Transient expression by lipid nanoparticle formulations

Abstract

The present invention relates to means and methods for transient expression of a protein of interest by non-viral formulations such as lipid nanoparticle (LNP) formulations.

Description

TRANSIENT EXPRESSION BY LIPID NANOPARTICLE FORMULATIONSFIELD OF INVENTION

[0001] The present invention relates to means and methods for transient expression of a protein of interest by non-viral formulations, such as lipid nanoparticle (LNP) formulations. In particular, the present invention relates to means and methods for transient expression in the mammary gland.BACKGROUND OF INVENTION

[0002] The production of proteins, in particular bioactive proteins, represents a major need in the industry and the general population. Unfortunately the activity of such proteins is often dependent upon complex post-translational processing and may thus require sophisticated eukaryotic expression systems.

[0003] In that context, the progress made over the years in the biotechnology field, recombinant DNA technology and gene transfer were significant. In particular, expression of recombinant proteins by the mammary gland, or cells thereof, has been reported.

[0004] Yang et al. (‘ ‘Adenoviral-mediated gene transfer into primary human and mouse mammary epithelial cells in vitro and in vivo” Cancer Letters 98 (1995) 9-17) reports a concentration-dependent adenoviral-mediated transfer expression of lacZ gene in human mammary epithelial cells in monolayer culture.

[0005] WO96 / 22379A2 teaches the expression of a nucleic acid (DNA) in the mammary gland of a ruminant mammal, including a step of infusing a liquid complex including a genetic construct into a ductal tree of the mammal.

[0006] W02004 / 034780A2 teaches the production of bioactive heterologous proteins; specifically of biopharmaceutical interest proteins using the mouse mammary gland as bioreactor and replication defective or helper dependent adenoviral vectors for gene transfer and expression of recombinant protein in milk.

[0007] WO2016 / 037297A1 teaches the infusion of an Adeno- Associated Virus (AAV) vector including an expression cassette for a recombinant fusion protein into the mammary gland of goats and recovery of milk from the animal.

[0008] However, viral-based techniques may induce inflammation and necessitate the production of substantial virus quantities, as reported by Russell et al. (Journal of Virology, May 2003, p. 5801-5809). Also, viral-based techniques are less prone to automatization, they may be labelled as a biohazard, and require significant optimization.

[0009] Hence, there remains a need for means and methods for protein expression in mammalian cells and mammalian hosts, with limited inflammation and immunologic reactions.

[0010] There also remains a need for means and methods for protein expression which do not require extensive surgery and which are compatible with animal well-being.

[0011] There also remains a need for means and methods for protein expression which are sufficiently stable over time, in particular over days.

[0012] In particular there remains a need for means and methods for protein expression which are compatible with industrialization or pre-industrialization production scales.

[0013] The invention has for purpose to meet the above-mentioned needs.SUMMARY

[0014] According to a first main embodiment, the invention relates to an in vitro method for producing proteins comprising a step of:a) providing a mammary gland epithelium, or cell thereof, for example previously obtained from a lactating mammal; b) bringing said epithelium or cell thereof in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest; c) optionally recovering the protein of interest.

[0015] According to a second main embodiment, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal; b) bringing the mammary gland epithelium of the lactating mammal, or cell thereof, in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

[0016] According to a third main embodiment, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal previously administered with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest, the said LNP or composition being brought in contact with all or part of the mammary gland epithelium, or cell thereof; b) recovering the protein of interest.

[0017] According to a fourth main embodiment, the invention relates to a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

[0018] According to a fifth main embodiment, the invention relates to an in vitro method for producing proteins comprising a step of a kit for producing proteins, comprising: (i) lipid nanoparticles (LNP), or a composition thereof, comprising amessenger RNA suitable for encoding a protein of interest, and (ii) means for administration to a mammary gland epithelium, or cell thereof.DEFINITIONS

[0019] In the present invention, the following terms have the following meanings:

[0020] “About” preceding a figure means plus or less 10% of the value of said figure.

[0021] “And / or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”).

[0022] “At least one” includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 250, 500, 750, 103,104, 105, 106, 107, 108, 109, 1010, 1011, 1012, 1013, 1014, 1015or more.

[0023] “Comprising”, “comprises” and “comprised of’ are used herein are synonymous with “including”, “includes” or “containing”, “contains”, and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. These terms also encompass “consisting of’.

[0024] The term “identity” when used in a relationship between the sequences of two or more polypeptides or of two or more nucleic acid molecules, refers to the degree of sequence relatedness between polypeptides or nucleic acid molecules, as determined by the number of matches between strings of two or more amino acid or nucleotide residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., “algorithms”). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York,1991; and Carillo et al., SIAM J. Applied Math. 48, 1073 (1988). Preferred methods for determining identity are designed to give the largest match between the sequences tested. Methods of determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG program package, including GAP (Devereux et al., Nucl. Acid. Res. \2, 387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., supra). The well-known Smith Waterman algorithm may also be used to determine identity. In one embodiment, the term identity is measured over the entire length of the sequence to which it refers.

[0025] “Nucleic acid” or “polynucleotide” refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. “Nucleic acid” or “Polynucleotides” include, without limitation single-and doublestranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double- stranded RNA, and RNA that is a mixture of single- and double- stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double- stranded regions. In addition, “Nucleic acid” or “polynucleotide” refers to triplestranded regions comprising RNA or DNA or both RNA and DNA. The term “nucleic acid” or “polynucleotide” also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications has been made to DNA and RNA; thus, “nucleic acid” or “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. “Polynucleotide” also embraces relatively short polynucleotides, often referred to as oligonucleotides.

[0026] As used herein, the term “messenger RNA (mRNA)” refers to a polyribonucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified mRNA. mRNA may contain one or more coding and non-coding regions. mRNA can be purified from natural sources, produced using recombinant expression systems and optionally purified, in vitro transcribed, or chemically synthesized. The term “messenger RNA (mRNA)” may thus encompass coding RNAs further comprise additional genetic elements, for example genetic elements that are required for RNA replication, for example self-replicating messenger RNAs.

[0027] As used herein, the terms “modification” and “modified” as such terms relate to the nucleic acids provided herein, include at least one alteration which preferably enhances stability and renders the mRNA more stable (e.g., resistant to nuclease digestion) than the wild-type or naturally occurring version of a nucleic acid (e.g a mRNA).

[0028] As used herein, the terms “stable” and “stability” as such terms relate to the nucleic acids of the present invention, and particularly with respect to the mRNA, refer to increased or enhanced resistance to degradation by, for example nucleases (i.e., endonucleases or exonucleases) which are normally capable of degrading such mRNA. Increased stability can include, for example, less sensitivity to hydrolysis or other destruction by endogenous enzymes (e.g., endonucleases or exonucleases) or conditions within the target cell or tissue, thereby increasing or enhancing the residence of such mRNA in the target cell, tissue, subject and / or cytoplasm. The stabilized mRNA molecules provided herein demonstrate longer half-lives relative to their naturally occurring, unmodified counterparts (e.g. the wild-type version of the mRNA). Also contemplated by the terms “modification” and “modified” as such terms related to the mRNA of the present invention are alterations which improve or enhance translation of mRNA nucleic acids, including for example, the inclusion of sequences which function in the initiation of protein translation (e.g., the Kozac consensus sequence). (Kozak, M., Nucleic Acids Res 15 (20): 8125-48 (1987)).

[0029] As used herein, the term “chemical modifications”, with respected to modified messenger RNAs, includes modifications which introduce chemistries which differ fromthose seen in naturally occurring mRNA, for example, covalent modifications such as the introduction of modified nucleotides, (e.g., nucleotide analogs, or the inclusion of pendant groups which are not naturally found in such mRNA molecules). In addition, suitable modifications include alterations in one or more nucleotides of a codon such that the codon encodes the same amino acid but is more stable than the codon found in the wildtype version of the mRNA.

[0030] “Protein”, “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified by, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labelling component. As used herein the term “amino acid” includes natural and / non-natural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetic s .

[0031] “T ransfection’ ’ refers to a process by which exogenous nucleic acid and / or protein are transferred or introduced into a host cell, in particular a host cell of eukaryotic origin. A “transfected” host cell is one which has been manipulated so as to incorporate the exogenous nucleic acid or protein. The cell includes the primary subject cell and its progeny. As used herein, “viral-mediated transfection” and “transduction” are considered as synonymous.

[0032] The term “vector” is meant to refer to any vehicle carrying a foreign nucleic sequence into a cell, for replication and / or expression. Hence, the term is susceptible to encompass both a replication vector and an expression vector; the two embodiments not being mutually exclusive.

[0033] The term “expression vector” is meant to encompass any vector which is susceptible to allow expression of a nucleic acid sequence within a given expression system; which may thus include transcription of the corresponding nucleic acid sequence, or alternatively both transcription and translation of the corresponding nucleic acidsequence. Such expression vectors generally include at least one - or more - promoter region (e.g. inducible or non-inducible promoter region) and at least one - or more - terminator and / or regulatory region, although not necessarily. For example, expression vectors may be part of expression systems including one or more than one (e.g. a plurality) of vector (e.g. as part of a dual expression system).

[0034] As used herein, the term “lipid” refers to any compound having a balance of hydrophobic and hydrophilic groups, which is capable of forming a bilayer such that a hydrophobic portion of the lipid material orients toward the bilayer while a hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of hydroxyl, 8hosphate, phosphono, carboxylic, sulfato, amino, sulfhydryl, guanadino, and other like groups. Hydrophobicity is conferred by the inclusion of groups that include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, aliphatic fluorocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic or heterocyclic group(s).

