Transfection compositions suitable for producing a biological product

Abstract

The present invention relates to transfection reagents compositions and to transfection compositions comprising such transfection reagents, suitable for producing a biological product. The present invention is directed to a transfection reagents composition and to transfection composition, suitable for the production of a biological product, wherein the transfection reagents composition comprises (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein R, Z and P+ are as defined in the description. The present invention also relates to in vitro or ex vivo uses of said at least one compound of general formula (I), to a method comprising the in vitro or ex vivo use of said composition, and a method for purifying, detecting and / or quantifying said at least one compound of general formula (I).

Description

[0001] TRANSFECTION COMPOSITIONS SUITABLE FOR PRODUCING A BIOLOGICAL PRODUCT

[0002] The present invention relates to transfection reagents compositions and to transfection compositions comprising such transfection reagents, suitable for producing a biological product. The present invention is directed to a transfection reagents composition and to transfection composition, suitable for the production of a biological product, wherein the transfection reagents composition comprises (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, wherein R, Z and P+are as defined in the description. The present invention also relates to in vitro or ex vivo uses of said at least one compound of general formula (I), to a method comprising the in vitro or ex vivo use of said composition, and a method for purifying, detecting and / or quantifying said at least one compound of general formula (I).

[0003] Viruses are small infectious particles that cannot replicate on their own but need to infect a cell in order to replicate. Viral particles (called virions) consist of a protein capsid or envelope and a genetic material inside. This genetic material is efficiently delivered to the infected host cells and thus modified viruses can be used as vectors to deliver desired nucleic acids to target cells in a process of viral transduction. As virus-based delivery represents a valuable tool in delivery of genetic material to various cell types and usually leads to good efficiency, there is a need to efficiently produce viruses that can later be utilized as vectors for virus-based delivery. Lentivirus (LV), Adenovirus (Ad), AAV (Adeno-Associated Virus) and retrovirus are the most common virus types that are usually produced in laboratory. To produce desired viral particles, cells are usually simultaneously co-transfected with several DNA plasmids. Some of the used plasmids code for proteins necessary to form viral capsid or envelope while the other ones encode a recombinant viral genome that will be delivered to target cells through the process of viral transduction.

[0004] While multiple viruses can be used as vectors, AAV is especially well suited for therapeutic applications. AAV is a small, non-enveloped and non-replicating virus with only two native genes, which makes it easy to work with from a vector-engineering standpoint. AAV elicits only a low immune response and has never been linked to disease in humans, and to date, AAV vectors have been safe for use in gene therapy. Engineered or recombinant, AAV vectors have no viral genes remaining, virtually eliminating the possibility that any viral genes will cause an adverse event in a patient. Lentiviral vectors, such as those based on human immunodeficiency virus (HIV), have gained attention due to their ability to transduce non-dividing cells. The general strategy in designing lentiviral vectors for gene therapy is based on the deletion and alteration of the native viral sequences to prevent the generation of replication-competent retroviruses. To achieve this, the lentivirus components are segregated into three or four different plasmid constructs with the goal of preventing the possibility of complete recombination to a fully replication competent lentivirus (RCL). In most cases, three additional plasmids provide the factors required for virus production and packaging (e.g., gag, pol, env). The envelope proteins are typically replaced by a heterologous viral glycoprotein, most commonly vesicular stomatitis virus G-protein (VSV-G), to modify the host range of the vector. An important safety feature is also the deletion of the promoter-enhancer region from the 3’ LTR preventing transcription from this region and subsequent viral replication (self-inactivating vector; SIN).

[0005] To produce viral particles, mammalian cells such as HEK-293, HEK-293T and derivatives, or HeLa and BHK cells are grown adherently in serum containing medium or in suspension with serum-free medium and are transfected with several plasmids.

[0006] Gene therapy aims to treat genetic diseases caused by absent, defective or dysregulated gene expression. Nucleic acid-based therapy targets wider therapeutic applications such as genetic diseases, immune diseases, cancers or viral infections. The therapy introduces copies of exogenous genes into living cells to induce the synthesis of the gene’s products. The gene transfer or delivery is the process of introducing such copies of exogenous genes into living cells and is achieved utilizing non-viral (transfection) or viral (transduction) delivery methods (Kulkarni et al., 2021).

[0007] The transduction can be performed by recombinant viruses which are able to infect multiple and selective tissues or target cells. Recombinant viruses can comprise AAV, LV, Ad, or oncolytic viruses.

[0008] The transfection is the process of introducing nucleic acids (e.g., DNA or RNA or their derivatives) into eukaryotic cells, using non-viral delivery systems. The transfection offers an alternative or a complementary method to the viral delivery system. Transfection can be applied in therapy through ex vivo or in vivo protocols. However, the main application of the transfection is to genetically modify cells in vitro to express or over-express exogenous proteins.

[0009] In the bioproduction field of biological products, transfection is used to generate stable cell clones or transient cells over-producing recombinant proteins, peptides or antibodies. The transfection is also particularly adapted to produce recombinant viruses such as AAV, LV or Ad to generate vectors suitable for in vivo or in vitro use. The recombinant virus production is based on a transient gene expression (TGE) system where all the components necessary to produce recombinant virus, i.e., recombinant viral particles or virus-like particles based on the virus are introduced by transfection, then expressed in situ into proteins or replicated into recombinant viral genome and finally are assembled to generate the recombinant viral particles or virus-like particles (Dobrowsky et al., 2021). The most widely used platform is the transient transfection of HEK293 cells for rAAV and LV production. Other platforms use baculovirus, Hela cells, BHK-21 cells, or insect cells.

[0010] The cell engineering to produce biological products (e.g., viruses, proteins, antibodies, peptides) consists of transfecting one or many expression vectors (e.g., plasmids, complementary DNA, mRNA, synthetic linear or closed linear DNA, DNA fragments) expressing in cells the different components necessary to produce the recombinant viruses, proteins, antibodies or peptides. The transfection can be achieved in adherent or suspension-adapted cells at high cell density cultured in media containing serum or in protein-free, chemically defined or completely synthetic media.

[0011] The recent successes of the cell and gene therapy, the rapid progression to many latestage clinical trials as well as the possible therapeutic applications to larger population of patients have highlighted the need to increase productivity for viral vectors (Bulcha et al., 2021).

[0012] The transfection for the TGE system represents the method of choice to produce recombinant viruses, such as AAVs or LVs. The calcium phosphate precipitation method, not scalable and reproducible, has been supplanted by cationic polymers or cationic lipids. Among the cationic polymers, the polyethylenimine (PEI) polymer is considered as the gold standard transfection reagent to produce recombinant viral vectors from HEK293 cells and derivatives.

[0013] PEI, offering the largest density of charged amino groups in a polymer backbone, can interact with the negatively charged nucleic acid, generating complex formations via polyelectrolyte interactions. Behr et al. has showed that the PEI was an effective polymer in transfection (Boussif et al., 1995) by initiating cell binding and triggering cell internalisation through endocytosis. Then, PEI, having buffering capacity at acidic pH, induces a ‘proton sponge’ activity resulting in vesicles swelling and endosomolysis ending by the release of DNA in the cytoplasm (Akinc et al., 2005; Sonawane et al., 2003). Both branched and linear PEI are efficient in transfection, but the linear topology was shown to be more efficient.

[0014] Innovative transfection technology that significantly improves titer and virus quality and reduces the production cost is an attractive solution for scale-up to satisfy the increasing demand of recombinant viruses for cell and gene therapy. Recently, new transfection reagents based on heterocyclic compounds grafted to cationic polymers have shown improved production yields of viral vectors, particularly AAV, when compared to production achieved with PEI (WO2021 / 023796; WO2021 / 023798).

[0015] However, lentiviral vectors bioproduction remains a challenge during scale-up, due to the inability to produce sufficient titres at the upstream level and to the low recoveries during the downstream processing (Perry and Rayal, 2021). Typical titres are in the range of 106to 107TU / mL at the upstream level.

[0016] Thus it is an object of the present invention to provide new versatile transfection reagents, in particular when provided in a composition, to produce a biological product such as viruses, virus-like particles, proteins, antibodies, peptides, preferably recombinant viruses such as AAVs and LVs, in both suspension and adherent cells, preferably mammalian cells such as HEK293T cells. In particular the present invention aims to provide more efficient transfection reagents to increase recombinant virus titres, particularly for lentiviral vectors, produced in HEK293 cell suspension platforms.

[0017] It is another object of the present invention to provide a method comprising the in vitro or ex vivo use of said new versatile transfection reagents.

[0018] It is another object of the present invention to provide the in vitro or ex vivo uses of said new versatile transfection reagents to transfect at least one nucleic acid molecule in a cell.

[0019] It is another object of the present invention to provide a method for purifying, detecting and / or quantifying said new versatile transfection reagents.

[0020] The present invention relates to a transfection composition suitable for the production of a biological product, wherein the composition comprises (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, and (ii) and acceptable excipient, buffering agent, cell culture medium, or transfection medium: wherein: - R is selected from the group consisting of the compounds having the following formulas

[0021] R1-R54: , wherein Wi, W2, W3, W4 and W5 may be identical or different, and represent H, a linear or branched, saturated or insaturated (C1-C10) alkyl, or a linear or branched, saturated or insaturated 0-(Ci-Cio) alkyl, and m represents an integrer between 1 and 10, preferably between 1 and 3;

[0022] - Z represents: -C=O-; or -(CH2)n-C(O)-, with n representing an integer between 1 and 3; - P+represents a graft cationic polymer selected from the group consisting of a linear or branched polyethyleneimine (PEI) and PEI dendrimers.

[0023] As defined herein, the term “tautomer1' refers to structural isomers differing only in the positions of hydrogen atoms and electrons. Examples of tautomers include, but are not limited to, ketone-enol, enamine-imine, amide-imidic acid, lactam-lactim, nitroso-oxime, ketene-ynol, amino acid, or phosphite-phosphonate.

[0024] As defined herein, the term “mesomet1' or “meso compound’ refers to a stereoisomer that has two or more chiral centers but is optically inactive.

