Antimicrobial peptides having liquid-liquid phase separation activities

Abstract

The present invention relates to antimicrobial peptides and their use for the treatment of bacterial infections. In particular, the inventors applied a variational autoencoder (VAE) model to design 40 peptides with potential liquid-liquid phase separation (LLPS) and antimicrobial activity. The finally selected 20 peptides having both antimicrobial and LLPS activities.

Description

[0001] ANTIMICROBIAL PEPTIDES HAVING LIQUID-LIQUID PHASE SEPARATION

[0002] ACTIVITIES

[0003] FIELD OF THE INVENTION:

[0004] The present invention is in the field of medicine, in particular synthetic biology and microbiology.

[0005] BACKGROUND OF THE INVENTION:

[0006] Antimicrobial resistance (AMR), one of the top 10 global health threats according to the World Health Organization (WHO), has been the cause of 1.27 million deaths in 2019 (1). AMR emerges when antimicrobials are no longer effective against pathogens, a risk that has been made evident since the golden age of antibiotics in the 1950s. With the current trend, AMR will threaten 10 million lives and bring about 90 trillion € in related costs by 2050 (1). Quick acquisition of AMR via horizontal gene transfer, particularly in hospitals, has led to the spread of resistant bacterial pathogens against which there is no effective antimicrobial strategy. Additionally, R&D of antibiotics is hampered by the current drought in natural product discovery and significant withdrawal of pharma companies. In 2021, compared to 4000 immuno-oncology compounds, there have been only 40 antimicrobials in clinical trials, none of which are potentially active against prioritized gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) or WHO critical threat pathogens (2).

[0007] Several approaches have been put forth such as natural product discovery, virtual docking, chemical libraries, and phages (2,3). Among these, ribosomal antimicrobial peptides (AMPs), the majority of which are membrane disruptive, can provide potential solutions to fight emergent multidrug-resistant pathogens with advantages including ease of development, broadspectrum activity, and, most importantly, lower emergence of resistance compared to other antimicrobials (4,5). Over 5,000 such AMPs have been described, encompassing both natural AMPs (from prokaryotes and eukaryotes including vertebrates) and human-engineered peptides via rational engineering and screening of random libraries and deep learning (6-9). However, AMPs potential is limited by rapid proteolysis under physiological conditions when used as oral drugs, leading to unsuccessful commercialization of such AMPs (6). This may be explained by their evolution under selection pressure within their niche environment largely devoid of humans. Hence, there is a strong need to develop protease-resistant AMPs and / or new classes of AMPs that circumvent this bottleneck.

[0008] SUMMARY OF THE INVENTION:

[0009] The present invention is defined by the claims. In particular, the present invention relates to antimicrobial peptides and their use for the treatment of bacterial infections.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] Main definitions:

[0012] As used herein, the terms “polypeptide”, “peptide”, and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides when discussed in the context of gene therapy refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.

[0013] As used herein, the term “antimicrobial peptide” has its general meaning in the art and refers to a peptide that has the ability to kill or inhibit the growth of bacteria. Antimicrobial peptides can act by disrupting the membrane integrity of the target microorganism, interfering with its metabolic processes, or modulating its immune response. In particular, the antimicrobial peptides of the present invention are synthetic peptides.

[0014] As used herein, the term “synthetic peptide” refers to a peptide that is not naturally occurring but is artificially synthesized in a laboratory setting. Synthetic peptides can be designed to mimic the structure or function of a natural peptide or to have novel properties or activities. Synthetic peptides can be synthesized by chemical methods, such as solid-phase synthesis, or by biological methods, such as recombinant DNA technology or in vitro translation. Synthetic peptides can be modified after synthesis by various methods, such as cyclization, conjugation, or cleavage. Synthetic peptides can have any length, ranging from dipeptides to polypeptides. As used herein, the term “polynucleotide” as used herein refers to polymers of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid (“DNA”), as well as triple-, double- and single-stranded ribonucleic acid (“RNA”). It also includes modified, for example by alkylation, and / or by capping, and unmodified forms of the polynucleotide. More particularly, the term “polynucleotide” includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA and mRNA, whether spliced or unspliced, any other type of polynucleotide which is an N- or C-glycoside of a purine or pyrimidine base, and other polymers containing normucleotidic backbones, for example, polyamide (e.g., peptide nucleic acids “PNAs”) and polymorpholino polymers, and other synthetic sequence-specific nucleic acid polymers providing that the polymers contain nucleobases in a configuration which allows for base pairing and base stacking, such as is found in DNA and RNA. In some embodiments, the polynucleotide comprises an mRNA. In other aspect, the mRNA is a synthetic mRNA. In some embodiments, the synthetic mRNA comprises at least one unnatural nucleobase. In some embodiments, all nucleobases of a certain class have been replaced with unnatural nucleobases (e.g., all uridines in a polynucleotide disclosed herein can be replaced with an unnatural nucleobase, e.g., 5-methoxyuridine). In some embodiments, the polynucleotide (e.g., a synthetic RNA or a synthetic DNA) comprises only natural nucleobases, i.e., A, C, T and G in the case of a synthetic DNA, or A, C, T, and U in the case of a synthetic RNA.

[0015] As used herein, the term "encoding" refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as, for example, a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a "polynucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.

[0016] As used herein, the expression “derived from” refers to a process whereby a first component (e.g., a first polypeptide), or information from that first component, is used to isolate, derive or make a different second component (e.g., a second polypeptide that is different from the first one).