[0035] As used herein, the term “hydrophobic chain” refers to a hydrophobic group or hydrocarbon chain comprised of alkyl, heteroalkyl, aryl, or heteroaryl moieties in any combination. Exemplary alkyl groups are a hydrocarbon or fluorocarbon group having from 6 to 24 carbon atoms, containing any number of branch points, cyclic structures or points of unsaturation alone such as the cis-9-octadecene (oleoyl) or cis,cis-9,12- octadecadiene (linoleyl) or in combination such as the oleyl and linoleyl. A hydrophobic group also includes the fused ring system of cholesterol and other sterols.

[0036] As used herein, the term “lipid nanoparticle” or “LNP” refers to a nano-sized transfer vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG- modified lipids).

[0037] As used herein, the term “nanoparticle size” or “nanoparticle diameter” refers to the hydrodynamic diameter as measured by dynamic light scattering; for example using a zeta sizer or nanoparticle tracking analysis (NTA).

[0038] As used herein, the term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at physiological pH. Such lipids include, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol and DMRIE.

[0039] A « lipid aggregate-forming cationic lipid » and the like refer, in the usual and customary sense, to a net positively charged lipid which can facilitate the formation of lipid aggregates. The term « lipid aggregate » refers to a lipid structure including a plurality of lipids or type of lipids, forming a higher order structure (e.g., secondary, tertiary or quaternary structure). Non-limiting examples of lipid aggregates include liposomes, unilamellar vesicles, multilamellar vesicles, micelles, amorphous aggregates, and the like. The lipid aggregates of the present invention can contain any suitable lipid, including cationic lipids, zwitterionic lipids, neutral lipids, or anionic lipids.

[0040] As used herein, the term “ionizable lipid” refers to any of a number of lipid species that are protonated at a low pH (i.e. lower than a physiological pH), which makes them positively charged, but remain neutral at physiological pH.

[0041] As used herein, the term “zwitterionic lipid,” refers to a lipid comprised of a zwitterionic head group and one or more hydrophobic chains linked through one or more covalent bonds.

[0042] As used herein, the term “zwitterionic head group” refers to a multi-functional group, which at a physiological pH is zwitterionic and at other lower pH within the same range is cationic. The cationic and negative charges are either permanent or pH dependent. The cationic nature may arise from any number of primary, secondary, tertiary or quaternary amines, guanadines or non-nitrogenous cations, e.g., phosphonium, sulfonium, etc. The anionic portion can consist of any number of sulfonates, phosphates, phosphonates or carboxylates.

[0043] As used herein, the term “neutral” refers to any of a number of lipid species which exist in an uncharged form. Such lipids include, for example diacylglyceride, tocopherol and cholesterol.

[0044] As used herein, the term “non-cationic lipid” refers to any neutral lipid as described above as well as anionic lipids. Examples of anionic lipids include cardiolipin, diacylphosphatidylglycerol, diacylphosphatidylserine and diacylphosphatidic acid.

[0045] As used herein, an « enhancer element » as provided herein refers to a compound (e.g. a peptide or otherwise) which facilitates transfection, thereby increasing transfection efficiency. Therefore, the transfection efficiency of a nucleic acid, polypeptide or complex thereof into a cell is higher in the presence of an enhancer element compared to the transfection efficiency in the absence of said enhancer element.

[0046] As used herein, the term “encapsulate” refers to coating of various substances within another material at sizes on the nano scale. The encapsulated material is referred to as the internal phase. The encapsulation material is known as the external phase, the shell, coating or membrane. Hence this term may refer to liponanoparticles having an external phase (membrane) including, for example phospholipids, and an internal phase (core).

[0047] As used herein, the term “mammary gland epithelium” refers to all or part of the epithelium forming the said mammary gland, including the epithelium comprising all or parts of the lactiferous duct system; the term may thus comprise all or parts of the epithelium and / or epithelial cells forming the ducts and ductal system. Ducts are generally composed of a double layer of cuboidal epithelial cells. Mammary ducts are lined by a single luminal layer of columnar or cuboidal epithelial cells surrounded by a discontinuous layer of contractile myoepithelial cells, in turn surrounded by a basement membrane. The ductal system is further composed of a small, central cistern and a branched network of blind ducts radiating from the cistern. The ducts terminate in groups of cells known as “buds” or “caps”. At the end of the ductal tree are terminal end buds (TEB) which allow the canals to grow and invade the breast stroma. Those buds do not secrete milk but comprise body cells which become internal lumen cells, and caps which become myoepithelial cells. In goats, the ductal system is relatively advanced at birth, with substantial branching already present. In mice, the mammary epithelium is rudimentary, consisting of only a few small ducts that grow allometrically until puberty.

[0048] As used herein, the term "ductal tree," is meant the branched network of tubular structures which conduct milk in a mammary gland.

[0049] As used herein, the term "streak canal" is meant the papillary duct at the lower end of the teat which leads to the ductal tree and which, typically in conjunction with a sphincter muscle, holds milk within the gland cistern between milkings.

[0050] As used herein, the term "milk protein", is meant any of the various proteins (e.g., P-casein, P -lactoglobulin and lactoferrin) which is secreted by the ductal tree and alveolar cells of a mammary gland.

[0051] As used herein, the term “rodent” includes mammals of the order Rodentia; in particular mammals belonging to the genus Mus or Rattus.

[0052] As used herein, the term “ruminant” includes mammals that are capable of acquiring nutrients from plant-based food by fermenting it in a specialized stomach (rumen) prior to digestion, principally through microbial actions. Ruminants include bovidae, in particular cattle, goats, sheep, giraffes, yaks, deer, antelope, and others.

[0053] As used herein, the term “bovid” includes any member of family Bovidae, which include hoofed mammals such as antelope, sheep, goats, and cattle, among others.

[0054] As used herein, the term “lactating mammal” is meant to refer to any mammal which is mature enough to be capable of producing milk. Hence, this term may encompass any mammal which is capable of producing milk; either with or without stimulation, regardless of the amount effectively produced.

[0055] As used herein, the term “secreted” refers to protein that is detected outside the target cell, in extracellular space. In particular, the protein may be detected in milk or a fraction thereof.

[0056] As used herein, the term “produced” in its broadest sense refers the translation of at least one mRNA into a protein (e.g. an enzyme, an antibody or an antigen-binding fragment thereof).

[0057] As used herein, the term “transfer vehicle” includes any of the standard pharmaceutical carriers, diluents, excipients and the like which are intended for use in connection with the administration of biologically active agents. In the sense of the present disclosure, lipid nanoparticles can be considered as transfer vehicles for messenger RNAs.DETAILED DESCRIPTION

[0058] The inventors have identified a novel approach for transient protein expression, which is considered as efficient expected to circumvent inflammation and immune responses commonly found with viral-based alternatives such as AAV-based protein expression.

[0059] This is achieved by injecting lipid nanoparticle (LNP) formulations containing a messenger RNA (mRNA) encoding a protein of interest into the mammary gland of a lactating mammal.

[0060] Advantageously, the administration of lipid nanoparticle (LNP) formulations into the ductal tree of the mammary gland is found to be compatible with production of proteins, without the disadvantages associated to previously reported viral vectors.

[0061] The resulting transient expression of the protein of interest is found to be compatible with pre-industrial and industrial application.

[0062] A proof-of-concept is provided, in the form of a mouse and goat model, as a validation of the versality of the approach.

[0063] This is surprising, because to the knowledge of the inventors, production of heterologous protein from the mammary gland, as a bioreactor, was never reported after local liponanoparticle (LNP)-based mRNA injections. In that regard, and in spite of a long-felt need, previous approaches relied mostly on viral vectors.

[0064] In particular, the inventors have now provided evidence that the production of heterologous proteins (e.g. antibodies), including therapeutic antibodies, can then be recovered from the produced milk in detectable quantities. Advantageously, the recovered heterologous proteins remain functional.

[0065] Hence, according to a first main embodiment, the invention relates to an in vitro method for producing proteins comprising a step of: a) providing a mammary gland epithelium, or cell thereof, for example previously obtained from a lactating mammal; b) bringing said epithelium or cell thereof in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest; c) optionally recovering the protein of interest.

[0066] According to a second main embodiment, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal; b) bringing the mammary gland epithelium of the lactating mammal, or cell thereof, in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

[0067] According to a third main embodiment, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal previously administered with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest, the said LNP or composition being brought in contact with all or part of a mammary gland epithelium, or cell thereof; b) recovering the protein of interest.

[0068] In particular, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal; b) bringing the mammary gland epithelium of the lactating mammal, or cell thereof, in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest; c) recovering the protein of interest.

[0069] A lactating mammal, according to the present disclosure and as developed hereafter, refers most preferably to a non-human mammal.

[0070] The mammal may be selected from the group consisting of a ruminant mammal or a non-ruminant mammal (e.g. a non-human non-ruminant mammal). According to some embodiments, the mammal is a ruminant mammal. According to some embodiments, the mammal is a non-ruminant mammal. According to some embodiments, the mammal is a rodent or a ruminant mammal; for example a mouse, a rat, cattle, a sheep, a goat, a rabbit.

[0071] According to some embodiments, the mammal belongs to the family of Bovidae; in particular selected from cattle, yaks, bisons, buffalos, antelopes, sheep and goats.

[0072] According to some embodiments, the mammal belongs to the genus Capra in particular selected from a domestic goat (Capra hirciis), Capra sibirica, Capra falconeri, Capra aegagrus, Capra cylindricornis, Capra caucasica, Capra ibex, Capra pyrenaica, Capra nubiana, Capra walie.

[0073] According to some embodiments, the mammal is a pregnant mammal; for example a pregnant rodent or pregnant ruminant mammal. According to some other embodiments, the mammal is a non-pregnant mammal; for example a non-pregnant rodent or pregnant ruminant mammal.