[0025] As defined herein, the term “racemate" or “racemic mixtures" refers to a mixture of two enantiomers in equal proportions.

[0026] As defined herein, the term “enantiomer1' refers stereoisomers that are mirror images, i.e. mirror image isomers.

[0027] As defined herein, the term “diastereomer1' refers to isomers of compounds with more than one chiral center that are not mirror images of one another.

[0028] As defined herein, the term “C1-C10 alkyl’ represents any monovalent radical of a linear or branched hydrocarbon chain comprising 1 to 10 carbon atoms. This term includes the term “C1- Cs alkyl”, which represents an alkyl group having 1 to 6 carbon atoms, or the term “C1-C4 alkyl”, which represents an alkyl group having 1 to 4 carbon atoms. Examples of suitable C1-C10 alkyl groups include, but are not limited to, C1-C4 alkyl groups such as methyl, ethyl, n-propyl, / '-propyl, n-butyl, / '-butyl, s-butyl or f-butyl, Ce-Cs alkyl groups such as n-hexyl, n-heptyl or n-octyl, as well as n-pentyl, 2-ethylhexyl, 3,5,5-trimethylhexyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl or n- octadecyl. Unless mentioned otherwise, said groups may be unsubstituted or substituted by one or more substituents such as, for example, halogen, alkyl, alkoxy, aryl, heteroaryl, haloalkyl, haloalkoxy, alkoxycarbonyl, alkanoyl, aroyl, formyl, nitrile, nitro, amido, alkylthio, alkylsulfinyl, alkylsulfonyl, arylthio, arylsulfinyl, arylsulfonyl, amino, alkylamino, arylamino, dialkylamino and diarylamino.

[0029] As defined herein, the term “acceptable excipient’ refers to any substance or combination of substances physiologically acceptable i.e., appropriate for its use in a composition in contact with a host, such as a eukaryotic cell or organism, especially a human, and thus is non-toxic. It can refer to a solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. Examples of suitable acceptable excipients include, but are not limited to, glucose, galactose, lactose, dextrose, maltose, mannitol, sucrose, trehalose, polyethyleneglycol, or pluronic acid. As defined herein, the term “buffer1’ refers to a buffer solution comprising a buffering agent. As defined herein, the term “buffering agent’ refers to an agent that adjusts, maintains or controls the pH of a solution. Buffering agents can be either the weak acid or weak base that would comprise a buffer solution. Examples of suitable buffering agents include, but are not limited to, sodium carbonate, sodium bicarbonate, sodium hydroxide, calcium bicarbonate, calcium citrate, sodium citrate, magnesium hydroxide, magnesium bicarbonate, potassium acetate, Tris acetate, sodium acetate, potassium phosphate monobasic, potassium carbonate, potassium bicarbonate, potassium citrate, or magnesium oxide.

[0030] As defined herein, the term “cell culture medium" or “transfection medium" refers to a medium containing serum, synthetic medium, animal-free component medium or chemically defined medium, in particular medium for maintaining cells alive, or for growing, for differentiating or for expanding cells, or for enhancing transfection.

[0031] “Excipients", “buffering agents", “cell culture mediums" and “transfection mediums" are in particular in accordance with those known in the art in the field of the invention.

[0032] As defined herein, the term “a biological product’ includes, but is not limited to, proteins; viruses, in particular recombinant viruses; antibodies and their fragments; peptides; nucleic acids; virus-like particles.

[0033] As defined herein, the term “transfection" refers to the introduction of a nucleic acid (e.g., DNA, or RNA or their derivatives) into eukaryotic cells and may be a stable or transient, standard or reverse transfection.

[0034] The above composition may be designated as a transfection reagents composition, wherein the compound defined under (i) above is a transfection reagent.

[0035] In a particular embodiment of the invention, the composition further comprises at least one, in particular one nucleic acid molecule to be transfected in a cell, preferably a eukaryotic cell, for the production of the biological product. Preferably said nucleic acid molecule is selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA / RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, a self-amplifying RNA (saRNA), a synthetic DNA, and an expression vector encoding said nucleic acid molecule, in particular a plasmid encoding said nucleic acid molecule or a plasmid expressing said nucleic acid molecule. Even more preferably, said nucleic acid molecule is a DNA or a mRNA.

[0036] When distinct nucleic acids are provided in the composition of the invention, they may be all DNA molecules or all RNA molecules or may be mixtures of DNA and RNA molecules or molecules comprising an association of DNA and RNA strands. Said nucleic acid molecule may be single stranded or double stranded, and may contain modified or unmodified nucleotide bases (nucleobases).

[0037] The terms “polynucleotide”, “nucleic acid’, “oligonucleotide”, and “nucleic acid molecule” are used interchangeably herein to designate these nucleic acid molecules.

[0038] The composition according to the invention may be used as a formulation of the nucleic acid molecule with the at least one compound of general formula (I) (including any of its particular embodiments disclosed herein) and the acceptable excipient, buffering agent, cell culture medium, or transfection medium, in accordance with the disclosure provided herein. It may alternatively comprise a cell culture or expanded cells, wherein prior to being provided as a culture and / or as expanded cells, isolated cells have been treated with said formulation for transfection. Otherwise stated, the composition of the invention encompasses, as an embodiment, a cell or a cell culture or expanded cells wherein said formulation has been introduced by transfection according to the invention. The cells are in particular eukaryotic cells, especially mammalian cells, preferably human cells. The cells may be dividing cells or non-dividing cells.

[0039] In a particular embodiment of the invention, the composition comprises one nucleic acid, in particular a single nucleic acid, preferably one plasmid DNA for transfection.

[0040] In a particular embodiment of the invention, the composition comprises multiple nucleic acid molecules, i.e., nucleic acid molecules having distinct composition or structure or both with regard to each other, preferably multiple DNA molecules, for co-transfection.

[0041] In a particular embodiment of the invention, the composition comprises multiple, i.e., distinct nucleic acids, in particular selected from the group consisting of multiple plasmid DNA, plasmid DNA and oligonucleotide, plasmid DNA and mRNA for co-transfection.

[0042] In a particular embodiment of the invention, the composition is suitable for the production of AAV and the composition comprises multiple nucleic acid molecules, in particular two or three nucleic acid molecules, preferably three plasmid DNAs suitable for the production of the AAV.

[0043] In another particular embodiment of the invention, the composition is suitable for the production of LV and the composition comprises multiple nucleic acid molecules, in particular three or four nucleic acid molecules, preferably four plasmid DNAs suitable for the production of the LV.

[0044] In a particular embodiment of the invention, the biological product is selected from the group consisting of:

[0045] (i) a biological product encoded by the nucleic acid from the composition, which is selected from a protein, a peptide, an antibody and a fragment thereof, (ii) a virus, in particular a recombinant virus, preferably a recombinant virus selected from the group consisting of an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus (Ad), an oncolytic virus, and a baculovirus, more preferably AAV or LV, even more preferably LV, and

[0046] (iii) a biological product which comprises virus-like particles.

[0047] According to the invention, AAV may be selected from the group consisting of an AAV of serotype 2 (AAV2), an AAV of serotype 5 (AAV5), an AAV of serotype 8 (AAV8) and an AAV of serotype 9 (AAV9) or any AAV serotype, preferably AAV may be selected from AAV2 or AAV9.

[0048] In a particular embodiment of the invention, the recombinant virus, especially the AAV, LV, Ad, oncolytic virus or baculovirus, such as one of the above-mentioned viruses, expresses a heterologous polypeptide, in particular a heterologous polypeptide of therapeutic interest.

[0049] The at least one preferred compound of general formula (I) as defined herein is one wherein R is selected from the group consisting of the compounds having the following formulas R1 , R2, R3, R4, R5, R6, R7, R8, R9, R10, R11 , R12, R13, R14, R15, R16, R17, R18, R19, R20,

[0050] R21 , R22, R23, R24, R25, R26, R27, R28, R29, R30, R31 , R32, R33, R34, R35, R36, R37, R38,

[0051] R39, R40, R41 , R42, R43, R44, R45, R46, R47, R48, R49, R50, R51 , R52, R53 and R54.

[0052] In a particular embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is selected from the group consisting of the compounds having the following formulas R1-R28:

[0053] In a preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is selected from the group consisting of the compounds having the following formulas R7, R8, R9, R12, R18 and R26:

[0054] In a more preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R represents a compound having the above- mentioned formula R7. In another particular embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is selected from the group consisting of the compounds having the following formulas R29-R53:

[0055] In another preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is selected from the group consisting of the compounds having the following formulas R50, R51 and R53:

[0056] In another particular embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is R54:

[0057] R54:

[0058] , wherein Wi, W2, W3, W4and W5may be identical or different, and represent H, a linear or branched, saturated or insaturated (C1-C10) alkyl, or a linear or branched, saturated or insaturated 0-(Ci-Cio) alkyl, and m represents an integrer between 1 and 10, preferably between 1 and 3.

[0059] In another preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein R is R54, wherein W1, W2, W3, W4and W5, which may identical or different, represent H, OH, or OMe.

[0060] In a preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein Z represents -C=O.

[0061] In a particular embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein the graft cationic polymer P+is a linear or branched PEI, preferably a linear PEI.

[0062] The graft cationic polymer P+of the invention may have a grafting ratio ranging from 5% to 50%, preferably from 20% to 35%, more preferably is 25%.

[0063] As defined herein, the term “grafting ratio" (in %) refers to the number of grafted monomers on primary, secondary or tertiary amino groups of P+by side chains as defined by the general formula (I), divided by the number of total monomers present in the original cationic polymer. The grafting ratio will depend upon the molecular weight of the cationic polymer, the chemical reactivity of the grafted side chains onto the polymer, or the obtained biological effect. Said grafting ratio may be determined by a measurement method well known in the art, for example by NMR.

[0064] The graft cationic polymer P+may have an average molecular weight (Mw) ranging from 1 kDa to 50 kDa, preferably from 5 kDa to 30 kDa or from 10 kDa to 25 kDa, in particular the graft cationic polymer P+has an Mw of 8, 10, 15, 22, 25 or 30 kDa, preferably of 10 or 22 kDa, more preferably 22 kDa. The graft cationic polymer P+can be associated with a counterion such as chloride, phosphate, citrate, acetate, propionate, carbonate, succinate, sulfonate, sulfate, or carboxylate.