[0017] As used herein, the “percent identity” between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described below. The percent identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm (Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology. 48 (3): 443-53.). The percent identity between two nucleotide or amino acid sequences may also be determined using for example algorithms such as EMBOSS Needle (pair wise alignment; available at www.ebi.ac.uk). For example, EMBOSS Needle may be used with a BLOSUM62 matrix, a “gap open penalty” of 10, a “gap extend penalty” of 0.5, a false “end gap penalty”, an “end gap open penalty” of 10 and an “end gap extend penalty” of 0.5. In general, the “percent identity” is a function of the number of matching positions divided by the number of positions compared and multiplied by 100. For instance, if 6 out of 10 sequence positions are identical between the two compared sequences after alignment, then the identity is 60%. The % identity is typically determined over the whole length of the query sequence on which the analysis is performed. Two molecules having the same primary amino acid sequence or nucleic acid sequence are identical irrespective of any chemical and / or biological modification. According to the invention a first amino acid sequence having at least 70% of identity with a second amino acid sequence means that the first sequence has 70; 71; 72; 73; 74; 75; 76; 77; 78; 79; 80; 81; 82; 83; 84; 85; 86; 87; 88; 89; 90; 91; 92; 93; 94; 95; 96; 97; 98; 99 or 100% of identity with the second amino acid sequence. As used herein, the term “mutation” has its general meaning in the art and refers to a substitution, deletion or insertion. In particular, the term "substitution" means that a specific amino acid residue at a specific position is removed and another amino acid residue is inserted into the same position. Within the specification, the mutation are references according to the standard mutation nomenclature.

[0018] As used herein, the term “liquid-liquid phase separation” or “LLPS” is a process in which a homogeneous solution of macromolecules, such as proteins, nucleic acids, or polysaccharides, undergoes spontaneous demixing into two coexisting phases: a dense, droplet-like phase that is enriched in specific macromolecules, and a dilute, background phase that contains the remaining components. The dense phase is often referred to as a condensate, and can exhibit liquid-like properties, such as fusion, fission, wetting, and deformation. LLPS is driven by the interplay of various molecular interactions, such as electrostatic, hydrophobic, and multivalent interactions, that favor the formation of dynamic and reversible bonds between macromolecules within the condensate. LLPS can be influenced by various factors, such as temperature, pH, salt concentration, and post-translational modifications, that modulate the balance between intra- and inter-molecular interactions.

[0019] As used herein, the term "subject", “host”, “individual” or “patient” refers to a mammal, typically a human being, male or female at any age that is in-need of a therapy.

[0020] As used herein, the term "bacterial infection" refers to a condition in which pathogenic bacteria invade the body of a subject and cause harm to the host's cells, tissues, organs, or systems. Bacterial infections can be acute or chronic, localized or systemic, and can affect various parts of the body, such as the skin, respiratory tract, urinary tract, gastrointestinal tract, blood, brain, bones, joints, or heart. Bacterial infections can be caused by different types of bacteria, such as gram-positive, gram-negative, anaerobic, aerobic, intracellular, extracellular, or multidrugresistant bacteria. Bacterial infections can be diagnosed by various methods, such as culture, microscopy, serology, molecular techniques, or biomarkers.

[0021] As used herein, the term “biofilm” has its general meaning in the art and refers to a complex community of microorganisms that adhere to a surface and are embedded in a matrix of extracellular polymeric substances, such as polysaccharides, proteins, and nucleic acids. Biofilms can form on various biotic or abiotic surfaces, such as teeth, medical devices, industrial pipes, or natural environments. Biofilms can protect the bacteria from environmental stresses, such as desiccation, pH changes, antibiotics, or host immune responses. Biofilms can also facilitate horizontal gene transfer and metabolic cooperation among the bacteria. Biofilms can cause various problems, such as dental caries, chronic infections, biofouling, or biocorrosion.

[0022] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular interval, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0023] Antimicrobial peptides of the present invention: The present invention relates to an antimicrobial peptides that comprises the consensus sequence of for formula (I)

[0024] MKxiKKx2FKx3LFKTFFKKGx4Gx5X6KKSx7Fx8X9XioSKxiiQxi2KKKxi3G xi4Wxi5xi6Xi7KD

[0025] (I) wherein

[0026] XI represents R, N, or A

[0027] X2 represents A or W

[0028] X3 represents F, S or A

[0029] X4 represents P, K or Q

[0030] X5 represents G, S or L

[0031] X6 represents F or Y

[0032] X7 represents V or S

[0033] X8 represents N or R

[0034] X9 represents K or V

[0035] XI 0 represents R or E

[0036] XI I represents T or S

[0037] X12 represents F, V or M

[0038] XI 3 represents F, L, A, or G

[0039] XI 4 represents G or I

[0040] XI 5 represents V or A

[0041] XI 6 represents G or A, and

[0042] XI 7 represents F or L.

[0043] According to the present invention, the antimicrobial peptide of the present invention is capable of inducing liquid-liquid phase separation.

[0044] In some embodiments, XI represents R.

[0045] In some embodiments, X2 represents A.

[0046] In some embodiments, X3 represents F.

[0047] In some embodiments, X4 represents P.

[0048] In some embodiments, X5 represents G.

[0049] In some embodiments, X6 represents F.

[0050] In some embodiments, X7 represents V.

[0051] In some embodiments, X8 represents N. In some embodiments, X9 represents K.

[0052] In some embodiments, XI 0 represents R.

[0053] In some embodiments, XI 1 represents T.

[0054] In some embodiments, X12 represents F.

[0055] In some embodiments, X13 represents F.

[0056] In some embodiments, X14 represents G.

[0057] In some embodiments, XI 5 represents V. In some embodiments, XI 6 represents G. In some embodiments, X17 represents F.

[0058] In some embodiments, the antimicrobial peptide of the present invention comprises an amino acid sequence having at least 90% of identity with the amino acid sequence of LI.3 AMP, L1.5AMP, L1.7AMP, L1.8AMP, L1.10AMP, LI. HAMP, L1.15AMP, L1.18AMP, L1.19AMP, L1.22AMP, L1.25AMP, L1.26AMP, L1.27AMP, L1.31AMP, L1.34AMP L1.35AMP, L1.36AMP, L1.39AMP or L1.40AMP (see Table 1).