[0074] According to some embodiments, the LNP or composition thereof is brought in contact with the mammary gland epithelium, or cell thereof, by administration in all or part of the ductal tree of the mammary gland.

[0075] According to some embodiments, the method for producing proteins includes a step of raising the lactating mammal under conditions sufficient for the protein to be present in the milk produced by the mammary gland; in particular raising the lactating mammal for at least 1 day before recovering the milk and / or protein thereof.

[0076] Milk, from treated lactating mammals, can be obtained, in particular, by conventional manual or mechanized milking methods. As used herein, the term “raising the lactating mammal” may thus refer to the minimum time required for the lactating mammal to express the protein encoded by the messenger RNA under conditions sufficient for the protein to be present in the milk produced by the mammary gland.

[0077] This minimum time may vary from one lactating mammal to the other, from a same species or from different species. This term may also depend on whether the production of milk in the lactating mammal is stimulated or not. Hence, this term may correspond to:(i) the minimal time required for the lactating mammal to express the protein encoded by the messenger RNA under conditions sufficient for the protein to be present in the milk produced by the mammary gland, while stimulating the lactation of the mammal; and / or(ii) the minimal time required for the lactating mammal to express the protein encoded by the messenger RNA under conditions sufficient for the protein to be present in the milk produced by the mammary gland, without stimulating the lactation of the mammal.

[0078] According to said embodiments of the methods, the lactating mammal (e.g. the cattle, or goat) is raised for at least 1, 2, 3, 4, 5, 6 or 7 day(s) following the administration of the LNP, or composition thereof, before recovering the milk and / or protein thereof.

[0079] According to some embodiments of the methods, the lactating mammal (e.g. the cattle, or goat) is raised for less than 15 days following the administration of the LNP, or composition thereof, before recovering the milk and / or protein thereof; for example less than 15, 14, 13, 12, 11 or 10 days following the administration of the LNP, or composition thereof, before recovering the milk and / or protein thereof.

[0080] According to some embodiments, the method for producing proteins may include more than one step of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof.

[0081] For example, according to said embodiments, the method for producing proteins may include more than one step (i.e. a plurality of steps) of administering the lipid nanoparticle, or composition thereof, in all or part of the ductal tree.

[0082] For example, a step of administering the lipid nanoparticle, or composition thereof, in all or part of the ductal tree may comprise administration in the nipple or the streak canal.

[0083] According to some embodiments, the method may include a plurality of steps of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof; wherein each step of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof, is distant from at least 2 days.

[0084] According to some embodiments, the method may include a plurality of steps of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof; wherein each step of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof, is distant of 10 or less than 10 days; for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0085] According to some embodiments, the method may include a plurality of steps of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof; wherein each step of bringing the mammary glandepithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof, is distant from 2 to 10 days; for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.

[0086] According to some embodiments, the method may further include a step of stimulating the production of milk by the lactating mammal, either prior to the administration of the lipid nanoparticles or compositions thereof, or after the administration of the lipid nanoparticles or compositions thereof, or both.

[0087] According to some embodiments, the method for producing proteins may include a step of recovering the milk from the lactating mammal thereby recovering the protein.

[0088] According to some embodiments, the milk is recovered on or after a reference time, following the administration of LNPs (or a composition thereof) according to the invention, to the lactating mammal; the reference time being selected from a group consisting of: 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h, 61h, 61h, 62h, 63h, 64h, 65h, 66h, 67h, 68h, 69h, 70h, 71h, 72h, 73h, 74h, 75h, 76h, 77h, 78h, 79h, 80h, 81h, 82h, 83h, 84h, 85h, 86h, 87h, 88h, 89h, 90h or more.

[0089] According to some embodiments, the milk is recovered until a reference time, following the administration of LNPs to the lactating mammal; the reference time being selected from a group consisting of: 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h,34h, 35h, 36h, 37h, 38h, 39h, 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h,52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h, 61h, 61h, 62h, 63h, 64h, 65h, 66h, 67h, 68h,69h, 70h, 71h, 72h, 73h, 74h, 75h, 76h, 77h, 78h, 79h, 80h, 81h, 82h, 83h, 84h, 85h, 86h,87h, 88h, 89h, 90h.

[0090] According to some embodiments, the milk is recovered at most 24h following the administration of LNPs (or a composition thereof) according to the invention, to the lactating mammal.

[0091] According to some embodiments, the milk is recovered until a reference time, following the administration of LNPs to the lactating mammal; the reference time rangingfrom about 24h post-administration to about 90h post-administration. When multiple administrations are made to the lactating mammal, the reference time may, preferably, be calculated based on the last administration to the lactating mammal.

[0092] In particular, the method for producing proteins may advantageously include a step of recovering the protein from the milk of the lactating mammal; either in the form of a milk or milk-containing composition, or separately from the milk (e.g. after a separation step).

[0093] According to some embodiments, the method for producing proteins may include a step of separating the protein of interest from the milk recovered.

[0094] According to said embodiments, a method for producing (heterologous) proteins according to the present disclosure may thus comprise the steps of:(i) recovering milk from the lactating mammal; and(ii) separating the (heterologous) protein from the milk, thereby recovering the protein.

[0095] Milk, from lactating mammals according to the invention, can be obtained by conventional methods of manual or mechanized milking.

[0096] According to some non-mutually exclusive embodiments, milk production may be modulated; for example milk production may be increased before, on, or after having brought the mammary gland epithelium of the lactating mammal, or cell thereof, of the lactating mammal, in contact with the lipid nanoparticle (LNP), or composition thereof, comprising the messenger RNA.

[0097] According to some embodiments, milk production is artificially induced or maintained. For example milk production may be chemically and / or hormonally induced.

[0098] Hence, according to some embodiments, the methods for producing a protein according to the invention may further include a step of inducing or promoting milkproduction in a non-lactating mammal, thereby providing a lactating mammal, or maintaining milk production in the lactating mammal.

[0099] As used herein, a “lactation-inducing” or “lactation-promoting” compound may refer to any compound, in particular any exogenously administered compound, that is susceptible to treat or prevention insufficient milk production and / or milk ejection. Such “lactation-inducing” or “lactation-promoting” compounds may or may not be naturally-occuring. Examples of such lactation-inducing or lactation-promoting compounds may be selected from the group consisting of: oxytocin, an estrogen compound (e.g. estradiol), a dopaminergic antagonist, a progestogene (e.g. progesterone) compound, prolactin, a steroid (e.g. corticosteroid, for example prednisolone), combinations thereof, and derivatives thereof.

[0100] According to some embodiments, milk production may be induced by administering to the lactating mammal a lactation-inducing or lactation-promoting compound. Hence according to some particular embodiments, the lipid nanoparticle (LNP), or composition thereof is co-administered with a lactation-inducing or lactationpromoting compound. According to some particular embodiments, the lipid nanoparticle (LNP), or composition thereof is administered before the lactation-inducing or lactationpromoting compound. According to some particular embodiments, the lipid nanoparticle (LNP), or composition thereof is administered after the lactation-inducing or lactationpromoting compound.

[0101] According to some embodiments, milk production is hormonally induced. According to some alternative embodiments, milk production is not hormonally induced. According to some embodiments, milk production is mechanically induced. According to some alternative embodiments, milk production is not mechanically induced.

[0102] Hence, according to some embodiments, the invention relates to a method for producing proteins, comprising a step of: a) inducing or promoting milk production in a non-lactating mammal, in particular by administering a lactation-inducing or lactation-promoting compound to the non-lactating mammal, thereby providing a lactating mammal;b) bringing the mammary gland epithelium of the lactating mammal, or cell thereof, of the lactating mammal in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

[0103] Hence, according to some embodiments, the invention relates to a method for producing proteins, comprising a step of: a) providing a lactating mammal previously administered with a (i) lactationinducing or lactation-promoting compound and a (ii) lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest, the said LNP or composition being brought in contact with all or part of a mammary gland epithelium, or cell thereof; b) recovering the protein of interest.

[0104] According to exemplary embodiments, the lactation-inducing or lactationpromoting compounds may be selected from the group consisting of an estrogen compound, a progestogene (e.g. progesterone) compound, a steroid compound, combinations thereof, and derivatives thereof.

[0105] According to exemplary embodiments, the lactation-inducing or lactationpromoting compounds may be selected from the group consisting of: estradiol, progesterone, prednisolone, combinations thereof, and derivatives thereof.

[0106] According to a fourth main embodiment, the invention relates to a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

[0107] According to a fifth main embodiment, the invention relates to a kit, in particular for producing proteins, comprising: (i) lipid nanoparticles (LNP), or a composition thereof, comprising a messenger RNA suitable for encoding a protein of interest, and (ii) means for administration to a mammary gland epithelium, or cell thereof.

[0108] According to a sixth main embodiment, the invention relates to a composition comprising (i) lipid nanoparticles (LNP) comprising a messenger RNA suitable for encoding a protein of interest, and (ii) a lactation-inducing or lactation-promoting compound.

[0109] According to a seventh main embodiment, the invention relates to a kit, in particular for producing proteins, comprising: lipid nanoparticles (LNP) comprising a messenger RNA suitable for encoding a protein of interest; a lactation-inducing or lactation-promoting compound, or composition thereof.Messenger RN s (mRN s)

[0110] The mRNAs of the invention may encode, for example, a secreted hormone, enzyme, receptor, antibody, antigen-binding fragment thereof, such as an antibody heavy or light chain or any other fragment thereof, polypeptide, peptide or any other protein of interest, including secreted and non- secreted proteins.

[0111] According to some embodiments, the mRNA is a modified mRNA.