[0065] In a particular embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein:

[0066] - R is selected from the group consisting of the compounds having the above-mentioned formulas R1-R54, in particular R is selected from the group consisting of the compounds having the above-mentioned formulas R1-R28, R29-R53 and R54, preferably R1-R28 and R29-R53, more preferably R7, R8, R9, R12, R18, R26, R50, R51 and R53, even more preferably R7; and / or

[0067] - Z represents: -C=O-; or -(CH2)n-C(O)-, with n representing an integer between 1 and 3, preferably Z represents -C=O; and / or

[0068] - P+represents a linear or branched PEI, preferably a linear PEI; and / or P+has a grafting ratio ranging from 5% to 50%, preferably from 20% to 35%, more preferably is 25%; and / or P+has an average molecular weight (Mw) ranging from 1 kDa to 50 kDa, preferably from 5 kDa to 30 kDa or from 10 kDa to 25 kDa, in particular the graft cationic polymer P+has an Mw of 8, 10, 15, 22, 25 or 30 kDa, preferably of 10 or 22 kDa, more preferably 22 kDa.

[0069] In a preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein:

[0070] - R is selected from the group consisting of the compounds having the above-mentioned formulas R7, R8, R9, R12, R18, R26, R50, R51 and R53, preferably R7; and / or

[0071] - Z represents: -C=O-; or -(CH2)n-C(O)-, with n representing an integer between 1 and 3, preferably Z represents -C=O; and / or

[0072] - P+represents a linear or branched PEI, preferably a linear PEI; and / or P+has a grafting ratio ranging from 20% to 35%, more preferably is 25%; and / or P+has an Mw of 8, 10, 15, 22, 25 or 30 kDa, preferably of 10 or 22 kDa, more preferably 22 kDa.

[0073] In a more preferred embodiment of the invention, the at least one preferred compound of general formula (I) as defined herein is one wherein:

[0074] - R represents R7; and / or

[0075] - Z represents -C=O; and / or

[0076] - P+represents a linear or branched PEI, preferably a linear PEI; and / or P+has a grafting ratio of 25%; and / or P+has an Mw of 10 or 22 kDa, preferably 22 kDa.

[0077] In a particular embodiment of the invention, the composition according to the invention comprises from 1 to 5, preferably at least two distinct compounds of general formula (I), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or an acceptable salt thereof. In another particular embodiment of the invention, the composition comprises one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, e.g., a single compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof. According to a particular embodiment of the invention, preferred compounds of formula (I) correspond to compounds 1 , 12, 13, 14, 15, 16, 18, 19, 21 , 22, 23, 24, 25, and any compound in the group of compounds 27 to 59, preferably compounds 1 , 12, 13, 14, 15, 16, 18, 23, 27, 30, 33, 44, 50, 51 , 52, 53, 54, 55, 56, 57, 58 and 59, more preferably compounds 1 , 14, 18, 30, 44, 52 and 55, even more preferably compound 55, as disclosed in Table 1 . In all these compounds, PEI refers to a linear PEI.

[0078] Table 1. Structures of preferred compounds of formula (I) of the invention.

[0079] Thus, in a particular embodiment of the invention, the at least one compound of general formula (I) is selected from the group consisting of the following compounds or mixtures thereof:

[0080]

[0081]

[0082] In a preferred embodiment of the invention, the at least one compound of general formula (I) is selected from the group consisting of the compounds 1 , 12, 13, 14, 15, 16, 18, 23, 27, 30, 33, 44, 50, 51 , 52, 53, 54, 55, 56, 57, 58 and 59. In a more preferred embodiment of the invention, the at least one compound of general formula (I) is selected from the group consisting of the compounds 1 , 14, 18, 30, 44, 52 and 55.

[0083] The present invention also relates to a method comprising the in vitro or ex vivo use of the composition according to the invention, wherein the method is for the production of (i) a biological product encoded by the nucleic acid from the composition, which is selected from a protein, a peptide, an antibody and a fragment thereof; or (ii) a biological product, which is a virus, in particular a recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, wherein said composition comprises multiple nucleic acid molecules for co-transfection; or (iii) a biological product which comprises virus-like particles, wherein said composition comprises multiple nucleic acid molecules for co-transfection.

[0084] Said method comprises the in vitro or ex vivo use of the composition according to the invention. Thus, all the above-mentioned embodiments directed to the definition of the composition according to the invention apply to the definition of the composition used in this method.

[0085] In a particular embodiment of the invention, said method is for the production of recombinant AAV, said composition comprising (i) at least one compound selected from the group consisting of compounds 1 , 14, 18, 30, 44, 52 and 55, preferably 55, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, and (iii) optionally a booster.

[0086] In another particular embodiment of the invention, said method is for the production of recombinant LV, said composition comprising (i) at least one compound selected from the group of compounds 1 , 14, 18, 30, 44, 52 and 55, preferably 55, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, and (iii) optionally a booster. As defined herein, the term “booster1’ refers to any molecule increasing transfection efficiency or biological product production.

[0087] In a particular embodiment of said method of the invention, said method is for the production of recombinant virus, said composition comprising a plurality of expression vectors such as plasmid vectors to transfect in eukaryotic cells, in particular in mammalian cells or in insect cells, preferably mammalian cells, more preferably adherent mammalian cells or suspension mammalian cells, even more preferably suspension and adherent HEK293T cells, wherein said vectors, in particular plasmids, are construct expressing viral structural sequences and transfer vector genome for virus or virus-like production and optionally expressing molecules of interest encoded by the transfer vector genome.

[0088] As defined herein, the term “adherent cells’’ refers to cells that need solid support for growth, and are thus anchorage-dependent. Examples of adherent cells include, but are not limited to, MRC-5 cells, HeLa cells, Vero cells, NIH-3T3 cells, L293 cells, CHO cells, BHK-21 cells, MCF-7 cells, A549 cells, COS cells, HEK 293 cells, Hep G2 cells, SNN-BE(2) cells, BAE-1 cells or SH-SY5Y cells.

[0089] As defined herein, the term “suspension cells’’ refers to cells that do not need solid support for growth, and are thus anchorage-independent. Examples of suspension cells include, but are not limited to, HEK293 cells and derivatives, CHO cells, NSO cells, LI937 cells, Namalawa cells, HL60 cells, WEHI231 cells, Yac 1 cells, Jurkat cells, THP-1 cells, K562 cells or LI266B1 cells.

[0090] The present invention also relates to a method for the preparation of recombinant adeno- associated virus (AAV) vector particles in a cell, wherein the cell is transfected with a transfection composition according to the invention, wherein the transfection composition comprises the following nucleic acid molecules: an AAV transfer nucleic acid molecule, in particular a plasmid (pTransfer) expressing therapeutic gene or reporter gene as GFP under control of a promoter; a Rep / Cap nucleic acid molecule, in particular a plasmid (pPackaging or pRC or pAAVRep / Cap) providing AAV viral functions and comprising a replication (Rep) gene and a capsid (Cap) gene from an AAV; and

[0091] - a helper nucleic acid molecule, in particular a plasmid (pHelper).

[0092] The present invention also relates to a method for the preparation of recombinant lentiviral vector particles in a cell, wherein the cell is transfected with a transfection composition according to the invention, wherein the transfection composition comprises the following nucleic acid molecules: - a vector plasmid comprising (i) lentiviral, especially HIV, cis-active sequences necessary for packaging, reverse transcription, and transcription, said vector plasmid further comprising optionally (ii) a nucleic acid molecule of interest, in particular of therapeutic interest, for example encoding a biological product suitable for treatment of a human being in need thereof under the control of regulatory expression sequences, especially a promoter;

[0093] - an envelope expression plasmid, in particular a VSV-G envelope expression plasmid comprising a nucleic acid molecule encoding a VSV-G envelope protein, wherein said nucleic acid molecule is under the control of regulating expression sequences, in particular regulatory expression sequences comprising a promoter; and

[0094] - one or more plasmid(s) for encapsidation, comprising lentiviral, especially HIV, gag and po / coding sequences, wherein the plasmid(s) encoding Gag and Pol polypeptides are suitable for the production of integration-competent vector particles or encode(s) modified gag-pol coding sequences suitable for the production of integration-deficient vector particles, wherein said lentiviral gag-pol or modified gag-pol coding sequence(s) is(are) under the control of regulating expression sequences.

[0095] The present invention also concerns a method for in vitro or ex vivo transfection of live cells comprising introducing in the cells the transfection composition according to the invention. Said live cells may be provided or maintained in medium containing serum, synthetic medium, animal-free component medium or chemically defined medium.

[0096] The present invention also concerns the in vitro or ex vivo use of the at least one compound of general formula (I) of the invention to transfect at least one nucleic acid molecule in a cell, in particular a eukaryotic cell, preferably a mammalian cell, more preferably an adherent mammalian cell or a suspension mammalian cell, even more preferably suspension and adherent HEK293T cells, wherein the at least one nucleic acid molecule is preferably selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA / RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, a self-amplifying RNA (saRNA), a synthetic DNA, more preferably a DNA or a mRNA, and an expression vector encoding said nucleic acid molecule, in particular a plasmid encoding said nucleic acid molecule or a plasmid expressing said nucleic acid molecule.

[0097] In a particular embodiment according to the invention, the invention relates to the in vitro or ex vivo use of at least one compound of general formula (I) of the invention to transfect at least one nucleic acid molecule in a cell, in particular a eukaryotic cell as previously defined, wherein the at least one compound of general formula (I) is selected from the group consisting of compounds 1, 13, 15, 16, 23, 27, 33, 47, 50, 51 , 52, 53, 55 and 58, preferably compound 55, and the at least one nucleic acid molecule is DNA.