[0059] In some embodiments, the antimicrobial peptide of the present invention comprises one or more conservative substitution(s). As used herein, the term “conservative substitution” is one in which an amino acid is substituted for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Amino acid substitutions may further be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine and valine; glycine and alanine; asparagine and glutamine; and serine, threonine, phenylalanine and tyrosine. Other groups of amino acids that may represent conservative changes include: (1) ala, pro, gly, glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. Other families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0060] In some embodiments, the antimicrobial peptide of the present invention comprises one or more substitution(s) by a non-natural amino acid. As used herein, the term “Non-natural amino acids” refers to non-genetically encoded amino acids, irrespective of whether they appear in nature or not. Non-natural amino acids that can be present in a variant of an amino acid sequence as defined herein include: P-amino acids; p-acyl-L-phenylalanine; N-acetyl lysine; O-4-allyl- L-tyrosine; 2-aminoadipic acid; 3 -aminoadipic acid; beta-alanine; 4-tert-butyl hydrogen 2- azidosuccinate; beta-aminopropionic acid; 2-aminobutyric acid; 4-aminobutyric acid; 2, 4,- diamino butyric acid; 6-aminocaproic acid; 2-aminoheptanoic acid; 2-aminoisobutyric acid; 3- aminoisobutyric acid; 2-aminopimelic acid; p-aminophenylalanine; 2, 3 -diaminobutyric acid; 2, 3 -diamino propionic acid; 2, 2Z-diaminopimelic acid; p-amino-L-phenylalanine; p-azido- L-phenylalanine; D-allylglycine; p-benzoyl-L-phenylalanine; 3 -benzothienyl alanine p- bromophenylalanine; t-butylalanine; t-butylglycine; 4-chlorophenylalanine; cyclohexylalanine; cysteic acid; D-citrulline; thio-L-citrulline; desmosine; epsilon-amino hexanoic acid; N-ethylglycine; N-ethylasparagine; 2-fluorophenylalanine; 3- fluorophenylalanine; 4-fluorophenylalanine; homoarginine; homocysteine; homoserine; hydroxylysine; allo-hydroxylysine; 3-(3-methyl-4-nitrobenzyl)-L-histidine methyl ester; isodesmosine; allo-isoleucine; isopropyl-L-phenylalanine; 3-methyl-phenylalanine; N- methylglycine; N-methylisoleucine; 6-N-methyllysine; O-methyl-L-tyrosine; N-methylvaline; methionin sulfoxide; 2-napthylalanine; L-3-(2-naphthyl)alanine; isoserine; 3 -phenyl serine; norvaline; norleucine; 5,5,5-trifluoro-DL-leucine; ornithine; 3 -chloro-tyrosine; N5- carbamoylornithine; penicillamine; phenylglycine; piperidinic acid; pyridylalanine; 1, 2, 3, 4- tetrahydro-isoquinoline-3-carboxylix acid; beta-2 -thienylalanine; y-carboxy-DL-glutamic acid; 4-fluoro-DL-glutamic acid; D-thyroxine; allo-threonine; 5 -hydroxy -tryptophan; 5-methoxy- tryptophan; 5 -fluoro-tryptophan; 3 -fluoro-valine. In some embodiments, the antimicrobial peptide of the present invention can further comprise a cell permeating peptide (CPP). The cell permeating peptide can assist in facilitating the entry of the antibiotic peptide into the target cell (i.e., bacterium). Various cell permeating peptides are known in the art. For example, additional CPPs known in the art can be found on online databases (i.e., http: / / crdd.osdd.net / raghava / cppsite ), in Oikawa et al., (Screening of a Cell- Penetrating Peptide Library in Escherichia coli: Relationship between Cell Penetration Efficiency and Cytotoxicity. ACS Omega 2018, 3, 16489-164), the disclosure of each is incorporated by reference in its entirety.

[0061] The peptides described herein can be prepared in a variety of ways known to one skilled in the art of peptide synthesis or variations thereon as appreciated by those skilled in the art. For example, synthetic peptides are prepared using known techniques of solid phase, liquid phase, or peptide condensation, or any combination thereof. Alternatively, the peptide of the present invention can be synthesized by recombinant DNA techniques well-known in the art and as described herein after.

[0062] Polynucleotides and host cells of the present invention:

[0063] A further object of the present invention relates to a polynucleotide that encodes one or more antimicrobial peptide(s) of the present invention.

[0064] In some embodiments, the polynucleotide of the present invention is a messenger RNA (mRNA).

[0065] A further object of the present invention relates to a vector, preferably an expression vector, containing the polynucleotide of the present invention.

[0066] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0067] In some embodiments, the vector of the present invention is a plasmid. As used herein, the term “plasmid” has its general meaning in the art and refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In some embodiments, the vector of the present invention is a viral vector (e.g., bacteriophages, replication defective retroviruses, adenoviruses and adeno-associated viruses).

[0068] In some embodiments, certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).

[0069] A further object of the present invention relates to a host cell that has been transfected, infected or transformed by a polynucleotide and / or a vector according to the present invention.

[0070] As used herein, the terms “host cell” and “recombinant host cell” are used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0071] Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids., which serve equivalent functions.

[0072] The recombinant expression vectors of the present invention comprise the polynucleotide of the present invention in a form suitable for expressing it in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector, the term “operably-linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequences in a manner that allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). As used herein, the term “regulatory sequence” is intended to includes promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a host cell. Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Typically, the regulatory sequence is a "promoter" sequence, which is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene which is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. Transcription promoters can include "inducible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), "repressible promoters" (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), and "constitutive promoters”.