[0112] The mRNA may optionally have chemical or biological modifications which, for example, improve the stability and / or half-life of such mRNA or which improve or otherwise facilitate protein production.

[0113] Exemplary modifications to an mRNA may include the depletion of a base (e.g., by deletion or by the substitution of one nucleotide for another) or modification of a base, for example, the chemical modification of a base. Modified bases contemplated by the invention may, in a non-exhaustive manner, consist of nucleotide analogs (i.e. chemically or enzymatically modified bases other than the four canonical nucleobases adenine (A), cytosine (C), guanine (G), and uracil (U)).

[0114] According to some embodiments, the mRNA is a modified mRNA, which may comprise or more nucleotide analogs; for example those selected from the group consisting of: N6-methyladenosine (m6A), 5 -methylcytidine (m5C), N7-methylguanosine (m7G), N4-acetylcytidine (ac4C), N5-formylcytidine (f^C), pseudouridine (T), and T-O- methylation (Nm).

[0115] Exemplary modifications to an mRNA may include the incorporation of nonnucleotide linkages or modified nucleotides into the mRNA sequences of the present invention (e.g., modifications to one or both the 3' and 5' ends of an mRNA molecule encoding a protein of interest). Such modifications include the addition of bases to an mRNA sequence (e.g., the inclusion of a poly A tail or a longer poly A tail), the alteration of the 3' UTR or the 5' UTR, complexing the mRNA with an agent (e.g., a protein or a complementary nucleic acid molecule), and inclusion of elements which change the structure of an mRNA molecule (e.g., which form secondary structures).

[0116] The lipid nanoparticle, or composition thereof, comprising the mRNA may further comprise a stabilizing reagent (i.e. a reagent that binds directly or indirectly to, and stabilize the mRNA, thereby enhancing residence time in the target cell). Exemplary stabilizing reagents include one or more proteins, peptides, aptamers, translational accessory protein, mRNA binding proteins, and / or translation initiation factors.

[0117] Optionally, the mRNA may be hybridized to one or more complementary nucleic acid molecules (e.g., DNA or RNA). When the mRNA is hybridized to a complementary nucleic acid molecule, the hybridization may be on all or part of the corresponding mRNA.

[0118] It will be understood that any of the modifications and means described herein, in particular those for enhancing the stability of mRNA may be used either alone or in combination.

[0119] The messenger RNA, according to the present disclosure is suitable for encoding a protein of interest, which may be an autologous or heterologous protein (i.e. a protein that is not normally expressed in the corresponding cell). When the protein of interest isan heterologous protein, it may for example consist of a protein that is a mutated form of an autologous protein.

[0120] The messenger RNA, according to the present disclosure is suitable for encoding a protein of interest that is not normally present in the milk of a corresponding lactating mammal; or alternatively that is not normally present in substantial amounts in the milk of a corresponding lactating mammal.

[0121] The messenger RNA, according to the present disclosure is suitable for encoding a protein of interest, for example an heterologous protein and / or a protein selected from the group consisting of: antigens, growth factors (e.g. Human growth hormone, epidermal growth factor, insulin-like growth factor, grain colony stimulating factor, macrophages, nerve growth factor), erythropoietin, coagulation factors and related proteins (e.g. FI, FIT, Fill, FIV, FV, FVI, FVII, FVIII, FIX, FX, FXI, FXII, FXIII, von Willebrand factor), antibodies or fragments thereof such as antigen-binding fragments thereof, cytokines (Ex Interleukin 6 or Interleukin 2), al-antitrypsin, human serum albumin, beta-globin, plasminogen activator of plasminogen, tumor suppressor proteins (Ex P53), protein C, interferons.

[0122] According to some exemplified embodiments, the messenger RNA, according to the present disclosure is suitable for encoding antibodies, fragments of antibodies and / or antigen-binding fragments. The methods of the invention are particularly suitable for the production of therapeutic antibodies or antigen-binding fragments thereof. Advantageously, such antibodies or fragments thereof (therapeutic or not) may retain their ability to bind specifically to their target. When the antibodies to be produced (and optionally recovered, for example recovered from the produced milk) are therapeutic, they may be advantageously humanized antibodies.

[0123] Examples of antigen-binding fragments may be selected from the group consisting of a Fab, a F(ab)’2, a single domain antibody, a ScFv, a Sc(Fv)2, a diabody, a triabody, a tetrabody, an unibody, a minibody, a maxibody, a small modular immunopharmaceutical (SMIP), minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody as an isolated complementarydetermining region (CDR), and fragments which comprise or consist of the VL or VH chains.

[0124] According to some embodiments, the messenger RNA may code for one, or more than one protein of interest; for example one or more than one antibody, or antigenbinding fragment.

[0125] According to some embodiments, the messenger RNA may code for one, or more than one immunoglobulin; for example IgG, IgM, IgE, IgA or IgD.

[0126] According to some embodiments, the messenger RNA may code for one or more than one therapeutic antibody, or antigen-binding fragment thereof. According to some embodiments, the messenger RNA may code for a polypeptide having at least 80% of sequence identity with a therapeutic antibody, or antigen-binding fragment thereof; for example 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity.

[0127] Examples of therapeutic antibodies which may be considered for production, according to the methods of the invention, may, in a non-exhaustive manner, consist of: Abagovomab, Abatacept, Abciximab, Abituzumab, Abrilumab, Actoxumab, Adalimumab, Adecatumab, Aducanumab, Aflibercept, Afutuzymab, Alacizumab, Alefacept, Alemtuzumab, Alirocumab, Altumomab, Amatixumab, Anatumomab, Anetumab, Anifromumab, Anrukinzumab, Apolizumab, Arcitumomab, Ascrinvacumab, Aselizumab, Atezolizumab, Atinumab, Altizumab, Atorolimumab, Avelumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Begelomab, Belatacept, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Bezlotoxumab, Biciromab, Bimagrumab, Bimekizumab, Bivatuzumab, Blinatumomab, Blosozumab, Bococizumab, Brentuximab, Briakimumab, Brodalumab, Brolucizumab, Bronticizumab, Canakinumab, Cantuzumab, Caplacizumab, Capromab, Carlumab, Catumaxomab, Cedelizumab, Certolizumab, Cetixumab, Citatuzumab, Cixutumumab, Clazakizumab, Clenoliximab, Clivatuzumab, Codrituzumab, Coltuximab, Conatumumab, Concizumab, Crenezumab, Dacetuzumab, Daclizumab, Dalotuzumab, Dapirolizumab, Daratumumab, Dectrekumab, Demcizumab, Denintuzumab, Denosumab, Derlotixumab, Detumomab,Dinutuximab, Diridavumab, Dorlinomab, Drozitumab, Dupilumab, Durvalumab, Dusigitumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Eldelumab, Elgemtumab, Elotuzumab, Elsilimomab, Emactuzumab, Emibetuzumab, Enavatuzumab, Enfortumab, Enlimomab, Enoblituzumab, Enokizumab,Enoticumab, Ensituximab, Epitumomab, Epratuzomab, Erlizumab, Ertumaxomab, Etaracizumab, Etrolizumab, Evinacumab, Evolocumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzomab, Fasimumab, Felvizumab, Fezkimumab, Ficlatuzumab, Figitumumab, Firivumab, Flanvotumab, Fletikumab,Fontolizumab, Foralumab, Foravirumab, Fresolimumab, Fulramumab, Futuximab, Galiximab, Ganitumab,Gantenerumab, Gavilimomab, Gemtuzumab, Gevokizumab, Girentuximab,Glembatumumab, Golimumab, Gomiliximab, Guselkumab, Ibalizumab, Ibritumomab,Icrucumab, Idarucizumab, Igovomab, Imalumab, Imciromab, Imgatuzumab, Inclacumab,Indatuximab, Indusatumab, Infliximab, Intetumumab, Inolimomab, Inotuzumab, Ipilimumab, Iratumumab, Isatuximab, Itolizumab, Ixekizumab, Keliximab, Eabetuzumab, Eambrolizumab, Lampalizumab, Lebrikizumab, Lemalesomab, Lenzilumab, Lerdelimumab, Lexatumumab, Libivirumab, Lifastuzumab, Ligelizumab,Lilotomab, Lintuzumab, Lirilumab, Lodelcizumab, Lokivetmab, Lorvotuzumab,Lucatumumab, Lulizumab, Lumiliximab, Lumretuzumab, Mapatumumab,Margetuximab, Maslimomab, Mavrilimumab, Matuzumab, Mepolizumab.Metelimumab, Milatuzumab, Minetumomab, Mirvetuximab, MitumomabMogamulizumab, Morolimumab, Motavizumab, Moxetumomab, Muromonab-CD3,Nacolomab, Namilumab, Naptumomab, Namatumab, Natalizumab, Nebacumab, Necitumumab, Nemolizumab, Nerelimomab, Nesvacumab, Nimotuzumab, Nivolumab,Nofetumomab, Obiltoxaximab, Obinutuzumab, Ocaratuzumab, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Olokizumab, Omalizumab, Onartuzumab, Ontuxizumab, Opicinumab, Oportuzumab, Oregovomab, Orticumab, Otelixizumab, Oltertuzumab, Oxelumab, Ozanezumab, Ozoralizumab, Pagibaximab, Palivizumab, Panitumumab, Pankomab, Panobacumab, Parsatuzumab, Pascolizumab, Pasotuxizumab,Pateclizumab, Patritumab, Pembrolizumab, Pemtumomab, Perakizumab, Pertuzumab, Pexelizumab, Pidilizumab, Pinatuzumab, Pintumomab, Polatuzumab, Ponezumab, Priliximab, Pritumumab, Quilizumab, Racotumomab, Radretumab, Rafivirumab, Ralpancizumab, Ramucirumab, Ranibizumab, Raxibacumab, Refanezumab,Regavirumab, Reslizumab, Rilonacept, Rilotumumab, Rinucumab, Rituximab, Robatumumab, Roledumab, Romosozumab, Rontalizumab, Rovelizumab, Ruplizumab, Sacituzumab, Samalizumab, Sarilumab, Satumomab, Secukimumab, Seribantumab, Setoxaximab, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Sirukumab, Sofituzumab, Solanezumab, Solitomab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Suvizumab, Tabalumab, Tacatuzumab, Tadocizumab, Talizumab, Tanezumab, Taplitumomab, Tarextumab, Tefibazumab, Telimomab aritox, Tenatumomab, Teneliximab, Teplizumab, Tesidolumab, TGN 1412, Ticlimumab, Tildrakizumab, Tigatuzumab, TNX-650, Tocilizumab, Toralizumab, Tosatoxumab, Tositumomab, Tovetumab, Tralokimumab, Trastuzumab, TRBS07, Tregalizumab, Tremelimumab, Trevogrumab, Tucotuzumab, Tuvirumab, Ublituximab, Ulocuplumab, Urelumab, Urtoxazumab, Ustekimumab, Vandortuzumab, Vantictumab, Vanucizumab, Vapaliximab, Varlimumab, Vatelizumab, Vedolizumab, Veltuzumab, Vepalimomab, Vesencumab, Visilizumab, Volocixumab, Vorsetuzumab, Votumumab, Zalutumimab, Zanolimumab, Zatuximab, Ziralimumab, Ziv-Aflibercept, and Zolimomab.Lipid nanoparticles (LNPs)