[0098] In another particular embodiment according to the invention, the invention relates to the in vitro or ex vivo use of at least one compound of general formula (I) of the invention to transfect at least one nucleic acid molecule in a cell, in particular a eukaryotic cell as previously defined, wherein the at least one compound of general formula (I) is selected from the group consisting of compounds 13, 16, 23, 33, 51, 52, 55 and 59, preferably from the group consisting of compounds 16, 33, 51, 55 and 59, more preferably compound 55, and the at least one nucleic acid molecule is mRNA.

[0099] The present invention also relates to a method for purifying, detecting and / or quantifying the at least one compound of general formula (I) as defined herein, wherein the at least one compound of general formula (I) is comprised in a liquid mixture comprising a biological matrix, wherein the biological matrix comprises a recombinant virus or virus-like particles produced using the at least one compound of general formula (I), wherein the method comprises the step of:

[0100] (a) performing an acidic hydrolysis of the liquid mixture by incubating the liquid mixture comprising the biological matrix in an aqueous solution comprising from 0.1% to 10% (v / v) hydrochloric acid (HCI) at a temperature ranging from 60°C to 1 10°C for a time period ranging from 2 hours to 24 hours, preferably an aqueous solution comprising 0.1 % (v / v) HCI at a temperature of 1 10°C for 2 hours or in an aqueous solution comprising 1 % (v / v) HCI at a temperature ranging from 60°C to 80°C for 2 hours, wherein said acidic hydrolysis does not degrade the at least one compound of general formula (I),

[0101] (b) purifying the reaction mixture obtained in step (a) in order to obtain a purified compound of general formula (I),

[0102] (c) detecting and / or quantifying the purified compound of general formula (I) obtained in step (b).

[0103] As defined herein, the term “purifying" means that other constituents of the solution are separated or removed to keep the solution of the transfection reagent only.

[0104] In a particular embodiment of the invention, before performing step (a), the method comprises a step of inactivating the recombinant virus or virus-like particles by heating the biological matrix at a temperature ranging from 1 10°C to 130°C for a time period ranging from 30 minutes to 4 hours, preferably at a temperature of 120°C for 30 minutes.

[0105] In a particular embodiment of the invention, step (b) is performed using ultrafiltration or centrifugation. In a particular embodiment of the invention, step (c) is performed using High-performance liquid chromatography (HPLC) or Ultra high-performance liquid chromatography (UHPLC) analytical technique, preferably UHPLC.

[0106] In a particular embodiment of the invention, the at least one compound of general formula (I) of step (c) is detected with a limit of detection (LCD) ranging from 0.4 ppm to 1 1 .5 ppm, and / or a limit of quantification (LOQ) ranging from 1 .4 ppm to 32.8 ppm.

[0107] In a particular embodiment of the invention, the at least one compound of general formula (I) is detectable in a biological matrix during the manufacturing process of the recombinant viruses, wherein the biological matrix is selected from the group consisting of a cell culture medium, in particular a culture medium of eukaryotic cells, in particular suspension cells or adherent cells, a buffer, a solution used during the manufacturing and purification process of recombinant viruses, and a final composition comprising purified viruses in a final formulation comprising an acceptable buffer and excipients.

[0108] In another particular embodiment of the invention, the at least one compound of general formula (I) is used during the manufacturing process of an advanced therapy medicinal product (ATMP) and is present in residual quantity (1 to 1000 ppm) with respect to transfection reagents provided in the manufacturing process and to other components of the liquid mixture.

[0109] The terms “cell culture medium", “suspension cells", “adherent cells", “buffet1', “a solution used during the manufacturing and purification process of recombinant viruses" and “an acceptable buffer and excipients" are defined according to the above definitions and in accordance with examples provided herein.

[0110] As defined herein, the expression “a solution used during the manufacturing and purification process of recombinant viruses" refers to any solution well known in the art that can be used during the manufacturing and purification process of recombinant viruses.

[0111] In a particular embodiment of the invention, the recombinant virus is selected from the group consisting of an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus, an oncolytic virus and a baculovirus, preferably is an AAV or a LV, more preferably is a LV.

[0112] In a particular embodiment of the invention, the at least one compound of general formula (I) is a compound selected from the group consisting of compounds 1 , 14, 18, 30, 44, 52 and 55, and the recombinant virus is an AAV.

[0113] In another particular embodiment of the invention, the at least one compound of general formula (I) is a compound selected from the group consisting of compounds 1 , 14, 18, 30, 44, 52 and 55, and the recombinant virus is a LV. In a particular embodiment of the invention, the ratio of the nucleic acid molecule:the compound of general formula (I) is 1 :1 , 1 :2, 1 :3 or 1 :4, preferably is 1 :1 or 1 :2.

[0114] In a particular embodiment of the invention, the quantity of the nucleic acid molecule ranges from 0.5 to 1 pg / million cells, preferably 1 pg / million cells.

[0115] Unless otherwise stated, all the above-mentioned embodiments may be combined together. Thus features which are described in the context of separate embodiments may be combined in a single embodiment.

[0116] Other features and advantages of the invention will be apparent from the examples which follow and will also be illustrated in the figures.

[0117] BRIEF DESCRIPTION OF THE FIGURES

[0118] Figure 1. Chemical structure of a compound of general formula (I).

[0119] Figure 2. Benchmark of different transfection reagents for LV production from suspension 293T cells in Freestyle™ F17 medium. Transfections were performed with 1 pg total DNA / million cells. LV production efficiency expressed as transducing unit (TU / mL). The following commercial or known reagents were used:

[0120] Transporter 5™ Transfection reagent (Polysciences), PEIMax (Polysciences), TransITO-Lenti (Mirus Bio), TranslT®-VirusGEN® (Mirus Bio), PEIpro® (Polyplus Transfection), FectoPRO® (Polyplus Transfection), jetPRIME® (Polyplus Transfection), and transfection reagent 1.42 (patent application WO2021 / 023796) and transfection reagent 2.22 (patent application WO2021 / 023798). The optimal ratios of DNA / transfection reagent (pg DNA:pL reagent) were 1 :4 (Transporter 5™), 1 :4 (PEIMax), 1 :3 (TranslT®-Lenti) 1 :1 (TranslT®-VirusGEN@), 1 :1 (PEIpro®), 1 :1 (FectoPRO®), 1 :2 (jetPRIME®), 1 :1 (transfection reagent 1.42) and 1 :1 (transfection reagent 2.22) .

[0121] Figure 3. Figure 3.a. Production of AAV-2 from suspension HEK-293T cells using compounds 1 , 44, 18, 55, 30, 52, 14 according to the invention, and FectoVIR®-AAV (Polyplus Transfection) as a commercial reference. Figure 3.b. Production of AAV-9 from suspension HEK-293 T cells using compounds 1 , 44, 18, 30, 52, 14, 55 according to the invention, and FectoVIR®-AAV (Polyplus Transfection) as a commercial reference. Figure 4. Productivity of lentivirus particles from suspension HEK-293T cells, expressed as transducing unit (TU / mL) and after transfection using 1 pg DNA / million cells. Figure 4.a. Production of lentivirus particles using compounds 1 , 14, 13, 54 according to the invention, and PEIpro® (Polyplus Transfection) as a commercial reference. Figure 4.b. Production of lentivirus particles using compounds 23, 21 , 15, 1 according to the invention, and jetPRIME® (Polyplus Transfection) as a commercial reference. Figure 4.c. Production of lentivirus using compounds 47, 50, 58, 59, 1 according to the invention, and jetPRIME® (Polyplus Transfection) as a commercial reference.

[0122] Figure 5. Production of lentivirus particles from adherent HEK-293T cells. Figure 5.a. Production of lentivirus particles with transfection performed at 1 pg DNA / million cells using compounds 44, 18, 30, 31 , 32, 1 according to the invention, and TranslT®-Lenti (Mirus Bio), jetPRIME® (Polyplus Transfection), and PEIpro® (Polyplus Transfection) as commercial references. Figure 5.b. Production of lentivirus particles with transfection performed at 0.75 pg DNA / million cells using compounds 58, 52, 30, 1 according to the invention, and TranslT®-Lenti (Mirus Bio), jetPRIME® (Polyplus Transfection), and PEIpro® (Polyplus Transfection) as commercial references. Figure 5.c. Production of lentivirus particles with transfection performed at 1 pg DNA / million cells using compounds 58, 52, 30, 1 according to the invention, and TranslT®-Lenti (Mirus Bio), jetPRIME® (Polyplus Transfection), and PEIpro® (Polyplus Transfection) as commercial references.

[0123] Figure 6. Production of lentivirus particles in HEK 293 F cells using compound 1 according to the invention, PEIpro® (Polyplus Transfection) as a commercial reference, compounds 58 and 59 according to the invention.

[0124] Figure 7. Production of lentivirus particles in suspension with different complexation buffers or cell culture media using compound 59 according to the invention.

[0125] Figure 8. Production of lentivirus particles in suspension with different complexation volumes using compound 1 according to the invention and jetPRIME® (Polyplus Transfection) as a commercial reference.

[0126] Figure 9. Production of lentivirus particles using diffent DNA amounts and different ratios [DNA:transfection reagent] using compound 55 according to the invention and PEIpro® (Polyplus Transfection) as a commercial reference. Figure 10. HPLC chromatograms after HCI treatment of compound 55 according to the invention in different conditions.

[0127] Figure 11. Determination of the linearity of the hydrolysis.

[0128] Figure 12. GFP pDNA transfection of HEK-293T cells with compounds 1 , 13, 15, 16, 23, 27, 33, 50, 51 , 53, 55 and 58 according to the invention, and comparative compounds 2, 4 and 20.

[0129] Figure 13. GFP pDNA transfection of HEK-293T cells with compounds 47 and 52 according to the invention.

[0130] Figure 14. GFP mRNA transfection of HEK-293T cells with compounds 13, 16, 23, 33, 51 , 52, 55 and 59 according to the invention, and comparative compounds 2, 4 and 20.

[0131] Experimental section

[0132] Material and Methods

[0133] Bioproduction

[0134] • Cell culture

[0135] HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cells were grown in suspension, in Freestyle™ F17 medium (ThermoFisher), supplemented with 8 mM glutamine, 100 ll / rnL of penicillin, 100 pg / mL of streptomycin and 0.1 % Pluronic. Cells were incubated at 37°C in a 8% CO2 in air atmosphere under agitation (130 rpm - orbital of 50 mm).