[0073] It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the present invention can be introduced into host cells to thereby produce the antimicrobial peptides of the present invention, encoded by the polynucleotides as described herein.

[0074] The recombinant expression vectors of the present invention can be designed for production of the peptides of interest in prokaryotic or eukaryotic cells. For example, the antimicrobial peptides of the present invention can be expressed in bacterial cells such as Escherichia coli or actinobacteria cells, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. In particular, the host cell of the present invention is a bacterial cell.

[0075] Expression of peptides in prokaryotes is most often carried with vectors containing constitutive or inducible promoters directing the expression of the peptides of interest. In some embodiment, the vector encodes for the peptides of interest that are fused to one or more heterologous sequences such as purification tags, for example: P-galactosidase, glutathione-S-transferase, green fluorescent proteins (GFP), and epitope tags such as FLAG, myc tag, poly histidine (e.g., 6HIS). In some embodiments, the fusion protein of the present invention comprises an AviTag® sequence. The AviTag® sequence (U.S. Pat. Nos. 5,932,433, 5,874,239 & 5,723,584) is a unique peptide, just 15 residues long, that is recognized by biotin ligase (Schatz P. J., 1993). Typically, the tags typically serve three purposes: (i) to increase expression of recombinant peptide; (ii) to increase the solubility of the recombinant peptide; and (iii) to aid in the purification of the recombinant peptide by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant peptide to enable separation of the recombinant peptide from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89) — not accurate, pETl la-d have N terminal T7 tag.

[0076] The vector of the present invention can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms “transformation” and “transfection” are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.

[0077] The host cell of the present invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) the antimicrobial peptides of the present invention. Accordingly, the invention further provides methods for producing the antimicrobial peptides of the present invention using the host cells of the present invention. In some embodiments, the method comprises culturing the host cell of the present invention (into which a recombinant expression vector encoding protein of the present invention has been introduced) in a suitable medium such that the antimicrobial peptide of the present invention is produced. In some embodiments, the method further comprises isolating the antimicrobial peptide of the present invention from the medium or the host cell.

[0078] Uses of the antimicrobial peptides:

[0079] The antimicrobial peptides of the present are particularly suitable for killing bacteria. In some embodiments, the antimicrobial peptides of the present invention are particularly suitable for therapeutic purposes. More particularly, the antimicrobial peptides of the present invention are particularly suitable for treating bacterial infections.

[0080] Thus, a further object of the present invention relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more antimicrobial peptide(s) of the present invention.

[0081] A further object of the present invention also relates to a method of therapy in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more polynucleotide(s) of the present invention.

[0082] In some embodiments, the subject can be human or any other animal (e.g., birds and mammals) (e.g., domestic animals such as cats and dogs; livestock and farm animals such as horses, cows, pigs, chickens, etc.). Typically said subject is a mammal including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, and mouse) and a primate (e.g., a monkey, chimpanzee, and a human). In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a farm animal or pet. In some embodiments, the subject is a human. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human child. In some embodiments, the subject is a human adult. In some embodiments, the subject is an elderly human. In some embodiments, the subject is a premature human infant.

[0083] In some embodiments, the bacteria may be Gram-positive or Gram-negative bacteria. Thus, bacteria may be of genus including, but not limited to Acetobacter, Acinetobacter, Actinomyces, Agrobacterium spp., Azorhizobium, Azotobacter, Anaplasma spp., Bacillus spp., Bacteroides spp., Bartonella spp., Bordetella spp., Borrelia, Brucella spp., Burkholderia spp., Calymmatobacterium, Campylobacter, Chlamydia spp., Chlamydophila spp., Clostridium spp., Corynebacterium spp., Coxiella, Ehrlichia, Enter obacter, Enterococcus spp., Escherichia, Francisella, Fusobacterium, Gardnerella, Haemophilus spp., Helicobacter, Klebsiella, Lactobacillus spp., Lactococcus, Legionella, Listeria, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium spp., Mycoplasma spp., Neisseria spp., Pasteur ella spp., Peptostreptococcus, Porphyromonas, Pseudomonas, Rhizobium, Rickettsia spp., Rodhalimaea spp., Rothia, Salmonella spp., Serratia, Shigella, Staphylococcus spp., Stenotrophomonas, Streptococcus spp., Treponema spp., Vibrio spp., Wolbachia, and Yersinia spp.

[0084] In some embodiments, the bacterial infection may be caused by bacteria including, but are not limited to Acetobacter aurantius, Acinetobacter baumannii, Actinomyces Israelii, Agrobacterium radiobacter, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Anaplasma phagocytophilum, Anaplasma marginale, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus mycoides, Bacillus stear other mophilus, Bacillus subtilis, Bacteroides fragilis, Bacteroides gingivalis, Bacteroides melaminogenicus (Prevotella melaminogenica), Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei, Burkholderia cepacia complex, Burkholderia cenocepacia, Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter fetus, Campylobacter jejuni, Campylobacter pylori, Chlamydia trachomatis, Chlamydophila. (such as C. pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani), Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii, Ehrlichia chaffeensis, Enterobacter cloacae, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenzae, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Listeria monocytogenes, Methanobacterium extroquens, Microbacterium multiforme, Micrococcus luteus, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium intracellulare, Mycobacterium leprae, Mycobacterium lepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma penetrans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, Pasteurella tularensis Peptostreptococcus, Porphyromonas gingivalis, Pseudomonas aeruginosa, Rhizobium Radiobacter, Rickettsia prowazekii, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis, Stenotrophomonas maltophilia, Streptococcus agalactiae, Streptococcus, avium, Streptococcus bovis, Streptococcus cricetus, Streptococcus faceium, Streptococcus faecalis, Streptococcus ferus, Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus salivarius, Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Treponema denticola, Vibrio cholerae, Vibrio comma, Vibrio parahaemolyticus, Vibrio vulnificus, Wolbachia, Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis.