[0128] The lipid nanoparticles are formulated to deliver one or more mRNA to one or more target cells.

[0129] The lipid nanoparticles (LNP), or composition thereof, according to the disclosure, comprise a messenger RNA suitable for encoding a protein of interest, as described above.

[0130] The messenger RNA is conjugated to the lipid nanoparticle (LNP), or is encapsulated within the lipid nanoparticle (LNP).

[0131] According to some embodiments, the messenger RNA is conjugated to the lipid nanoparticle (LNP). According to some embodiments, the messenger RNA is encapsulated within the lipid nanoparticle (LNP).

[0132] The lipid nanoparticles (LNP) of the present invention may comprise at least one lipid selected from the group consisting of: a cationic lipid, an ionizable lipid, a noncationic lipid, or conjugate thereof.

[0133] The lipid nanoparticles (LNP) of the present invention may comprise at least one lipid selected from the group consisting of: a cationic lipid, an ionizable lipid, or conjugate thereof.

[0134] According to some embodiments, the lipid nanoparticles (LNP) of the present invention comprise: at least one cationic lipid or ionizable lipid; at least one phospholipid; at least one sterol, for example at least one cholesterol.

[0135] According to some embodiments, the messenger RNA is conjugated to the lipid nanoparticle (LNP). According to some embodiments, the messenger RNA is encapsulated within the lipid nanoparticle (LNP).

[0136] According to some embodiments, the lipid nanoparticles (LNP) of the present invention comprise at least one conjugated lipid; in particular at least one PEGylated lipid.

[0137] According to some embodiments, the lipid nanoparticles (LNP) of the present invention comprise: at least one cationic lipid or ionizable lipid; at least one conjugated lipid, for example at least one PEGylated lipid; at least one phospholipid; at least one sterol, for example at least one cholesterol.

[0138] According to some embodiments, the lipid nanoparticles (LNP) of the present invention comprise:at least one cationic lipid or ionizable lipid; at least one conjugated lipid for example at least one PEGylated lipid; at least one phospholipid; cholesterol.

[0139] According to some embodiments, the lipid nanoparticles (LNP) of the present invention comprise: at least one cationic lipid or ionizable lipid; at least one PEGylated lipid; at least one phospholipid; at least one cholesterol.

[0140] Examples of suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides).

[0141] Also contemplated is the use of polymers as transfer vehicles, whether alone or in combination with other transfer vehicles. Suitable polymers may include, for example, polyacrylates, poly alkylcyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, dendrimers and polyethylenimine.

[0142] Examples of cationic lipids include those described in WO 2010 / 053572 or US4897355 or US 5171678 or US5334761 or WO 2010 / 042877 or WO2005 / 121348.

[0143] In some embodiments, the cationic lipid or ionizable lipid is selected from the group consisting of: N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (“DOTMA”) dioleoylphosphatidyl-ethanolamine (“DOPE”), 5- carboxyspermylglycinedioctadecylamide (“DOGS”), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (“DOSPA”), l,2-Dioleoyl-3-Dimethylammonium- Propane (“DODAP”), 1 ,2-Dioleoyl-3-Trimethylammonium- Propane (“DOTAP”), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (“DSDMA”), 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”), 1 ,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane (“DLinDMA”), 1 ,2-dilinoleyloxy-N,N-dimethyl-3- aminopropane (“DLenDMA”), N-dioleyl-N,N-dimethylammonium chloride (“DODAC”), N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”), N-(l,2- dimyristyloxypropyl-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12- octadecadienoxy)propane (“CLinDMA”), 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethyl l-l-(cis,cis-9',l-2'-octadecadienoxy)propane (“CpLinDMA”), N,N-dimethyl-3,4-dioleyloxybenzylamine (“DMOBA”), l,2-N,N'-dioleylcarbamyl-3- dimethylaminopropane (“DOcarbDAP”), 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine (“DLinDAP”), l,2-N,N-Dilinoleylcarbamyl-3- dimethylaminopropane (“DLincarbDAP”), 1 ,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (“DLinCDAP”), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]- dioxolane (“DLin-K-DMA”), 2, 2-dilinoleyl-4-dimethylaminoethyl[ 1,3] -dioxolane( “DLin-K-XTC2-DMA ”), 2-(2,2-di((9Z,12Z)-octadeca-9,12-dien-l-yl)-l,3-dioxolan-4- yl)-N,N-dimethylethanamine ( “DLin-KC2-DMA ”), Nl-[2-((lS)-l-[(3- aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4- di[oleyloxy] -benzamide (MVL5), N4-cholesteryl-spermine (GL67), (6Z,9Z,28Z,31Z)- heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino) butanoate (DLin-MC3- DMA;MC3), di((Z)- non-2- en-1- yl) 9-((4- (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), heptadecan-9- yl 8-((2- hydroxyethyl) (8-(nonyloxy)-8- oxooctyl)amino)octanoate (Lipid 5), heptadecan-9- yl 8- ((2- hydroxyethyl)(6- oxo-6-(undecyloxy)hexyl)amino) octanoate (Lipid H (SM-102)), ((4- hydroxybutyl)azanediyl)bis(hexane-6,l- diyl)bis(2- hexyldecanoate) (ALC-0315), l,l'-((2-(4-(2-((2-(bis(2- hydroxy dodecyl)amino)ethyl) (2- hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl) bis(dodecan-2- ol) (C12-200), tetrakis(8- methylnonyl) 3,3',3",3"'-(((methylazanediyl) bis(propane-3,l diyl))bis(azanetriyl))tetrapropionate (3060110), (azanetriyl))tetrapropionate (3060110), 3,6- bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine- 2,5- dione (cKK- E12), 3,6- bis(4-(bis((9Z,12Z)-2- hydroxyoctadeca-9,12- dien-l-yl)amino)butyl)piperazine-2,5-dione (OF-02), (((3,6- dioxopiperazine-2,5- diyl)bis (butane-4,1- diyl))bis(azanetriyl))tetrakis(ethane-2,l- diyl) (9Z,9'Z,9"Z,9"'Z,12Z,12'Z,12"Z,12"'Z)- tetrakis (octadeca-9,12-dienoate) (OF-Deg-Lin), (((3,6- dioxopiperazine-2,5- diyl)bis(butane-4,l- diyl))bis(azanetriyl))tetrakis (butane-4,1- diyl) (9Z,9'Z,9"Z,9"'Z, 12Z,12'Z,12"Z,12"'Z)- tetrakis (octadeca-9,12- dienoate) (OF-C4-Deg- Lin), N1,N3 ,N5- tris(3-(didodecylamino)propyl)benzene-l,3,5- tricarboxamide (TT3), Hexa(octan- 3- yl) 9,9',9",9"',9"",9"'"-((((benzene-l,3,5- tricarbonyl)ris(azanediyl)) tris (propane-3, 1- diyl))tris(azanetriyl))hexanonanoate (FTT5), ethyl 5,5- di((Z)- heptadec-8-en-l-yl)-l-(3- (pyrrolidin-1- yl)propyl)-2,5-dihydro-lH-imidazole-2-carboxylate (A2- Iso5-2DC18); 2- [(polyethylene glycol)-2000]-N,N- ditetradecylacetamide; P- sitosterol, (3S,8S,9S,10R,13R,14S,17R)-17-( (2R,5R)-5- ethyl-6- methylheptan-2- yl)-10,13- dimethyl-2,3,4,7,8,9,10,l l,12,13,14,15,16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-ol (ALC-0159); bis(2-(dodecyldisulfa-nyl)ethyl) 3,3'-((3- methyl-9- oxo-10- oxa-13,14- dithia-3,6- diazahexacosyl)azanediyl) dipropionate (BAME-O16B); 2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13- dimethyl- 17-((R)-6- methylheptan-2- yl)-2,3,4,7,8,9,10,l l,12,13,14,15,16,17-tetradecahydro-lH- cyclopenta[a]phenanthren-3-yl)oxy)carbonyl)amino)-N,N-bis(2- hydroxy ethyl)- N- methylethan- 1-aminiumbromide (BHEM-Cholesterol); 1,1 '-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-l- yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200); 3,6-bis(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5-dione (cKK-E12); 3P-[N-(N',N'- dimethylaminoethane)- carbamoyl]cholesterol (DC-Cholesterol); 2,3-dioleyloxy- N-[2- (sperminecarboxamido)ethyl]- N,N- dimethyl- 1- propanaminium trifluoroacetate (DOSPA); 1,2- dioleoyl-3- trimethylammonium- propane (DOTAP); 1,2- distearoyl-sn- glycero-3-phosphocholine (DSPC); Ethylphosphatidylcholine (ePC); 1,2-dimyristoyl- rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2000-DMG); Nl,N3,N5-tris(3- (didodecylamino)propyl)benzene-l,3,5-tricarboxamide (TT3); or combinations thereof.