[0136] HEK-293T (ATCC® CRL-3216™): Human embryonic kidney cell is a highly transfectable derivative of human embryonic kidney 293 cells and expresses the SV40 T-antigen. HEK-293T cells are widely used for recombinant virus production, gene expression and protein production.

[0137] • Recombinant AAV production

[0138] HEK-293T cells were seeded at 1 x 106cells / mL in 28.5 mL of Freestyle™ F17(ThermoFisher) supplemented with 8 mM L-glutamine, 100 Ll / rnL of penicillin, 100 pg / mL of streptomycin and 0.1 % Pluronic in 125 mL flask Erlenmeyer. Cells were incubated at 37°C in a 8% CO2 in air atmosphere under agitation (130 rpm - orbital of 50 mm). Recombinant Adeno Associated Viruses (rAAVs) were produced in HEK-293T cells, seeded at 1 x 106cells / mL and cultivated for 24 hours at 37°C, 8% CO2 before being co-transfected with 3 plasmids, a pRC2-9 vector expressing Rep and Cap from CellBiolabs, a pHelper pALD-X80 vector from Aldevron®, and pAAV-GFP (catalog number AAV-400, Cell BIOLABS, INC.) transfer vector expressing the GFP under control of a CMV promoter. Plasmids were diluted at a 2:2:1 mass ratio (for respectively pRC2-9:pAAV-GFP:pHelper) in 1.5 mL of non-supplemented culture medium at a total amount of 1 pg DNA per millions cells, then transfection reagent was added onto the diluted DNA (ratio 1 pL per pg of total DNA), mixed with a vortex, and incubated for 30 minutes at room temperature. Transfection complexes were added onto the cells and then the Erlenmeyer flask was incubated for 72 hours at 37°C in a 8% COs in air atmosphere under agitation (130 rpm - orbital of 50 mm).

[0139] Transfected cells were harvested 3 days after transfection and centrifugated for 5 minutes at 1000 rpm, the supernatant was discarded, and the pellet was resuspended in 2 mL of PBS. Cells were then lysed to liberate rAAVs using 3 successive freeze / thaw cycles at -80°C and 37°C. Then, 2 mL of the lysate were collected and centrifuged 30 minutes at 14000 rpm to separate rAAVs from cell debris. Supernatants containing rAAVs were then analyzed.

[0140] The transducing unit titer (TU / mL) was determined by using recombinant Adeno Associated Viruses expressing the GFP reporter gene after infection of permissive cells, HT-1080, or CHO- K1 , depending on the produced rAAV serotype, in 96-well plate. Briefly, permissive cells were seeded at 7 x 103cells per well and incubated 4 hours at 37°C in a 5% CO2 in air atmosphere. Harvested rAAVs were then serial diluted in supplemented culture medium and added on permissive cells as a replacement of the previous culture medium used. The GFP expression was analyzed by flow cytometry 72 hours after transduction to determine the transducing units.

[0141] The capsid titer (VP / mL) was determined by using AAV Titration ELISA kit (catalog number PRAAV2 / PRAAV5 / PRAAV8 / PRAAV9, PROGEN INC), following the manufacturer recommendations and protocol and according to the serotype of the recombinant Adeno Associated Viruses produced and analyzed.

[0142] The genome titer (VG / mL) was determined by qPCR (QuantStudio™3 - ThermoFisher). Plasmid DNA coding for GFP was used from a concentration of 2 x 108copies / pL to 2 x 102copies / pL to generate a standard curve using primers (Qiagen) targeting GFP reporter gene expressed by the rAAV produced, and the SensiFAST probe Lo-ROX kit (Ozyme). Recombinant LV production

[0143] HEK-293T cells were seeded at 1 x 106cells / mL in 28.5 mL of Freestyle™ F17 supplemented with 8 mM L-glutamine, 100 ll / rnL of penicillin, 100 pg / mL of streptomycin and 0.1% Pluronic in 125 mL flask Erlenmeyer. Cells were incubated at 37°C in a 8% COs in air atmosphere under agitation (130 rpm - orbital of 50 mm).

[0144] Recombinant Lentivirus (LVs)) were produced in HEK-293T cells, seeded at 1 x 106cells / mL and cultivated for 24h at 37°C, 8% CO2 before being co-transfected with the 4 plasmids of the pALD- Lenti system (Aldevron®) at a ratio of 1 :2:1 :3 of pALD-Rev, pALD-GagPol, pALD-VSV-G, and pALD-Lenti-EGFP, respectively, and a total DNA amount of 1 pg per million cells.

[0145] Plasmids were diluted in 1 .5 mL of non-supplemented culture medium, then transfection reagent was added onto the diluted DNA (ratio 1 pL per pg of total DNA), mixed with a vortex, and incubated for 30 minutes at room temperature. Transfection complexes were added onto the cells and then the Erlenmeyer flask was incubated for 72 hours at 37°C in an 8% CO2 in air atmosphere under agitation (130 rpm - orbital of 50 mm).

[0146] Recombinant lentiviruses were harvested by centrifugation at 1300 g for 15 minutes at 4°C. Supernatant containing the virus was then aliquoted and stored at 4°C.

[0147] The transducing unit titer (TU / mL) was determined by using recombinant lentivirus expressing the GFP reporter gene after infection of permissive cells, HT-1080, or in 96-well plate. Briefly, HT- 1080 were seeded at 7 x 103cells per well and incubated 3 hours at 37°C in a 5% CO2 in air atmosphere. Harvested LVs were then serial diluted in supplemented culture medium and added on permissive cells as a replacement of the previous culture medium used. The GFP expression was analyzed by flow cytometry 72 hours after transduction to determine the transducing units.

[0148] EXAMPLES

[0149] Example 1. General route for the synthesis of grafted polymers compounds of formula (I; of the invention (4)

[0150] Amine (1 ) and derivatives are engaged in a coupling reaction with the corresponding acid chloride or carboxylic acid (2) to afford esters (3). The ester saponification yields to the corresponding acid (4), which is grafted to the PEI backbone (P+) by generation of amide functions using DMTMM (4- (4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride) in NMM (4-Methylmorpholine) coupling reagent.

[0151] General Procedure A:

[0152] In an adapted round bottom flask, the amine (1 ) (1.05 eq.) was diluted in CH2CI2 (0.6 M) and triethylamine (1 .5 eq.) was added at 0°C. The acyl chloride (1 eq.) was then added dropwise over 10 minutes using a dry dropping funnel and the reaction mixture was stirred at 0°C for 15 minutes, then at room temperature for 15 minutes. The mixture was quenched with H2O and poured into 1 N HCI and the layers were separated. The aqueous layer was extracted with DCM (Dichloromethane). The combined organic layers were washed with saturated NaCI, dried over Na2SC>4, filtered and evaporated to give the product as a pale orange oil.

[0153] To a solution of the ester (3), obtain in the previous step, in ethanol was added NaOH 5 M and the reaction mixture was stirred at room temperature for 40 minutes.

[0154] The mixture was concentrated to remove the ethanol and the residue was acidified with 3 M HCI leading to precipitation. The mixture was filtered on a glass frit and the solid was washed with H20, then Et2O and dried under high vacuum for 2 days. The product was obtained as a white solid.

[0155] General procedure B:

[0156] Acyl chloride (1.1 eq.) was added dropwise to a solution of amine (1 ) (1 eq.) and Et3N (2.2 eq.) in CH2CI2(0.3 M) at 0°C and the mixture was stirred until completion.

[0157] The mixture was quenched with H2O and extracted with CH2CI2. The combined organic layers were dried over Na2SO4, filtered and evaporated under reduced pressure to give the product.

[0158] If needed, the product can be purified by flash chromatography over silica gel using CH2CI2 / MeOH as eluent.

[0159] The product was retaken in Ethanol and NaOH 1 M was added and the reaction was stirred at room temperature until completion (approximately 1 hour).

[0160] The mixture was evaporated to remove the EtOH and the residue was neutralized with HCI 3 N until pH 5-6.

[0161] The product was then purified by reverse phase chromatography using water and acetonitrile.

[0162] General Procedure C:

[0163] Carboxylic acid (2) (1 eq.) and DIPEA (N,N-Diisopropylethylamine) (3 eq) were stirred in DMF(Dimethylformamide). HATLI (1 -[Bis(dimethylamino)methylene]-1 H-1 ,2,3-triazolo[4,5- b]pyridinium 3-oxid hexafluorophosphate) (1.2 eq.) was added and after 15 minutes, the amine (1 ) (1.05 eq.) was added and the reaction is stirred for 16 hours. DMF was evaporated and the mixture was extracted using saturated NaHCO3and ethyl acetate. The organic layer was washed with saturated NaCI and dried over Na2SO4 and evaporated under reduced pressure. The product was then purified by silica gel chromatography. The obtained ester compound was then saponified using NaOH in ethanol.

[0164] General Procedure D:

[0165] In an adapted round bottom flask, PEI (1 g, 1 eq.) was added and diluted with H2O (20 mL). NMM (4-Methylmorpholine) (2 eq.) was then added followed by the carboxylic acid (x eq. according to the grafting percentage needed) in MeOH (80 mL). After stirring 10 minutes at 30°C, DMTMM (4- (4,6-Dimethoxy-1 ,3,5-triazin-2-yl)-4-methylmorpholinium chloride) (2 eq.) was added and the mixture was stirred at the required temperature for 12-16 hours.

[0166] After evaporation of MeOH, the residue was diluted with H2O (80 mL) and HCI 3 M (approximately?.5 mL) was slowly added until pH 2 and solubilization. The solution was then purified by TFF (Tangential flow filtration) against 20 mM HCI (Sartocon

[0167] Slice 200 30 kDa) or by dialysis against 50 mM HCI.

[0168] The product was then lyophilized for 5 days to give a white to off-white powder.

[0169] Example 2. Syntheses of compounds of formula (I) of the invention, i.e., compounds 1, 12- 16, 18, 19, 21-25 and 27-59.