[0085] In some embodiments, the bacterial infection is caused by intracellular bacteria. As used herein, the term “intracellular bacteria” refers to bacteria that can invade and replicate inside the cells of a host organism, such as animal or human cells. Some intracellular bacteria live in specialized compartments called vacuoles, while others escape into the cytoplasm of the host cell. Intracellular bacteria can cause chronic and sometimes fatal infections, as they are able to evade the immune system and resist antibiotics. Examples of intracellular bacteria include but are not limited to Chlamydia, Rickettsia, Coxiella, Legionella, Mycobacterium, Francisella, Brucella, and Listeria.

[0086] In some embodiments, the antimicrobial peptides of the present invention are particularly suitable for treating bacterial infection caused by gram-negative ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enter obacter spp.)

[0087] In some embodiments, the bacteria are drug resistant. As used herein, the term “drug resistant bacteria” refers to bacteria that have developed the ability to survive exposure to antibiotics that would normally kill them or inhibit their growth. Drug resistance can arise through genetic mutations or by acquiring resistance genes from other bacteria through horizontal gene transfer. Drug resistant bacteria pose a serious threat to human health, as they can cause infections that are difficult or impossible to treat with available drugs. Some examples of drug resistant bacteria are methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), extended-spectrum beta-lactamase (ESBL)-producing Enter obacter iaceae, carbapenem-resistant Enter obacter iaceae (CRE), multi drug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). In some embodiments, the bacterial infection may cause conditions such as, but are not limited to pneumonia, tuberculosis, meningitis, diarrhoeal diseases, formation of biofilm, sepsis, listeriosis, gastroenteritis, toxic shock syndrome, hemorrhagic colitis, hemolytic uremic syndrome, Lyme Disease, gastric and duodenal ulcers, human ehrlichiosis, pseudomembranous colitis, cholera, salmonellosis, cat scratch fever, necrotizing fasciitis (GAS), streptococcal toxic shock syndrome, nosocomial and community associated infections, atherosclerosis, sudden infant death syndrome (SIDS), wound infection, septicemia, gastrointestinal disease, hospital- acquired endocarditis and blood stream infections.

[0088] In some embodiments, the antimicrobial peptides of the present invention are particularly for treating so-called biofilm-associated infections (BAI), which are surface-attached cellular agglomerates of bacteria. Biofilms contributes significantly to bacterial resistance to conventional antibiotics. Biofilms are associated to various pathological conditions in humans such as cystic fibrosis, colonization of indwelling medical devices and dental plaque formation and wounds. Biofilm formation occurs when microbial cells adhere to each other and are embedded in a matrix of extracellular polymeric substance (EPS) on a surface. The growth of microbes in such a protected environment that is enriched with biomacromolecules (e.g. polysaccharides, nucleic acids and proteins) and nutrients allow for enhanced microbial crosstalk and increased virulence.

[0089] In another aspect, there is provided a method of removing a biofilm. The method comprising administration of an effective amount of a peptide as described herein. In some embodiments, the biofilm may occur on surfaces. The term “surface” refers to any surface whether medical or industrial, that provides an interface between a fluid, such as a liquid or air, and a solid. The interface between fluid and solid can be intermittent, and can be caused by flowing or stagnant fluid, aerosols, or other means for air-borne fluid exposure. A surface refers, in some examples, to a plane whose mechanical structure is compatible with the adherence of bacteria. In the context of the peptides and methods described herein, the terminology “surface” encompasses the inner and outer aspects of various instruments and devices, both disposable and nondisposable, medical and non-medical. Examples of non-medical uses include the hull of a ship, dockyard, food processors, mixers, machines, containers, water tanks, water filtrations, purification systems, preservatives in food industries, personal care products such as shampoo, cream, moisturizer, hand sanitizer, soaps and the like. Examples of medical uses include the entire spectrum of medical devices. Such “surfaces” may include the inner and outer aspects of various instruments and devices, whether disposable or intended for repeated uses. Examples include the entire spectrum of articles adapted for medical use, including scalpels, needles, scissors and other devices used in invasive surgical, therapeutic or diagnostic procedures; implantable medical devices, including artificial blood vessels, catheters and other devices for the removal or delivery of fluids to patients, artificial hearts, artificial kidneys, orthopaedic pins, plates and implants; catheters and other tubes (including urological and biliary tubes, endotracheal tubes, peripherally insertable central venous catheters, dialysis catheters, long term tunneled central venous catheters, peripheral venous catheters, short term central venous catheters, arterial catheters, pulmonary catheters, Swan-Ganz catheters, urinary catheters, peritoneal catheters), urinary devices (including long term urinary devices, tissue bonding urinary devices, artificial urinary sphincters, urinary dilators), shunts (including ventricular or arterio-venous shunts); prostheses (including breast implants, penile prostheses, vascular grafting prostheses, heart valves, artificial joints, artificial larynxes, otological implants), vascular catheter ports, wound drain tubes, hydrocephalus shunts, pacemakers and implantable defibrillators, dental implants, filings, dentures and the like. Other examples will be readily apparent to practitioners in these arts. Surfaces found in the medical environment also include the inner and outer aspects of pieces of medical equipment, medical gear worn or carried by personnel in the health care setting. Such surfaces can include counter tops and fixtures in areas used for medical procedures or for preparing medical apparatus, tubes and canisters used in respiratory treatments, including the administration of oxygen, of solubilised drugs in nebulisers and of aesthetic agents. Also included are those surfaces intended as biological barriers to infectious organisms in medical settings, such as gloves, aprons and face-shields. Commonly used materials for biological barriers may be latex-based or non-latex based. An example for a non-latex based biological barrier material may include vinyl. Other such surfaces can include handles and cables for medical or dental equipment not intended to be sterile. Additionally, such surfaces can include those non-sterile external surfaces of tubes and other apparatus found in areas where blood or body fluids or other hazardous biomaterials are commonly encountered. In some embodiments, the biofilm may be comprised on catheters and medical implants.