[0144] In some embodiments, the cationic lipid or ionizable lipid is as described in Billingsley (“Ionizable Lipid Nanoparticle-Mediated mRNA Delivery for Human CAR T Cell Engineering”. Nano Lett. 2020; 20(3): 1578-1589) or W02021077067A1.

[0145] According to some embodiments, the cationic lipid or ionizable lipid is 1, 1 '-[[2- [2- [4- [2- [[2- [2- [Z?A(2-hydroxy tetradecyl) amino] ethoxy] ethyl] (2- hydroxytetradecyl)amino]ethyl]-l-piperazinyl]ethoxy]ethyl]imino]Z?A-2-tetradecanol corresponding compound C14-4 (CAS Number 2639634-80-1):

[0146] Examples of ionizable cationic lipid are further described in U.S. provisional patent application 61 / 617,468.

[0147] Also contemplated are cationic lipids such as the dialkylamino-based, imidazole- based, and guanidinium-based lipids. For example, certain embodiments are directed to a composition comprising one or more imidazole-based cationic lipids.

[0148] According to some embodiments, the cationic or ionizable lipid may be selected from the group consisting of: SM-102, ALC-0315, ALC-0159, C14-4 and combinations thereof.

[0149] According to some embodiments, the cationic or ionizable lipid may be selected from the group consisting of: SM-102, ALC-0315, ALC-0159, and combinations thereof.

[0150] According to some embodiments, the cationic or ionizable lipid may be selected from the group consisting of: ALC-0315, ALC-0159, and combinations thereof.

[0151] According to some embodiments, the cationic or ionizable lipid may be selected from the group consisting of: SM-102, ALC-0315, and combinations thereof.

[0152] According to some embodiments, the cationic or ionizable lipid comprise or consist of: SM-102. According to some embodiments, the cationic or ionizable lipid comprise or consist of: ALC-0315.

[0153] The use of cholesterol-based cationic lipids is also contemplated by the present invention. Such cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids. Suitable cholesterol-based cationic lipids include, for example, N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N- oleylamino-propyl)piperazine (DC-Chol).

[0154] In addition, several reagents are commercially available to enhance transfection efficiency. Suitable examples include LIPOFECTIN (DOTMA:DOPE) (Invitrogen, Carlsbad, Calif.), LIPOFECTAMINE (DOSPA:DOPE) (Invitrogen), LIPOFECTAMINE2000. (Invitrogen), FUGENE, TRANSFECTAM (DOGS), and EFFECTENE.

[0155] In some embodiments, the cationic or ionizable lipids are selected from the group consisting of: distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl- phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N- maleimidomethyl)-cyclohexane-l -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl- phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1- trans PE, l-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or a combination thereof.

[0156] According to some embodiments, the lipid nanoparticles (LNP) may comprise phospholipids, sterols, sterol derivatives, and / or polyethylene glycol (PEG) -modified lipids.

[0157] According to some embodiments, the lipid nanoparticles (LNP) may comprise phospholipids selected from the group consisting of: phosphatidylcholine, phosphatidylethanolamine, and derivatives thereof.

[0158] According to some embodiments, the cationic or ionizable lipids may be in combination with one or more phospholipids, sterols, sterol derivatives, and / or polyethylene glycol (PEG)-modified lipids.

[0159] According to some embodiments, the lipid nanoparticles (LNP) may comprise: at least one cationic lipid or ionizable lipid, in particular selected from: SM-102, ALC-0315 and / or ALC-0159; optionally at least one PEGylated lipid, in particular PEG2000-DMG; at least one phospholipid, in particular DSPC; at least one cholesterol.

[0160] According to some embodiments, the lipid nanoparticles (LNP) may comprise: at least one ionizable lipid selected from: SM-102, ALC-0315 and / or ALC-0159; optionally at least one PEGylated lipid, in particular PEG2000-DMG; at least one phospholipid, in particular DSPC; at least one cholesterol.

[0161] According to some embodiments, the lipid nanoparticles (LNP) may comprise: at least one ionizable lipid selected from: SM-102 and / or ALC-0315; optionally at least one PEGylated lipid, in particular PEG2000-DMG; at least one phospholipid, in particular DSPC; at least one cholesterol.

[0162] According to some embodiments, compositions comprising lipid nanoparticles of the invention may be supplemented by one or more of the following ingredients: potassium chloride, monobasic potassium phosphate, sodium chloride, dibasic sodium phosphate dihydrate, sucrose tromethamine (tris), tromethamine HC1, acetic acid, sodium acetate.

[0163] Advantageously, the LNP compositions according to the invention may not contain any adenovirus or AAV vector.

[0164] According to some embodiments, lipid nanoparticles according to the invention may be characterized by an average particle size ranging from about 5 nm to about 1000 nm.

[0165] According to some embodiments, lipid nanoparticles according to the invention may be characterized by an average particle size of less than or equal to about lOnm, 50nm, lOOnm, 250nm, 500nm, lOOOnm, or comprising and / or spanning the above numerical ranges.

[0166] In some embodiments, the lipid nanoparticles may be characterized by an average particle size of from about 50nm to 150nm or from about 50nm to about 250 nm.

[0167] In some embodiments, at least 50%, 75%, 80%, 90% (or ranges including and / or spanning the above percentages) of the lipid nanoparticles present have a particle size distribution equal to or less than about: 20nm, 40nm, 60nm, 80nm, lOOnm, l lOnm, 120nm, 130nm, 140nm, 160nm, 180nm, 200nm, 300nm, 400nm, 500nm, or a range including and / or spanning the above values of nm.

[0168] In some embodiments, the lipid nanoparticles have an average particle size of less than or equal to about lOnm, 50nm, lOOnm, 250nm, 500nm, lOOOnm, or comprising and / or spanning the above numerical ranges. In some embodiments, at least 90% of the lipid nanoparticles present have a nanoparticle size distribution equal to or less than about: 20nm, 40nm, 60nm, 80nm, lOOnm, l lOnm, 120nm, 130nm, 140nm, 160nm, 180nm, 200nm, 300nm, 400nm, 500nm, or a range comprising and / or spanning the foregoing values of nm.BRIEF DESCRIPTION OF THE DRAWINGS

[0169] Figure 1. Percentage of positive cells for mCherry over the time in a gMEC cell line (goat mammary epithelial cell) (1A) and goat fibroblasts (IB). Twenty-four hours after transfection with the liponanoparticles (LNPs) containing mRNA encoding for mCherry protein, the cells were counted every day, by microscopy, to estimate the number of positive cells for mCherry fluorescence. Three fields of cell were counted for each timepoint and each ratio, and the value represent mean + / -SD. The Y-axis represents the percentage of positive cells and the x-axis represents the time after transfection, from Oh to 240h.

[0170] Figure 2. Map of injection for each mouse. Injection of LNP containing mRNA was done in two mice per map of injection (injected in the same position if the gland is “open”) and the whole experiment is done in triplicate (3 different maps), resulting in a 6 mice experiment, A: positive control LNP+mRNA coding for firefly luciferase, C-D-E- F: LNP+mRNA coding for luciferase, mRNA: free mRNA coding for luciferase. Luciferase expression and activity was assessed 24h (picture represented here), 48h and 72h after the injection of the LNP by in vivo imaging system (IVIS) analysis. Luciferin (substrate of luciferase) was injected to mice prior to IVIS analysis and the luminescent signal was recorded.

[0171] Figure 3. Responsive gland to transfection with five sets of luciferaseencoding mRNA liponanoparticles. Each one of the five LNPs (LNP-A, LNP-C, LNP- D, LNP-E, LNP-F) were tested from left to right. For each condition, the number of responsive glands, 24h, 48h and 72h after injection of the LNP + mRNA coding for the luciferase, was calculated after luciferin injection into the mice and luminescent signal measurement by using IVIS instrument (6 mice in total, 1 gland per sample per mouse). The cut-off for positivity corresponds to the mean of the values obtained for Mock mouse +3*SD (data not shown). LNP-A: positive control LNP+mRNA coding for luciferase, LNP-C / D / E / F: tested LNP+mRNA coding for luciferase, N.mRNA : free mRNA coding for luciferase.

[0172] Figure 4. Average radiance of mammary gland after transfection. The average radiance of each gland was calculated by IVIS 24h, 48h and 72h after injection of the LNP + mRNA of luciferase, and after injection of luciferin in the mouse. The average radiance were represented in loglO. LNP-A: positive control LNP+mRNA coding for luciferase, LNP-C / D / E / F: tested LNP+mRNA coding for luciferase, N.mRNA: free mRNA coding for luciferase. The discontinuous line represents the cutoff for positivity, and corresponds to the mean of the values obtained for Mock mouse +3*SD (data not shown). Each point represents the value of one gland and was repeated in six mice (6 values in total), the mean is calculated + / - SD. Statistical analysis used is ANOVA where *:p<0.05 and ***:p<0.001 versus N.mRNA.