[0170] Synthesis of compound 1

[0171] Following general procedure D with 1 .i as the carboxylic acid

[0172] 1H NMR (400 MHz, D2O) 5 7.4-6.5 (m, 4H), 4.53-4.30 (m, 1 H), 4.1 -2.6 (m, 20.42H), 2.1 -1.1 ppm (m, 4H).

[0173] Synthesis of compound 1.i

[0174] Following general procedure A with ethyl isonipecotate and 4-methoxybenzoyl chloride.

[0175] 1H NMR (400 MHz, MeOD) 5 7.44-7.36 (m, 2H), 7.07-6.99 (m, 2H), 4.54-4.43 (m, 1 H), 3.87 (s, 3H), 3.83-3.75 (m, 1 H) 3.21 -3.11 (m, 1 H), 3.04-2.92 (m, 1 H), 2.43-2.36 (m, 1 H), 2.05-1.95 (m, 1 H), 1 -901 .80 (m, 1 H), 1 .73-1 .56 ppm (m, 2H).

[0176] Synthesis of compound 12

[0177] Following general procedure D with 12.i (0.35 eq.) as the carboxylic acid. Before the purification, the solvents were evaporated and TFA (Trifluoroacetic acid) was added at 0°C and the mixture was stirred at room temperature for 3 hours. Water was added and the product was then purified by dialysis against HCI 50 mM.1H NMR (400 MHz, D2O) 5 7.35-6.60 (m, 4H), 4.50-4.26 (m, 1 H), 3.93-2.56 (m, 21 .1 H), 2.07-0.33 ppm (m, 6H).

[0178] Synthesis of compound 12.i

[0179] Following general procedure A using ethyl isonipecotate and 4-hydroxybenzoic acid.

[0180] Following this step, the product was retaken in a mixture of THF(Tetrahydrofuran) and H2O (1 :1 ) and triethylamine (2.5 eq.) and DMAP(4-(Dimethylamino)pyridine) (0.2 eq) were added and the solution was cooled to 0°C. Boc2O (1.1 eq.) was then added and the solution was stirred until completion. The product was then purified by reverse phase chromatography.

[0181] 1H NMR (400 MHz, CDCI3) 6 7.54-7.38 (m, 2H), 7.28-7.21 (m, 2H), 4.64-4.40 (m, 1 H), 3.92-3.68 (m, 1 H), 3.18-3.04 (m, 2H), 2.70-2.60 (m, 1 H), 2.09-1 .59 ppm (m, 4H).

[0182] Synthesis of compound 13

[0183] Following general procedure D with 13.i (0.5 eq.) as the carboxylic acid

[0184] 1H NMR (400 MHz, D2O) 5 7.24-6.61 (m, 4H), 4.44-2.44 (m, 28.2H), 1 .95 - 0.88 ppm (m, 4H).

[0185] Synthesis of compound 13.i

[0186] Following general procedure B using ethyl isonipecotate and 2-(4-hydroxyphenyl)acetyl chloride1H NMR (400 MHz, MeOD) 6 7.01 -6.86 (m, 2H), 6.72-6.57 (m, 2H), 4.25-4.19 (m, 1 H), 3.82-3.76

[0187] (m, 1 H), 3.54 (s, 2H), 3.13-2.78 (m, 1 H), 2.78-2.66 (m, 1 H), 2.47-2.38 (m, 1 H), 1 .92-1 .71 (m, 1 H), 1.69-1.58 (m, 1 H), 1.55-1.32 (m, 1 H), 1.32-1.18 ppm (m, 1 H).

[0188] Synthesis of compound 14

[0189] Following general procedure D with 14.i (0.5 eq.) as the carboxylic acid

[0190] 1H NMR (400 MHz, D2O) 6 7.25-6.54 (m, 4H), 4.52-0.55 ppm (m, 31 H).

[0191] Synthesis of compound 14.i

[0192] Following general procedure C using ethyl isonipecotate and 3-(4-Hydroxyphenyl)propionic acid

[0193] 1H NMR (400 MHz, MeOD) 5 7.07-7.02 (m, 2H), 6.75-6.68 (m, 2H), 4.39-4.27 (m, 1 H), , 3.83-3.76 (m, 1 H), 3.08-2.99 (m, 1 H), 2.95-2.88 (m, 1 H), 2.87-2.73 (m, 2H), 2.73-2.60 (m, 2H), 2.63-2.48 (m, 1 H), 1 .90-1 .76 (m, 2H), 1 .54-1 .27 ppm (m, 2H).

[0194] Synthesis of compound 15

[0195] Following general procedure D with 15.i (0.3 eq.) as the carboxylic acid

[0196] 1 H NMR (400 MHz, D2O) 6 7.34-6.51 (m, 4H), 4.56-0.71 ppm (m, 35.7H). Synthesis of compound 15.i

[0197] Following general procedure C using ethyl isonipecotate and 4-(4-Hydroxyphenyl)butanoic acid

[0198] 1H NMR (400 MHz, MeOD) 5 7.07-6.96 (m, 2H), 6.80-6.65 (m, 2H), 4.45-4.35 (m, 1 H), 3.82-3.72 (m, 1 H), 3.08-2.97 (m, 1 H), 2.74-2.68 (m, 1 H), 2.56-2.48 (m, 2H), 2.42-2.28 (m, 3H), 1.93-1.78 (m, 4H), 1 .66-1 .46 ppm (m, 2H).

[0199] Synthesis of compound 16

[0200] Following general procedure D with 16.i (0.3 eq.) as the carboxylic acid

[0201] 1H NMR (400 MHz, D2O) 5 7.54-6.31 (m, 4H), 4.53-4.35 (m, 1 H), 4.10-2.54 (m, 30.4H), 2.18-1 .24 ppm (m, 4H).

[0202] Synthesis of compound 16.i

[0203] Following general procedure A using ethyl isonipecotate and 3-methoxybenzoyl chloride

[0204] 1H NMR (400 MHz, MeOD) 5 7.31 -7.22 (m, 1 H), 6.96-6.90 (m, 1 H), 6.88-6.81 (m, 2H), 4.38-4.29 (m, 1 H), 3.72 (s, 3H), 3.63-3.57 (m, 1 H), 3.08-2.99 (m, 2H), 2.58-2.48 (m, 1 H), 1 .97-1 .89 (m, 1 H), 1 .82-1 .73 (m, 1 H), 1 .65 - 1 .51 ppm (m, 2H). Synthesis of compound 18

[0205] Following general procedure D with 18.i (0.4eq.) as the carboxylic acid

[0206] 1H NMR (400 MHz, D2O) 5 7.01 -6.53 (m, 3H), 4.50-2.56 (m, 24.91 H), 2.01 -1 .26 ppm (m, 4H).

[0207] Synthesis of compound 18.i

[0208] Following general procedure A using ethyl isonipecotate and 3,4-Dimethoxybenzoyl chloride

[0209] 1H NMR (400 MHz, DMSO-d6) 5 12.30 (s, 1 H), 7.04-6.83 (m, 3H), 4.32-3.66 (m, 8H), 3.13-2.89 (m, 2H), 2.62-2.45 (m, 1 H), 1 .96-1 .74 (m, 2H), 1 .56-1 .43 ppm (m, 2H).

[0210] Synthesis of compound 19

[0211] Following general procedure D with 19.i (0.4eq.) as the carboxylic acid

[0212] 1H NMR (400 MHz, D2O) 6 6.94-5.56 (m, 5H), 4.63-0.94 ppm (m, 22.05H).

[0213] Synthesis of compound 19.i Following general procedure A using ethyl isonipecotate and 1 ,3-Benzodioxole-5-carbonyl chloride

[0214] 1H NMR (400 MHz, DMSO-d6) 5 12.30 (s, 1 H), 7.02-6.82 (m, 3H), 6.07 (s, 2H), 4.34-3.49 (m, 2H), 3.12-2.89 (m, 2H), 2.61 -2.51 (m, 1 H), 1.91 -1.74 (m, 2H), 1.60-1.39 ppm (m, 2H).

[0215] Synthesis of compound 21

[0216] Following general procedure D using 21 .i (0.35 eq) as the carboxylic acid.

[0217] 1H NMR (400 MHz, D2O) 6 7.61 -7-12 (m, 5H), 4.58-4-48 (m, 1 H), 4.19-2.55 (m, 19.41 H), 2.12- 1 .1 1 ppm (m, 4H).

[0218] Synthesis of compound 21.i

[0219] Following general procedure B using ethyl isonipecotate and benzoyl chloride.

[0220] 1H NMR (400 MHz, CDCI3) 5 7.42-7.31 (m, 5H), 4.53-4.42 (m, 1 H), 3.12-3.01 (m, 3H), 2.62-2.54 (m, 1 H), 1 .94-1 .72 ppm (m, 4H). Synthesis of compound 22

[0221] Following general procedure D with 22. i (0.4 eq.) as the carboxylic acid

[0222] 1H NMR (400 MHz, D2O) 5 8.22-6.13 (m, 7H), 4.51 -0.72 ppm (m, 22.42H). Synthesis of compound 22.i

[0223] Following general procedure A using ethyl isonipecotate and 2-Naphthoyl Chloride

[0224] 1H NMR (400 MHz, DMSO-d6) 5 12.33 (s, 1 H), 8.08-7.90 (m, 4H), 7.63-7.54 (m, 2H), 7.52-7.48 (m, 1 H), 4.43-4.28 (m, 1 H), 3.67-3.56 (m, 1 H), 3.19-2.95 (m, 2H), 2.59-2.52 (m, 1 H), 2.02-1.73 (m, 2H), 1.63-1.50 ppm (m, 2H).

[0225] Synthesis of compound 23

[0226] Following general procedure D with 24. i (0.35 eq.) as the carboxylic acid.

[0227] 1H NMR (400 MHz, D2O) 5 7-52-6.81 (m, 4H), 4.55-4.45 (m, 1 H), 4.1 1 -2.54 (m, 19.21 H), 1 -98- 1 .21 ppm (m, 4H).