[0090] In some embodiments, the antimicrobial peptides is administered with a second therapeutic agent that may be administered separately or together with the antimicrobial peptide of the present invention. In some embodiments, the second therapeutic agent may be a further or different antimicrobial agent. In some embodiments, the antimicrobial agent may include, but is not limited to an antifungal, antiviral, antibacterial or an antibiotic and an anti-parasite. In some embodiments, the antimicrobial is an antibiotic. In some embodiments, the antibiotic may include, but is not limited to Ampicillin, Bacampicillin, Carbenicillin Indanyl, Mezlocillin, Piperacillin, Ticarcillin, Amoxicillin-Clavulanic Acid, Ampicillin-Sulbactam, Benzylpenicillin, Cioxacillin, Dicloxacillin, Methicillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin Tazobactam, Ticarcillin Clavulanic Acid, Nafcillin, Cephalosporin I Generation, Cefadroxil, Cefazolin, Cephalexin, Cephalothin, Cephapirin, Cephradine, Cefaclor, Cefamandol, Cefonicid, Cefotetan, Cefoxitin, Cefprozil, Ceftinetazole, Cefuroxime, Loracarbef, Cefdinir, Ceftibuten, Cefoperazone, Cefixime, Cefotaxime, Cefpodoxime proxetil, Ceftazidime, Ceftizoxime, Ceftriaxone, Cefepime, Azithromycin, Clarithromycin, Clindamycin, Dirithromycin, Erythromycin, Lincomycin, Troleandomycin, Cinoxacin, Ciprofloxacin, Enoxacin, Gatifloxacin, Grepafloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Sparfloxacin, Trovafloxacin, Oxolinic acid, Gemifloxacin, Pefloxacin, Imipenem-Cilastatin, Meropenem, Aztreonam, Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Streptomycin, Tobramycin, Paromomycin, Teicoplanin, Vancomycin, Demeclocycline, Doxycycline, Methacycline, Minocycline, Oxytetracycline, Tetracycline, Chlortetracycline, Mafenide, Silver Sulfadiazine, Sulfacetamide, Sulfadiazine, Sulfamethoxazole, Sulfasalazine, Sulfisoxazole, Trimethoprim- Sulfamethoxazole, Sulfamethizole, Rifabutin, Rifampin, Rifapentine, Linezolid, Streptogramins, Quinopristin Dalfopristin, Bacitracin, Chloramphenicol, Fosfomycin, Isoniazid, Methenamine, Metronidazol, Mupirocin, Nitrofurantoin, Nitrofurazone, Novobiocin, Polymyxin, Spectinomycin, Trimethoprim, Colistin, Cycloserine, Capreomycin, Ethionamide, Pyrazinamide, Para-aminosalicyclic acid, Erythromycin ethyl succinate, Miconazole, Ketoconazole, Clotrimazole, Econazole, Bifonazole, Butoconazole, Fenticonazole, Isoconazole, Oxiconazole, Sertaconazole, Sulconazole, Tioconazole, Fluconazole,

[0091] Itraconazole, Isavuconazole, Ravuconazole, Posaconazole, Voriconazole, Terconazole,

[0092] Terbinafine, Amorolfme, Naftifine, Butenafine, Anidulafungin, Caspofungin, Micafungin, Benzoic acid, Ciclopirox, Tolnaftate, Undecylenic acid, Flucytosine, or 5-fluorocytosine, Griseofulvin, Haloprogin and combinations thereof. In some embodiments, the antibiotic may include, but is not limited to nalidixic acid, gentamicin, erythromycin, streptomycin and kanamycin. Compositions:

[0093] A further object of the present invention relates to a composition comprises an amount of one or more antimicrobial peptide(s) of the present invention.

[0094] A further object of the present invention relates to a composition comprises an amount of one or more polynucleotides(s) of the present invention.

[0095] In some embodiments, the composition of the present invention is a pharmaceutical composition.

[0096] Thus a further object of the present invention relates to a pharmaceutical composition comprising one or more antimicrobial peptide(s) of the present invention and a pharmaceutically acceptable carrier.

[0097] Thus a further object of the present invention relates to a pharmaceutical composition comprising one or more polynucleotides(s) of the present invention and a pharmaceutically acceptable carrier.

[0098] As used herein, the term “pharmaceutical composition” refers to a composition described herein, or pharmaceutically acceptable salts thereof, with other agents such as carriers and / or excipients. The pharmaceutical compositions as provided herewith typically include a pharmaceutically acceptable carrier.

[0099] As used herein, the term “pharmaceutically acceptable carrier” includes any and all solvents, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's Pharmaceutical-Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980) discloses various carriers used in formulating pharmaceutical compositions and known techniques for the preparation thereof.

[0100] Pharmaceutically acceptable carriers that may be used in these compositions include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene- block polymers, polyethylene glycol and wool fat. For use in administration to a patient, the composition will be formulated for administration to the patient. The compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. The used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. Sterile injectable forms of the compositions of this invention may be aqueous or an oleaginous suspension. These suspensions may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as carboxymethyl cellulose or similar dispersing agents that are commonly used in the formulation of pharmaceutically acceptable dosage forms including emulsions and suspensions. Other commonly used surfactants, such as Tweens, Spans and other emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms may also be used for the purposes of formulation. The compositions of this invention may be orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include, e.g., lactose. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Alternatively, the compositions of this invention may be administered in the form of suppositories for rectal administration. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, beeswax and polyethylene glycols. The compositions of this invention may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, including diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the compositions may be formulated in a suitable ointment containing the active component suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds of this invention include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the compositions can be formulated in a suitable lotion or cream containing the active components suspended or dissolved in one or more pharmaceutically acceptable carriers. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2- octyl dodecanol, benzyl alcohol and water. Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Patches may also be used. The compositions of this invention may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well- known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents.