[0173] Figure 5A / B. in vitro confirmation of LNPs transfection ability. Quantification of human IgG (Figure 5Aa & Figure5Ba) and antigen-binding IgG (Figure5Ab & Figure5Bb), by ELISA, present in the supernatant of transfected cells. In Y-axis, the amount of quantified immunoglobulin is expressed in ng / ml. (Figure5A) Analysis of cell supernatants of HC11 transfected with 1 or 2 pg LNPs (SM102 or ALC0315), 24h and 48h post transfection. Results shown as mean + / -SEM of 5 technical replicates of one experiment. From left to right: Control (Ctrl); after transfection in combination with SM102 1 pg after 24h; with SM102 1 pg after 48h; after transfection in combination with SM1022 pg after 24h; after transfection in combination with SM102 2 pg after 48h; with ALC0315 1 pg after 24h; with ALC0315 1 pg after 48h; with ALC0315 2 pg after 24h; with ALC0315 2 pg after 48h; (Figure5B) Analysis of cell supernatants of gMC transfected with 1 or 2 pg LNPs (SM102 or ALC0315), 24h post transfection. (Ba) Results shown as mean + / -SEM of 2 independent experiments. From left to right: Control (Ctrl); SM102 1 pg; SM1022 pg; ALC0315 1 pg; ALC0315 2 pg.

[0174] Figure 6: in vivo mAb production by using LNPS. (A) Quantification of human IgG in the milk, 24h and 36h after injection in one udder of a lactating goat of Img or 2mg of LNPs (SM-102 & ALC-0315). From left to right for each condition: SM-102 2mg; SM102 1 mg; ALC-0315 2 mg; ALC-0315 1 mg (B) Daily milk volume (bar), anti- TNFa IgG concentration in the daily milk (dot) 24h after injection of 2 mg of the LNPSM-102 in one udder of a lactating goat. For the Human IgG and anti-TNFa measures, results shown as mean + / -SEM of 2 technical replicates of one experiment.

[0175] Figure 7: in vitro screening of different LNP compositions. Different goat Mammary Cells (internal gMC references: 39089; 93554; 95654) were transfected with 2 ug of three different LNPs carrying mRNA coding for a GFP protein: Control, LNP preparations 1 to 10 and as reference SM-102. After 24h, a cytometry analysis is performed on the cells to determine the percentage of positive cells (A) and the efficiency of transfection (Mean Fluorescent Intensity) (B). Results shown as mean + / -SEM of 3 independent experiments. Each tested condition corresponds, from left to right, to the gMC39089, gMC93554 and gMC95654 cell preparations.

[0176] Figure 8: Stability of two LNP preparations. Cl 4-4 and SM-102 were incubated in PBS 37°C during the indicated time. Then gMC93554 were transfected with 2ug of these pre-incubated LNPs carrying mRNA of GFP C14-4 and as reference SM- 102, in the right panel. After 24h, a cytometry analysis is performed on the cells to determine the percentage of positive cells. Results shown as mean + / -SEM of 2 independent experiments, unless for 16h and 20h for SM-102, which was performed once.

[0177] Figure 9: in vitro validation of a LNP transfection efficiency. Quantification of human IgG by ELISA, present in the supernatant of transfected cells. Analysis of cell supernatants of three different cell lines (from left to right, gMC95654 and gMC93554, goat fibroblasts: F25EA) transfected with 1 or 2 pg LNP9, 72h post transfection. Results shown as mean + / -SEM of 2 technical replicates of one experiment.

[0178] Figure 10: in vivo mAb production by using C14-4 as carrier of IgG mRNA. Daily milk volume (bar), anti-TNFa IgG concentration in the daily milk (black dot) and total human IgG (white square) produced by day 24h after injection of 8 mg of the LNP9 in one udder of a lactating goat. For the Human IgG and anti-TNFa measures, results shown as mean + / -SEM of 3 technical replicates of one experiment.EXAMPLES

[0179] The present invention is further illustrated by the following examples.Materials and Methods

[0180] Cell culture

[0181] Goat mammary epithelial cells were cultured in DMEM High glucose (Gibco, 119650), FBS 10% (Pan Biotech, P30-3031) and 1% Antibiotic antimycotic (Gibco, 15240-062). The medium is supplemented with lOng / mL of Epithelial growth factor (EGF) (Sigma, E9644) and insulin lOug / mL. Goat fibroblasts cells were cultured in DMEM Low glucose (Gibco, 31885), FBS 10% (Pan Biotech, P30-3031) and 1% Antibiotic antimycotic (Gibco, 15240-062). When the cells reached 90% confluency they were detached by trypsin (Gibco, 25200056) and seeded in a new flask or wells with fresh medium. The cells are cultured at 38,5°C, with 6% CO2.

[0182] Transfection

[0183] The cells are seed at 100 000 cells per well of a 24-w plate in 400uL of complete medium 24 hours before the transfection. The day of the transfection the mRNA is prepared using Lipofectamine™ MessengerMAX™ Transfection Reagent (Thermofisher, LMRNA001) following the provider instructions. The mRNA encapsulated inside the reagent codes for the mCherry protein. Cells are transfected with lug of mRNA. The following days fluorescent is analyzed by fluorescent microscope.

[0184] According to a second alternative protocol LNPs are loaded with two mRNAs coding for light chain and heavy chain (ratio 1:1) of an antibody. The cells are seed at 150 OOOcells per well of a 12-w plate in ImL of complete medium 24h before transfection. The day of transfection the medium is replaced by fresh medium, and cells are treated with 1 or 2ug of LNP, during 24h or 48h.

[0185] Mice experiment

[0186] Three weeks and eight days prior to injection, Balb / c mice are bred to produce pregnant, and therefore lactating, females. After three weeks, the mice give birth, and the pups remain with the mother for eight days. After this period, the mothers are anesthetizedwith 2-4% isoflurane in oxygen via a nose cone. Eye lubricant is also applied. Meloxicam (5-10 mg / kg) is administered subcutaneously for analgesia. The extremities of the mice are taped, and the injection zone is cleaned with alcohol swabs prior to injection of 10-20 pl of methylene blue, 1 pg of free mRNA or 1 pg of mRNA loaded in different LNP formulations into the nipple using a 50 pL metal syringe (33G). mRNA remain identical and code for luciferase protein. Twenty-fourhours after injection of the LNP, luciferin is injected intraperitoneally in the mice, prior to IVIS analysis. The mice are followed accordingly to the animal ethic guideline and by veterinarians.

[0187] ELlSA Test

[0188] Two types of enzyme-linked immunosorbent assay (ELISA) were performed. The first was designed to detect human IgG antibodies, while the second was a functional ELISA targeting the antigen TNF-alpha. The human IgG ELISA was performed according to the providers recommendation (RDB3257, RD-Biotech). Samples are diluted at different factors (1, 5, 25, 125, 625, 3125) in PBS before being analysed. For the functional TNF-alpha ELISA, plates were coated with lOOuL (lug / mL) of antigen of the produce antibody (TNF-alpha) overnight at 4°C. After three washes with PBS-Tween 0.1% and blocking with blocking buffer (5% BSA PBS-Tween 0.1%). The ELISA can be performed. A standard curve is made using originator antibody (from 50ng / mL to 4.4ng / mL) and dilution of the samples are chosen according to results of the human IgG ELISA. Samples and standards are diluted in 0.2% BSA PBS-Tween 0.1%. Only the dilution that fit to the standard curve are used to calculate the final concentration of the samples.

[0189] Goat experiment

[0190] For the first experiment (example 5 - see hereafter), three lactating goats were selected for injections in each udder (right or left): a control goat (no injection or PBS injection), a SM-102 goat (receiving 1 mg or 2 mg of LNPs), and an ALC-0315 goat (receiving 1 mg or 2 mg of LNPs). Milk was collected the day before injection, then twice daily (morning and evening) for one week. Milk volumes were recorded and presence of human and anti-TNFa antibodies in the milk was analysed by ELISA. The injections werenot considered invasive, as they were not systemic. The animals were closely monitored throughout the study, and blood and milk analyses were performed to confirm that no animal suffering occurred.

[0191] For the second experiment (example 8 - see hereafter), two lactating goats were selected for injection: a control goat (no injection or PBS injection) and a treated goat (receiving 8mg of the LNP C14-4 preparation). Before injection, the udder of the goat was washed four times with PBS 37°C, then after injection a massage was performed to increase the dissemination of the solution containing the LNP. Milk was collected the day before injection, then twice daily (morning and evening) for one week. Milk volumes were recorded and presence of human and anti-TNFa antibodies in the milk was analysed by ELISA.Results

[0192] Example 1 : Transfection in cellulo

[0193] Different ratios of MessengerMAX and mRNA quantity were tested, ranging from 0.5:1 to 6:1 in two different cell lines: gMEC cells and goat fibroblasts. After 24 hours, and each day, the number of fluorescent cells is counted (Figure 1). The 3:1 and 6:1 ratio show similar values, as well as the 1:1 and 0.5:1 ratio. Cell morphology was further analyzed. By optical microscopy no excessive cell death was observed. For the 3:1 ratio in both cell lines, the percentage of fluorescent cells is above 80% for the first two days, after which it gradually decreases. The same trend is observed with the 6:1 ratio, except for the fibroblasts, where the fluorescence remains above 80% for the first four days. This experiment has shown that gMEC cells and fibroblasts can be efficiently transfected with mRNA and express the protein of interest.

[0194] Example 2: Mice experiment

[0195] To assess dissemination of liquid into the mammary gland after intraductal injection, blue methylene was injected into the mammary gland through the ductal canal.The observation of the blue dye, after animal dissection, shows that the injection was located in the mammary gland lumen.

