition for use as described above, optionally together with packaging materials.

[0086] Further aspects relate to the use of the bacteriophages provided herein in methods for preventing and / or treating bacterial infections and for avoiding bacterial proliferation. [Brief description of the drawings]

[0087] [Figure 1A] In vitro DNA manipulation, Gibson assembly. [Figure 1B] PTO (phosphorothioate) linkages provide protection from T5 exonuclease in in vitro DNA manipulation. [Figure 2A]Inhibition of T5 exonuclease by PTO binding. [Figure 2B] Enzymatic assays showed digestion with T5 exonuclease, which protected the eight PTO bonds from invagination by T5 exonuclease. [Figure 3A] Genetically modified phage T7_T3p48 (replacement of the tail fiber responsible for binding from the natural T7 tail fiber to the tail fiber of T3 phage) with altered host range. In a spot assay, T7_T3p48 phage on bacteria 613 (specific for T3 and T7). On the top and left are T7 and T3 phages from stocks produced by conventional methods as a control, and on the right is the genetically modified synthetic phage T7_T3gp48 produced by Gibson assembly from four sections of the T7 phage genome and T3gp48 (tail fiber gene sequence of T3) and then expressed in vitro. [Figure 3B] Genetically modified phage T7_T3p48 (exchange of the tail fiber responsible for binding from the natural T7 tail fiber to the tail fiber of T3 phage) with altered host range. In the spot assay, T7_T3p48 phage on bacteria W3110 (specific for T7 only). On the top and left are T7 and T3 phages from a stock produced by conventional methods as a control, and on the right is the genetically modified synthetic phage T7_T3gp48 produced by Gibson assembly from four sections of the T7 phage genome and T3gp48 (tail fiber gene sequence of T3) and then expressed in vitro. [Figure 4A]T7 phage produced by in vitro DNA assembly and cell-free protein expression. Gel image DNA assembly T7 phage from four fragments. Lanes 1-4 are PCR amplified sections of the T7 genome, lane 5 is an equimolar ratio of T7 genome sections used for Gibson assembly, and lane 6 is an equimolar ratio of T7 genome sections after Gibson assembly reaction. Additional bands indicate successful assembly of full-length T7 phage genome, lane 7 is the reference T7 phage genome, and the ladder is 1 kb Extend Ladder (New England Biolabs). [Figure 4B] T7 phage produced by in vitro DNA assembly and cell-free protein expression. In the spot assay, in T7 phage synthesis, the top left is T7 GA+txtl, i.e., synthetic T7 phage produced by Gibson assembly of genome from four fragments and then expressed in vitro. The top right is T7 positive control, i.e., T7 phage from a phage stock produced by conventional methods. The bottom right is T7 GA-txtl, i.e., T7 phage genome assembled by Gibson assembly from four fragments (no in vitro expression). The bottom left is T7 GA negative control+txtl, i.e., a combination of four sections of T7 phage genome without Gibson assembly reaction and subsequent in vitro expression reaction (no assembled full-length genome). [Diagram 5] T5 phage produced by in vitro DNA assembly from six 20 kb fragments and cell-free protein expression (T5_synthetic). Spot assay: top, T5 positive control, i.e. T5 phage produced by conventional method, left, T5 GA+txtl, i.e. synthetic T5 phage produced by Gibson assembly (GA) from six fragments followed by in vitro expression reaction, right, negative control T5 GA-txtl, i.e. T5 phage genome produced by Gibson assembly from six fragments without subsequent in vitro expression reaction. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0088] (Production of Escherichia coli Phage According to the Present Invention) The T7 coliphage has a genome with duplicated sequences at its termini and a size of 40 kbp, which is produced entirely synthetically by the method according to the invention.

[0089] As a first step, the template was methylated with DAM-methylase.

[0090] Four nucleic acid segments of approximately 10 kbp in size were then amplified in a PCR reaction. The first and last primers on the genome were modified and differed from the other primers in that the first eight base pairs exhibit phosphorothioate bonds in the backbone, and only had a phosphodiesterase backbone. In detail, the primers used for the ends of the genome have phosphorothioate bonds (in the backbone) between the first eight bases, and the remaining nucleotides are linked by (regular) phosphodiester bonds.

[0091] After the PCR reaction, the methylated template DNA was removed by the enzyme Dpnl, and the excess primers and removed template were removed by a PCR clean-up kit.

[0092] The four DNA fragments were added in a 1:1:1:1 stoichiometric ratio and assembled with Gibson master mix.

[0093] The DNA was then added to the cell-free expression system, an Escherichia coli S30 cell extract produced according to the protocol of E. Falgehnauer et al. (E. Falgenhauer, S. von Schoenberg, C. Meng, A. Mueckl, K. Vogele, Q. Emslander, C. Ludwig, F. C. Simmel, ChemBioChem 2021, 22, 2805).

[0094] The mixture was incubated at 29° C. for 8 h.

[0095] The completely synthesized and produced bacteriophage was then confirmed by plaque assay.