[0101] In some embodiments, the antimicrobial peptide or polynucleotide of the present invention can be conjugated to at least one other molecule. Typically, said molecule is selected from the group consisting of polynucleotides, polypeptides, lipids, lectins, carbohydrates, vitamins, cofactors, and drugs.

[0102] In some embodiments, the antimicrobial peptide or polynucleotide of the present invention is formulated with lipidoids. The synthesis of lipidoids has been extensively described (see Mahon et al., Bioconjug Chem. 2010 21 : 1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci US A. 2011 108: 12996-3001). While these lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561- 569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105: 11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107: 1864-1869; Leuschner et al., Nat Biotechnol. 2011 29: 1005-1010), the present disclosure describes their formulation and use in delivering polynucleotides.

[0103] In some embodiments, the antimicrobial peptide or polynucleotide of the present invention is formulated using one or more lipid-based structures that include but are not limited to liposomes, lipoplexes, or lipid nanoparticles (Paunovska, Kalina, David Loughrey, and James E. Dahlman. "Drug delivery systems for RNA therapeutics." Nature Reviews Genetics (2022): 1-16).

[0104] Liposomes are artificially prepared vesicles which can primarily be composed of a lipid bilayer and can be used as a delivery vehicle for the administration of pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which can be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which can be between 50 and 500 nm in diameter. Liposome design can include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations. As a non-limiting example, liposomes such as synthetic membrane vesicles are prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372. In some embodiments, the liposomes are formed from 1, 2-di oleyloxy -N,N- dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (as described in US20100324120) and liposomes which can deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.). The polypeptide of polynucleotide of the present invention can be encapsulated by the liposome and / or it can be contained in an aqueous core which can then be encapsulated by the liposome (see International Pub. Nos. W02012031046, W02012031043, W02012030901 and W02012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684).

[0105] In some embodiments, the polynucleotide of the present invention is formulated with stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6: 1438- 1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2: 1002- 1007; Zimmermann et al., Nature. 2006 441 : 111-114; Heyes et al. J Contr Rel. 2005 107:276- 287; Semple et al. Nature Biotech. 2010 28: 172-176; Judge et al. J Clin Invest. 2009 119:661- 673; deFougerolles Hum Gene Ther. 2008 19: 125-132; U.S. Patent Publication No US20130122104). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1,2- dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2- distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2- dilinolenyloxy-3 -dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0106] In some embodiments, the polynucleotide of the present invention is formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930. Lipid nanoparticle formulations typically comprise a lipid such as , in particular, an ionizable cationic lipid, and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. The lipid can be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2- DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In some embodiments, the lipid is a cationic lipid such as, but not limited to, DLin-DMA, DLin-D- DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid can be the lipids described in and / or made by the methods described in US Patent Publication No. US20130150625. As a non-limiting example, the cationic lipid can be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-l- yloxy]methyl}propan-l-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en- l-yloxy]-2-{[(9Z)-octadec-9-en-l-yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3 -[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]-2-

[0107] [(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3- [(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy ] -2- { [(9Z, 12Z)-octadeca-9, 12 -di en- 1 - yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof. Nanoparticle formulations of the present disclosure can be coated with a surfactant or polymer in order to improve the delivery of the particle. In some embodiments, the nanoparticle is coated with a hydrophilic coating such as, but not limited to, PEG coatings and / or coatings that have a neutral surface charge. The hydrophilic coatings can help to deliver nanoparticles with larger payloads such as, but not limited to, polynucleotides within the central nervous system. As a non-limiting example nanoparticles comprising a hydrophilic coating and methods of making such nanoparticles are described in US Patent Publication No. US20130183244.

[0108] The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

[0109] FIGURES:

[0110] Figure 1. Activity screening against B. subtilis. AJ Peptides with antimicrobial activity. B / Peptides with no antimicrobial activity. Associated slowed / stopped growth curves for B. subtilis. All (including control) AMPs were produced using CFPS and no peptide purification was carried out prior to the activity test. Growth curves (OD600 0-0.45 over time 4-20 h for all) are the average of n = 3 independent experiments.

[0111] Figure 2 showing the LLPS activity of the 40 synthetic peptides.

[0112] EXAMPLE: Methods:

[0113] Generator variational autoencoder (VAE)

[0114] We used VAEs because they have previously been used for de novo AMP design (Dean SN, Walper SA. Variational autoencoder for generation of antimicrobial peptides. ACS Omega. 2020;5:20746-20754. doi: 10.1021 / acsomega.0c00442'). The generative VAE consists of an encoder, a latent vector, and a decoder. The encoder feeds the input data (one-hot encoded amino acid letter of peptides) into a latent vector that is an information bottleneck, and the decoder aims to reconstruct the input data from the latent vector. Training the VAE and minimizing the difference between the input data and the reconstructed data acts twofold; the encoder learns to map the training dataset into a lower-dimensional space and the decoder learns to generate samples similar to the training data from any vector in the latent space. Thus, each peptide in the training dataset lands on a point in the multi-dimensional latent space. Picking vectors from the empty regions in this space and feeding them into the decoder yield peptide sequences that share the same grammar but are novel and not seen in the training dataset. After pretraining and training (transfer learning), we generated new AMPs by sampling from the latent space using different strategies in particular by exploring the neighborhood of a control functional AMP (e.g. LI AMP), gradient descent, or random sampling.