[0196] After the validation of the injection method, five samples were injected into the mammary glands of mice, following the described procedure in the figure 2 and according to the previously established protocol. Five types of LNPs were compared, all containing the same mRNA coding for the luciferase, alongside mRNA-free and a positive control (LNP-A comprising a combination of a SM-102 ionizable lipid, PEG2000-DMG, DSPC and Cholesterol) (Figure 2).

[0197] After 24, 48, and 72 hours, the radiance of each gland was analyzed using IVIS (Figure 3). A gland with a radiance superior to the mean of the radiance of the mock mouse (data not shown) + 3*SD is considered as responsive. We observed that the highest number of responsive glands was obtained after 24 hours in all treatments, followed by a decrease at 48 hours. The signal after 72 hours became mainly close to the threshold. For the positive control and LNP D and E 6 injected glands responded to the treatment after 24 hours, while only 5 glands responded for LNP C, F and N.mRNA. In terms of signal intensity, after 24h, LNP-E, provided the highest signal (4.82 p / s / cm2 / sr), followed by LNP D (4.50 p / s / cm2 / sr) and C (4.01 p / s / cm2 / sr). The positive control LNP-A shows an intermediary average radiance of 4.58 p / s / cm2 / sr. All these signals decrease after 48h and 72h. (Figure 4).

[0198] Example 3: Induction of lactation in goats after administration of a LNP a RNA coding for a protein of interest.

[0199] The following protocol can be followed, on or after administration of the LNP preparation, and prior to milk and / or protein recovery. Briefly, an estrogen compound, a progestogene compound, and a steroid compound are administered or co-administered to a lactating goat. The administration or co-administration can advantageously be considered along with a mechanical stimulation (e.g. massage), over a plurality of days. A corresponding exemplary schedule may thus comprise or consist of the one described herebelow (see Table 1).

[0200] Table 1 : Example of a lactation-promoting protocol after the first LNP administration and prior to milk collection.

[0201] The amounts administered to lactating goats may be as shown hereafter:Estradiol: 0.25 mg / kg; Progesterone: 0.75 mg / kg; Prednisolone: 0.4 mg / kg; Massage of the mammary gland / udder: 5 minutes.

[0202] Example 4: Production of antibodies in cellulo

[0203] Two gold standards LNPs, Pfizer®-BioNTech®’s ALC-0315 and Modema®’s SM-102, were selected. The particles carried two mRNAs in a 1:1 ratio: the light chainand heavy chain of Adalimumab. The LNP-mRNA particles were tested in mouse mammary epithelial cells (HC11 cell line) and in goat mammary cells (gMC), isolated for mammary gland biopsy, to confirm their ability to transfect mammary cells, in particular goat mammary cells, and to express a functional anti-TNFa mAb. Twenty-four and 48 hours after treatment, the supernatants were collected and analysed for the presence of human IgG in HC11 transfected cells (Figure 5Aa) or gMCs cells (Figure 5Ba) and anti-TNFa mAb in HC11 transfected cells (Figure 5Ab) or gMCs cells (Figure 5Bb) by using specific ELISA. In both cell types, the LNP SM-102 performed better than ALC-0315, inducing higher production of human IgG. The amount of LNP used did not significantly impact expression levels. Moreover, the produced antibodies were also able to bind TNFa and remain functional.

[0204] Example 5: Goat experiment

[0205] Analysis of the milk of the udders injected with 1 or 2 mg of lipid nanoparticles. Milk of the udder injected with 2 mg of SM-102 contained recombinant antibodies after 24h post-inoculation (Figure 6A). Results for the other LNPs show variability but remain detectable in the produced milk. SM-102 2mg samples show presence of Human IgG at 24h and 36h (Figure 6A). For the functionality ELISA, the IgG concentration follows a coherent kinetic profile and the ability to bind to its antigen (Dotted black line on Figure 6B). In this sample, the IgG concentration started at approximately 6 ng / mL at 24 hours and decreased slowly, reaching nearly 0 at 96 hours post inoculation. No issues were assessed with milk production (although data for 60 and 72 hours were not recorded). These results show the production of an active antibody, in the milk of goat, after inoculation with LNPs carrying light and heavy chain of an antibody.

[0206] Example 6: screening of lipid nanoparticles on mammary gland cells

[0207] Ten lipid nanoparticles (LNPs) with different compositions were used to transfect gMC cells. Their efficiency of transfection was estimated by using two parameters: the percentage of positive cells for GFP (Figure 7A) and the MFI (Mean fluorescent intensity) corresponding to the efficiency (Figure 7B) were analyzed by cytometry. SM- 102 is the reference LNP. In term of percentage of positive cell SM-102 and C14-4 showthe best transfection rate between 60 and 80%. The main difference between both LNPs is the MFI. It can be determined that C14-4 shows a better efficiency than SM-102, depending on the cell type from 1 000 000 to 2 000 000 fluorescent intensity, whereas 1 000 000 is the maximum for the reference SM-102 . It has been observed that C14-4 is more stable than SM-102. Indeed after 24h of preincubation in PBS at 37°C, C14-4 is able to transfect perfectly cells, whereas SM-102 might decrease between 6h and 16h. Overall it is shown that the tested LNPs are efficient and compatible with the expression of heterologous proteins in the mammary gland of goats, with C14-4 providing the most optimal level of expression under the tested conditions (Figure 8).

[0208] Example 7: validation of C14-4 transfection efficacy and IgG production

[0209] C14-4 efficiency for GFP-encoding mRNA transfection was assessed. To confirm its efficiency to carry two mRNAs, the LNP was loaded with light and heavy chain mRNA of adalimumab (ratio 1:1). Three different cell lines (2 gMCs and 1 goat fibroblasts) were transfected with 1 or 2 ug of LNP. The production of IgG was analyzed by ELISA anti-human IgG after 72h. The three cell lines were able to production human IgG, between 30ng / mL to lOOng / mL (Figure 9). These results confirm the ability of LNPs to carry a plurality of functional mRNAs, that lead to the production of human IgG after transfection in vitro.

[0210] Example 8: optimized goat experiment

[0211] Following the injection of 8mg of C14-4 carrying mRNA, in the udder of a lactating goat, for adalimumab, the presence of recombinant antibodies is assessed in the milk (Figure 10). Milk samples, from the udder injected with C14-4 (8mg / dose), show presence of human IgG from 24h and 96h. For the functionality ELISA, the IgG concentration follows a coherent kinetic profile and the ability to bind to its antigen. In this sample, the IgG concentration started at approximately 60 ng / mL at 24 hours and exhibits a peak at 36h with a concentration approximately between 80-100 ng / mL. No issues were assessed with milk production. These results show an improved production of an active antibody, in the milk of goat, after inoculation with the tested LNPs carryinglight and heavy chain of an antibody. The results show an increased yield + / - 15 times and an increased duration by + / - 3 days.

[0212] Overall, these results demonstrate that LNP-mediated transfection of immunoglobulin-encoding nucleic acids in a mammary gland epithelium can be associated to measurable and durable immunoglobulin expression in vivo, and efficient recovery of the recombinant polypeptides in the milk of the animal.

Claims

CLAIMS1. An in vitro method for producing proteins comprising a step of: a) providing a mammary gland epithelium, or cell thereof; b) bringing said epithelium or cell thereof in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest; c) optionally recovering the protein of interest.

2. A method for producing proteins, comprising a step of: a) providing a lactating mammal; b) bringing the mammary gland epithelium of the lactating mammal, or cell thereof, in contact with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest.

3. A method for producing proteins, comprising a step of: a) providing a lactating mammal previously administered with a lipid nanoparticle (LNP), or composition thereof, comprising a messenger RNA suitable for encoding a protein of interest, the said LNP or composition being brought in contact with all or part of a mammary gland epithelium, or cell thereof; b) recovering the protein of interest.

4. The method for producing proteins according to any of claims 2 or 3; wherein the mammal is selected from the group consisting of a rodent or a ruminant mammal.

5. The method for producing proteins according to any of claims 2 to 4; wherein the LNP or composition thereof is brought in contact with the mammary gland epithelium, or cell thereof, by administration in all or part of the ductal tree of the mammary gland.

6. The method for producing proteins according to any of claims 2 to 5; which includes a step of raising the lactating mammal under conditions sufficient for the protein to be present in the milk produced by the mammary gland; in particular raising the lactating mammal for at least 1 day before recovering the milk and / or protein thereof.

7. The method for producing proteins according to any of claims 2 to 6; which includes a step of recovering the milk from the lactating mammal thereby recovering the protein.

8. The method for producing proteins according to any of claims 6 or 7; which includes a step of separating the protein of interest from the milk recovered.

9. The method for producing proteins according to any of claims 2 to 8, which includes a plurality of steps of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof.

10. The method for producing proteins according to claim 9, wherein each step of bringing the mammary gland epithelium, or cell thereof, in contact with the lipid nanoparticles or compositions thereof, is distant from 2 to 10 days.

11. The method for producing proteins according to any one of claims 2 to 10, comprising a step of administering to the lactating mammal a lactation-inducing or lactation-promoting compound selected from the group consisting of an estrogen compound, a progestogene compound, a steroid compound, combinations thereof, and derivatives thereof.

12. The method for producing proteins according to any one of the preceding claims, wherein the lipid nanoparticles (LNP) comprise at least one lipid selected from the group consisting of: a cationic lipid, an ionizable lipid, or conjugate thereof.

13. The method for producing proteins according to any one of the preceding claims, wherein the lipid nanoparticle (LNP) comprises: at least one cationic lipid or ionizable lipid;at least one phospholipid; at least one sterol.

14. The method for producing proteins according to any one of the preceding claims, wherein the mammal is a rodent or a ruminant mammal.

15. The method for producing proteins according to any one of the preceding claims, wherein the mammal is a bovid.

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