[0096] (Providing T7 phage containing T3 phage tail fibers) Stepwise / sequential assembly of modified bacteriophage genomes is shown by example in T7 phage, where the tail fiber gene sequence of T7 phage was exchanged with the tail fiber gene sequence of T3 phage (gene T3p48).

[0097] Based on the design of the T7 phage, this phage T7_T3p48 can be assembled from three fragments of 10-12 kb, one fragment of 3.5 kb, and the T3p48 gene (1.7 kb).

[0098] In the sequential approach, three fragments (A1, A2, A3) are first assembled into one section (A), which is then joined with the other two fragments in a second Gibson assembly step to form the complete phage genome (AB).

[0099] The complete phage genome (AB) is used in a cell-free reaction to obtain genetically modified phages.

[0100] Successful replacement of the tail fiber gene is demonstrated by plaque assays in two different hosts.

[0101] Host 1 is DSM 613, which allows both T7 and T3 phages to replicate. Both wild-type and modified phages produce plaques on this host (Figure 3A).

[0102] On the second host, E. coli W3110, only the T7 phage could grow, whereas T3 did not produce plaques (Fig. 3B).

[0103] The modified phage T7_T3p48 is also unable to replicate on this host, which defines the successful exchange of the tail fibers responsible for binding to the host and further defines the host specificity.

Claims

1. (a) providing a viral or bacteriophage nucleic acid template to be amplified; (b) amplifying said viral or bacteriophage nucleic acid template using polymerase chain reaction to amplify different nucleic acid segments, wherein a primer used at the 5' end of at least one of said viral or bacteriophage nucleic acid templates optionally introduces a nucleic acid exonuclease inhibitor modification, in particular a phosphorothioate bond, between the first nucleotide at the 5' end of at least one of said corresponding nucleic acid segments; (c) invaginating with a 5'-3' exonuclease, in particular T5 exonuclease, to provide a sticky-ended nucleic acid segment; (d) annealing the attached nucleic acid segments; (e) ligating the annealed nucleic acid segments to provide an amplified filamentous bacteriophage nucleic acid; 1. A method for in vitro amplification of viral or bacteriophage nucleic acid, in particular a bacteriophage genome, comprising:

2. The method of claim 1 , wherein the bacteriophage nucleic acid is selected from the group consisting of DNA, RNA and variants thereof.

3. The method according to claim 1 or 2, wherein the different nucleic acid segments have the same nucleic acid length, preferably between about 5 kbp and about 30 kbp, in particular about 10 kbp.

4. The method according to any one of claims 1 to 3, wherein the stoichiometric ratio of the attached nucleic acid segments annealed in step (d) is 1:5, in particular 1:

1.

5. The method according to any one of claims 1 to 4, wherein the provided amplified viral or bacteriophage nucleic acid is modified, in particular by nucleic acid deletions, insertions and / or point mutations.

6. methylating said viral or bacteriophage nucleic acid template prior to the amplification step (b); digesting the methylated viral or bacteriophage nucleic acid template after the amplification step (b); The method of claim 5 , comprising:

7. (a) providing a viral or bacteriophage nucleic acid, in particular a bacteriophage genome, preferably a viral or bacteriophage nucleic acid or genome provided by the method according to any one of claims 1 to 6; (b) providing a cell-free expression system and optionally adding at least one protein and / or a nucleic acid encoding a protein that enhances or improves the reaction, preferably selected from the group consisting of cofactors, chaperones, polymerases, transcription regulators and / or any mixtures thereof; (c) combining said viral or bacteriophage nucleic acid with said cell-free expression system; (d) incubating the combined viral or bacteriophage nucleic acid and the cell-free expression system under conditions suitable for expression and self-assembly of the bacteriophage; 2. A method for expressing and self-assembling a virus or bacteriophage in a cell-free expression system, comprising:

8. The method according to claim 7, wherein the cell-free expression system is host-independent and is preferably selected from a cell lysate or an artificial expression system.

9. The method according to claim 8, wherein the cell lysate is derived from a microorganism, in particular E. coli, yeast, insect, mammalian, plant and / or is artificial.

10. The modified bacteriophage (i) providing a modified bacteriophage genome; adding to the bacteriophage a modified or unmodified protein to be assembled, the protein being different from the corresponding protein of the bacteriophage; adding to the bacteriophage a nucleic acid encoding a modified or unmodified protein to be assembled, the protein being different from the corresponding protein of the bacteriophage; (v) inhibiting expression of a protein of the bacteriophage by the nucleic acid molecule; and / or Any combination of steps (i) to (v) Step (vi) The method according to any one of claims 7 to 9, wherein the composition is produced by applying at least one step selected from the group consisting of:

11. A virus, in particular a bacteriophage, provided by the method according to any one of claims 1 to 10.

12. A completely in vitro method for the amplification and presentation of synthetic bacteriophages.

13. 13. A virus, in particular a bacteriophage, according to claim 11 or 12, for use in the pharmaceutical, chemical, biotechnological, agricultural and / or food industries.

14. A virus, particularly a bacteriophage, for use in a method for the prevention and / or treatment of a bacterial infection.

15. Use of a virus, in particular a bacteriophage, according to claim 11 or 12 for avoiding bacterial proliferation.

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