[0115] Cell-free production and activity test of AMPs

[0116] DNA fragments encoding AMPs were designed with T7 promoter as disclosed in Pandi, Amir, et al. "Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides." Nature Communications 14.1 (2023): 7197. B. subtilis V 79 was used as representative of Gram-positive bacteria. From LB agar plates into LB medium, three overnight cultures for each strain were made from three different colonies and grown while shaking at 37 °C. The next day, each was subcultured in LB (1 : 1000) and grown while shaking at 37 °C to OD ~ 1. Cells were diluted in LB to 104 cfu mL-1, and 16 pL of diluted cells were added to wells of a 384-well plate (Greiner Bio-One, #781185) in which 4 pL of the cell-free reaction mix (with AMPs produced) had been added beforehand. OD600 was measured every 10 min in a plate reader (Tecan Infinite® 200 PRO) shaking at 37 °C for 20 h. Growth curves were analysed for AMPs impairing bacterial growth. We analysed plates both by visual investigation of the microplates after 20 h as well as by visual analysis of the growth curves looking for stopped or slowed growth plotted OD600 over time compared to the controls. Results:

[0117] We applied our previous variational autoencoder (VAE) model (Pandi, Amir, et al. "Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides." Nature Communications 14.1 (2023): 7197), to design 40 peptides with potential LLPS and antimicrobial activity from LI AMP (SEQ ID NO:1) disclosed in Pandi, Amir, et al.

[0118] "Cell-free biosynthesis combined with deep learning accelerates de novo-development of antimicrobial peptides." Nature Communications 14.1 (2023): 7197. This was achieved through gradient descent exploration of the latent space (using VAE’s decoder) at the vicinity of L1AMP. We identified 20 peptides having both antimicrobial and LLPS activities (Figures 1 and 2)

[0119] We also assessed the antimicrobial activity of AMPs against E. coli and B. subtilis (as representatives of gram-negative and gram-positive bacteria, respectively) at two different concentrations of 25 and 125 pM, without bacteria (only peptide solution), and with bacteria. We showed that AMPs, by inducing LLPS, enhance their ability to encapsulate and kill bacteria within droplets or act collectively to kill bacteria. This collective action boosts antimicrobial potency and may protect AMPs from degradation or enzymatic digestion in physiological conditions (Data not shown).

[0120] Table 1: synthetic peptides of the present disclosure. REFERENCES:

[0121] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

1. CLAIMS:

1. An antimicrobial peptide that comprises the consensus sequence of for formula (I)MKx 1KKX2FKX3LFKTFFKKGX4GX5X6KKSX7FX8X9X 10 SKx 11 Qx 12KKKx 13 G xl4Wxl5xl6xl7KD (I) whereinXI represents R, N, or AX2 represents A or WX3 represents F, S or AX4 represents P, K or QX5 represents G, S or LX6 represents F or YX7 represents V or SX8 represents N or RX9 represents K or VXI 0 represents R or EXI I represents T or SXI 2 represents F, V or MXI 3 represents F, L, A, or GXI 4 represents G or IXI 5 represents V or AXI 6 represents G or A, andXI 7 represents F or L.

2. The antimicrobial peptide according to claim 1 that is capable of inducing liquid-liquid phase separation.

3. The antimicrobial peptide according to claim 1 or 2 wherein XI represents R.

4. The antimicrobial peptide according to claim 1 or 2 wherein X2 represents A.

5. The antimicrobial peptide according to claim 1 or 2 wherein X3 represents F.

6. The antimicrobial peptide according to claim 1 or 2 wherein X4 represents P.

7. The antimicrobial peptide according to claim 1 or 2 wherein X5 represents G.

8. The antimicrobial peptide according to claim 1 or 2 wherein X6 represents F.

9. The antimicrobial peptide according to claim 1 or 2 wherein X7 represents V.

10. The antimicrobial peptide according to claim 1 or 2 wherein X8 represents N.

11. The antimicrobial peptide according to claim 1 or 2 wherein X9 represents K.

12. The antimicrobial peptide according to claim 1 or 2 wherein X10 represents R.

13. The antimicrobial peptide according to claim 1 or 2 wherein XI 1 represents T.

14. The antimicrobial peptide according to claim 1 or 2 wherein X12 represents F.

15. The antimicrobial peptide according to claim 1 or 2 wherein X13 represents F.

16. The antimicrobial peptide according to claim 1 or 2 wherein X14 represents G.

17. The antimicrobial peptide according to claim 1 or 2 wherein XI 5 represents V.

18. The antimicrobial peptide according to claim 1 or 2 wherein X16 represents G.

19. The antimicrobial peptide according to claim 1 or 2 wherein X17 represents F.

20. The antimicrobial peptide according to any one of claims 1 to 20 wherein the amino acid sequence having at least 90% of identity with the amino acid sequence of L1.3AMP, L1.5AMP, L1.7AMP, L1.8AMP, L1.10AMP, LI. HAMP, L1.15AMP, L1.18AMP, L1.19AMP, L1.22AMP, L1.25AMP, L1.26AMP, L1.27AMP, L1.31AMP,L1.34AMP L1.35AMP, L1.36AMP, L1.39AMP or L1.40AMP (SEQ ID NO: 4, 6, 8, 9, 11, 12, 16, 19, 20, 23, 26, 27, 28, 32, 35, 36, 37, 40 and 41).

21. A polynucleotide that encodes one or more antimicrobial peptide(s) according to any one of claims 1 to 20.

22. The polynucleotide according to claim 1 that is a messenger RNA (mRNA).

23. A host cell that has been transfected, infected or transformed by the polynucleotide according to claim 21 or 22.

24. A method of treating a bacterial infection in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the antimicrobial peptide according to any one of claims 1 to 20 or the polynucleotide of claim 22.

25. A method of removing a biofilm on a surface comprising administration of an effective amount of the antimicrobial peptide according to any one of claims 1 to 20.

26. A composition comprising an amount of one or more antimicrobial peptide(s) according to any one of claims 1 to 20 or the polynucleotide according to claim 21 or 22.

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