[0228] Synthesis of compound 24

[0229] Following general procedure D with 24. i (0.4eq.) as the carboxylic acid

[0230] 1H NMR (400 MHz, D2O) 5 7.39-6.89 (m, 4H), 4.52-4.33 (m, 1 H), 4.1 1 -2.54 (m, 18H), 1.95-1.31 ppm (m, 4H). Synthesis of compound 24.i

[0231] Following general procedure A using ethyl isonipecotate and 4-fluorobenzoyl chloride

[0232] 1H NMR (400 MHz, CDCI3) 6 7.44-7.32 (m, 2H), 7,16-7.01 (m, 2H), 4-61 -3.62 (m, 2H), 3.12-3.02 (m, 2H), 2.66-2.49 (m, 1 H), 2.13-1.61 ppm (m, 4H).

[0233] Synthesis of compound 25

[0234] Following general procedure D with 25. i (0.4eq.) as the carboxylic acid

[0235] 1H NMR (400 MHz, D2O) 6 7.50-6.91 (m, 4H), 4.56-4.46 (m, 1 H), 4.36-1 .03 (m, 21 .53H).

[0236] Synthesis of compound 25.i

[0237] Following general procedure B using ethyl isonipecotate and 2-fluorobenzoyl chloride

[0238] 1H NMR (400 MHz, DMSO-d6) 5 7.55-7.20 (m, 4H), 4.24-4.17 (m, 1 H), 3.36-2.28 (m, 1 H), 3.04- 2.95 (m, 2H), 2.12-2.04 (m, 1 H), 1 .83-1 .61 (m, 2H), 1 .58-1 .35 ppm (m, 2H).

[0239] Synthesis of compound 27

[0240] Following general procedure D with 27. i (0.4 eq.) as the carboxylic acid1H NMR (400 MHz, D2O) 6 7.52-6.80 (m, 3H), 4.59-4.42 (m, 1 H), 4.16-2.66 (m, 17.48H), 1.97- 1 .37 ppm (m, 4H).

[0241] Synthesis of compound 27.i

[0242] Following general procedure A using ethyl isonipecotate and 2,6-difluorobenzoyl chloride

[0243] 1H NMR (400 MHz, DMSO-d6) 5 12.39 (s, 1 H), 7.60-7.50 (m, 1 H), 7.31 -7.15 (m, 2H), 4.41 -4.35 (m, 1 H), 3.46-3.29 (m, 1 H), 3.24-2.92 (m, 2H), 2.62-2.52 (m, 1 H), 2.02-1.74 (m, 2H), 1.57-1.32 ppm (m, 2H).

[0244] Synthesis of compound 28

[0245] Following general procedure D with 28. i (0.4eq.) as the carboxylic acid

[0246] 1H NMR (400 MHz, D2O) 5 7.03-6.63 (m, 3H), 4.57-0.92 ppm (m, 22.03H).

[0247] Synthesis of compound 28.i

[0248] Following general procedure A using ethyl isonipecotate and 3,5-difluorobenzoyl chloride

[0249] 1H NMR (400 MHz, DMSO-d6) 5 12.36 (s, 1 H), 7.39-7.30 (m, 1 H), 7.24-7.09 (m, 2H), 4.234-4.22 (m, 1 H), 3.53-3.40 (m, 1 H), 3.15-2.90 (m, 2H), 2.62-2.51 (m, 1 H), 1.97-1.72 (m, 2H), 1.61 -1.44 ppm (m, 2H). Synthesis of compound 29

[0250] Following general procedure D with 29. i (0.4 eq.) as the carboxylic acid

[0251] 1H NMR (400 MHz, D2O) 5 7.33-6.78 (m, 3H), 4.52-4.31 (m, 1 H), 4.16-2.54 (m, 16.73H), 2.02- 1 .39 ppm (m, 4H).

[0252] Synthesis of compound 29.i

[0253] Following general procedure A using ethyl isonipecotate and 3,4-Difluorobenzoyl chloride

[0254] 1H NMR (400 MHz, DMSO-d6) 5 12.31 (s, 1 H), 7.60-7.42 (m, 2H), 7.29-7.23 (m, 1 H), 4.33-4.20 (m, 1 H), 3.57-3.44 (m, 1 H), 3.17-2.88 (m, 2H), 2.62-2.52 (m, 1 H), 1.97-1.72 (m, 2H), 1.63-1.41 ppm (m, 2H).

[0255] Synthesis of compound 30

[0256] Following general procedure D with 30. i (0.4eq.) as the carboxylic acid

[0257] 1H NMR (400 MHz, D2O) 5 7.41 -7.20 (m, 1 H), 7-0.9-6.75 (m, 2H), 4.54-4.41 (m, 1 H), 4.16-2.46 (m, 17.4H), 1 .98-1 .38 ppm (m, 4H).

[0258] Synthesis of compound 3O.i Following general procedure A using ethyl isonipecotate and 2,4-difluorobenzoyl chloride

[0259] 1H NMR (400 MHz, DMSO-d6) 5 12.33 (s, 1 H), 7.57-7.42 (m, 1 H), 7.41 -7.31 (m, 1 H), 7.22-7.14 (m, 1 H), 4.43-4.26 (m, 1 H), 3.41 -3.32 (m, 1 H), 3.24-2.88 (m, 2H), 2.58-2.52 (m, 1 H), 1.97-1.74 (m, 2H), 1.56-1.38 ppm (m, 2H).

[0260] Synthesis of compound 31

[0261] Following general procedure D with 31 .i (0.4 eq.) as the carboxylic acid

[0262] 1H NMR (400 MHz, D2O) 5 7.22-6.89 (m, 3H), 4.56-4.39 (m, 1 H), 4.04-2.65 (m, 17.7H), 2-0.11 .45 ppm (m, 4H).

[0263] Synthesis of compound 31.i

[0264] Following general procedure A using ethyl isonipecotate and 2,5-Difluorobenzoyl chloride

[0265] 1H NMR (400 MHz, DMSO-d6) 5 12.33 (s, 1 H), 7.41 -7.30 (m, 3H), 4.37-4.29 (m, 1 H), 3.39-3.32 (m, 1 H), 3.20-2.88 (m, 2H), 2.59-2.52 (m, 1 H), 2.04-1 .70 (m, 2H), 1 .55-1 .41 ppm (m, 2H).

[0266] Synthesis of compound 32 Synthesis of compound 32.i

[0267] Following general procedure A using ethyl isonipecotate and 2,3-difluorobenzoyl chloride

[0268] 1H NMR (400 MHz, DMSO-d6) 5 12.42-12.26 (m, 1 H), 7.56-7.47 (m, 1 H), 7.34-7.26 (m, 1 H), 7.25- 7.20 (m, 1 H), 4.39-4.31 (m, 1 H), 3.47-3.25 (m, 1 H), 3.23-2.90 (m, 2H), 2.60-2.52 (m, 1 H), 2.04- 1 .71 (m, 2H), 1 .61 -1 .35 ppm (m, 2H).

[0269] Synthesis of compound 33

[0270] Following general procedure D with 33. i (0.4 eq.) as the carboxylic acid

[0271] 1H NMR (400 MHz, D2O) 5 7.44-6.88 (m, 4H), 4.49-0.79 ppm (m, 22.6H).

[0272] Synthesis of compound 33.i

[0273] Following general procedure A using ethyl isonipecotate and 4-chlorobenzoyl chloride

[0274] 1H NMR (400 MHz, DMSO-d6) 5 12.32 (s, 1 H), 7.55-7.47 (m, 2H), 7.46-7.38 (m, 2H), 4.37-4.22 (m, 1 H), 3.58-3.27 (m, 12H), 3.22-2.84 (m, 2H), 2.63-2.51 (m, 1 H), 1.97-1.71 (m, 2H), 1.57-1.44 ppm (m, 2H).

[0275] Synthesis of compound 34 Following general procedure D with 34. i (0.42eq.) as the carboxylic acid

[0276] 1H NMR (400 MHz, D2O) 5 7.61 -6.85 (m, 4H), 4.52-0.87 ppm (m, 22.6H).

[0277] Synthesis of compound 34.i

[0278] Following general procedure A using ethyl isonipecotate and 4-bromobenzoyl chloride

[0279] 1H NMR (400 MHz, DMSO-d6) 5 12.32 (s, 1 H), 7.77-7.58 (m, 2H), 7.50-7.28 (m, 2H), 4.36-4.23 (m, 1 H), 3.57-3.45 (m, 1 H), 3.15-2.88 (m, 2H), 2.63-2.52 (m, 1 H), 2.04-1.68 (m, 2H), 1.58-1.46 ppm (m, 2H).

[0280] Synthesis of compound 35

[0281] Following general procedure D with 35. i (0.4eq.) as the carboxylic acid

[0282] 1H NMR (400 MHz, D2O) 5 7.61 -6.91 (m, 3H), 4.4- 0.31 ppm (m, 22.2H).

[0283] Synthesis of compound 35.i

[0284] Following general procedure A using ethyl isonipecotate and 3,4-dichlorobenzoyl chloride

[0285] 1H NMR (400 MHz, DMSO-d6) 5 12.32 (s, 1 H), 7.85-7.56 (m, 2H), 7.41 -7.36 (m, 1 H), 4.34-4.23 (m, 1 H), 3.53-3.44 (m, 1 H), 3.23-2.85 (m, 2H), 2.63-2.52 (m, 1 H), 2.04-1.68 (m, 2H), 1.59-1.46 ppm (m, 2H). Synthesis of compound 36

[0286] Following general procedure D with 36. i (0.4 eq.) as the carboxylic acid

[0287] 1H NMR (400 MHz, D2O) 6 7.17-6.49 (m, 3H), 4.46-0.74 ppm (m, 26.7H).

[0288] Synthesis of compound 36.i

[0289] Following general procedure A using ethyl isonipecotate and 3,5-dimethylbenzoyl chloride

[0290] 1H NMR (400 MHz, DMSO-d6) 5 12.30 (s, 1 H), 7.08-7.05 (m, 1 H), 6.98-6.93 (m, 2H), 4.34-4.26 (m, 1 H), 3.59-3.49 (m, 1 H), 3.1 1 -2.88 (m, 2H), 2.57-2.52 (m, 1 H), 2.30 (s, 6H), 1.98-1.71 (m, 2H), 1 .54-1 .41 ppm (m, 2H).

[0291] Synthesis of compound 37

[0292] Following general procedure D with 37. i (0.4eq.) as the carboxylic acid

[0293] 1H NMR (400 MHz, D2O) 6 7.20-6.85 (m, 2H), 4.52-0.98 ppm (m, 21 .12H).

[0294] Synthesis of compound 37.i

[0295] Following general procedure B using ethyl isonipecotate and 3,4,5-Trifluorobenzoyl chloride1H NMR (400 MHz, DMSO-d6) 5 12.33 (s, 1 H), 7.52-7.35 (m, 2H), 4.33-4.20 (m, 1 H), 3.55-3.43 (m, 1 H), 3.17-2.90 (m, 2H), 2.63-2.52 (m, 1 H), 1.96-1.67 (m, 2H), 1.65-1.43 ppm (m, 2H).

[0296] Synthesis of compound 38

[0297] Following general procedure D with 38. i (0.4eq.) as the carboxylic acid

[0298] 1H NMR (400 MHz, D2O) 5 7.26-6.96 (m, 1 H), 4.57-0.87 ppm (m, 22.66H).

[0299] Synthesis of compound 38.i

[0300] Following general procedure B using ethyl isonipecotate and 2,3,4,5-Tetrafluorobenzoyl chloride

[0301] 1H NMR (400 MHz, DMSO-d6) 5 12.35 (s, 1 H), 7.57 (s, 1 H), 4.36-4.27 (m, 1 H), 3.52-3.38 (m, 1 H), 3.18-2.90 (m, 2H), 2.61 -2.52 (m, 1 H), 2.04-1 .69 (m, 2H), 1 .62-1 .37 ppm (m, 2H).

[0302] Synthesis of compound 39

[0303] Following general procedure D with 39. i (0.4 eq.) as the carboxylic acid

[0304] 1H NMR (400 MHz, D2O) 5 4.56-4.40 (m, 1 H), 4.05-2.60 (m, 22.3H), 2.13-1 .29 ppm (m, 4H).

[0305] Synthesis of compound 39.i Following general procedure B using ethyl isonipecotate and pentafluorobenzoyl chloride

[0306] 1H NMR (400 MHz, DMSO-d6) 5 12.39 (s, 1 H), 4.37-4.30 (m, 1 H), 3.62-3.45 (m, 1 H), 3.23-2.97 (m, 2H), 2.62-2.54 (m, 1 H), 2.04-1 .74 (m, 2H), 1 .58-1 .30 ppm (m, 2H).

[0307] Synthesis of compound 40

[0308] Following general procedure D using 40. i (0.35 eq.) as the carboxylic acid

[0309] 1H NMR (400 MHz, D2O) 5 8.99-8.84 (m, 2H), 8.21 -8.08 (m, 2H), 4.68-4.52 (m, 1 H), 4.42-3.1 1 (m, 28.97H), 2.18-1.45 ppm (m, 4H).

[0310] Synthesis of coumpound 4O.i

[0311] Following general procedure B using ethyl isonipecotate and isonicotinoyl chloride

[0312] 1H NMR (400 MHz, D2O) 5 8.93 (d, J = 6Hz, 2H), 8.1 1 (d, J = 6Hz, 2H), 4.43-4.34 (m, 1 H), 3.56- 3.48 (m, 1 H), 3.33-3.21 (m, 1 H), 3.19-3.09 (m, 1 H), 2.85-2.71 (m, 1 H), 2.20-2.08 (m, 1 H), 1.96- 1 .85 (m, 1 H), 1 .78-1 .67 ppm (m, 2H).

[0313] Synthesis of compound 41

[0314] Following general procedure D with 41 .i (0.34 eq.) as the carboxylic acid

[0315] 1H NMR (400 MHz, D2O) 5 8.71 -8.60 (m, 1 H), 8.28-8.22 (m, 1 H), 7.84-7.71 (m, 2H), 4.54-4.46 (m, 1 H), 4.1 1 -2.65 (m, 23.68H), 2.21 -1 .33 ppm (m, 4H). Synthesis of compound 41.i

[0316] Following general procedure B using ethyl isonipecotate and picolinoyl chloride

[0317] 1H NMR (400 MHz, DMSO-d6) 5 8.59-8.55 (m, 1 H), 7.93-7.88 (m, 1 H), 7.52-7.41 (m, 2H), 4.26- 4.16 (m, 1 H), 3.55-3.44 (m, 1 H), 3.07-2.92 (m, 2H), 2.13-2.05 (m, 1 H), 1.83-1.77 (m, 1 H), 1.68- 1 .62 (m, 1 H), 1 .60-1 .41 ppm (m, 2H).

[0318] Synthesis of compound 42

[0319] Following general procedure D with 42. i (0.54 eq.) as the carboxylic acid

[0320] 1H NMR (400 MHz, D2O) 5 8.91 -8.87 (m, 1 H), 8.84-8.80 (m, 1 H), 8.61 -8.56 (m, 1 H), 8.12-8.06 (m, 1 H), 4.55-4.47 (m, 1 H), 4.10-2.83 (m, 26.78H), 2.09-1 .49 ppm (m, 4H).

[0321] Synthesis of compound 42.i

[0322] Following general procedure B using ethyl isonipecotate and nicotinoyl chloride

[0323] 1H NMR (400 MHz, DMSO-d6) 5 8.63 (dd, J = 4.8, 1 .7 Hz, 1 H), 8.57 (dd, J = 2.3, 0.9 Hz, 1 H), 7.80 (dt, J = 7.8, 1 .9 Hz, 1 H), 7.47 (ddd, J = 7.8, 4.9, 0.9 Hz, 1 H), 4.22-4.15 (m, 1 H), 3.49-3.41 (m, 1 H), 3.10-2.98 (m, 2H), 2.16-2.04 (m, 1 H), 1.85-1.77 (m, 1 H), 1.72-1.65 (m, 1 H), 1.54-1.47 ppm (m, 2H). Synthesis of compound 43

[0324] Following general procedure D with 43. i (0.35 eq.) as the carboxylic acid

[0325] 1H NMR (400 MHz, D2O) 5 8.92-8.48 (m, 3H), 4.56-4.45 (m, 1 H), 4.08-2.83 (m, 23.23H), 2.15- 1 .43 ppm (m, 4H).

[0326] Synthesis of compound 43. i

[0327] Following general procedure B using ethyl isonipecotate and pyrazine-2-carbonyl chloride

[0328] 1H NMR (400 MHz, DMSO-d6) 5 8.82 (d, J = 1 .6 Hz, 1 H), 8.74 (d, J = 2.6 Hz, 1 H), 8.68 (dd, J = 2.6, 1.6 Hz, 1 H), 4.37-4.27 (m, 1 H), 3.65-3.54 (m, 1 H), 3.16-3.08 (m, 1 H), 3.07-2.95 (m, 1 H), 2.52- 2.41 (m, 1 H), 1.95-1.87 (m, 1 H), 1.80-1.73 (m, 1 H), 1.61 -1.47 ppm (m, 2H).

[0329] Synthesis of compound 44

[0330] Following general procedure D with 44. i (0.35eq.) as the carboxylic acid

[0331] 1H NMR (400 MHz, D2O) 5 9.16-9.09 (m, 1 H), 8.94-8.86 (m, 1 H), 7.68-7.60 (m, 1 H), 4.52-4.43 (m, 1 H), 4.07-2.72 (m, 30.12H), 2.40-1 .19 ppm (m, 4H).

[0332] Synthesis of compound 44.i Following general procedure B using ethyl isonipecotate and pyrimidine-4-carbonyl chloride

[0333] 1H NMR (400 MHz, DMSO-d6) 5 9.24 (d, J = 1 .5 Hz, 1 H), 8.95 (d, J = 5.1 Hz, 1 H), 7.63 (dd, J = 5.1 , 1.5 Hz, 1 H), 4.24-4.17 (m, 1 H), 3.52-3.35 (m, 2H), 3.13-2.93 (m, 2H), 2.25-2.18 (m, 1 H), 1.93- 1.63 (m, 1 H), 1.65-1.39 (m, 2H),

[0334] Synthesis of compound 45

[0335] Following general procedure D with 45. i (0.35 eq.) as the carboxylic acid

[0336] 1H NMR (400 MHz, D2O) 58.86-8.78 (m, 2H), 7.62-7.54 (m, 1 H), 4.54-4.45 (m, 1 H), 4.1 1 -2.76 (m,

[0337] 23.1 H), 2.09-1.01 ppm (m, 4H).

[0338] Synthesis of compound 45.i

[0339] Following general procedure C using ethyl isonipecotate and Pyrimidine-2-carboxylic acid

[0340] 1H NMR (400 MHz, DMSO-d6) 5 8.89 (d, J = 5.0 Hz, 2H), 7.59 (t, J = 5.0 Hz, 1 H), 4.32-4.23 (m, 1 H), 3.23-3.13 (m, 1 H), 3.07-2.89 (m, 2H), 2.49-2.40 (m, 1 H), 1.95-1.86 (m, 1 H), 1.71 (m, 1 H), 1.56-1.41 ppm (m, 2H).

[0341] Synthesis of compound 46

[0342] Following general procedure D with 46. i (0.42eq.) as the carboxylic acid

[0343] 1H NMR (400 MHz, D2O) 59.27-9.16 (m, 1 H), 7.95-7.87 (m, 2H), 4.59-4.47 (m, 1 H), 4.09-2.65 (m, 24.1 H), 2.00-1.48 ppm (m, 4H). Synthesis of compound 46.i

[0344] Following general procedure B using ethyl isonipecotate and pyridazine-3-carbonyl chloride

[0345] 1H NMR (400 MHz, DMSO-d6) 5 9.33-9.26 (m, 1 H), 7.89-7.79 (m, 2H), 4.36-4.29 (m, 1 H), 3.56-

[0346] 3.48 (m, 1 H), 3.18-2.99 (m, 2H), 2.47-2.38 (m, 1 H), 1.96-1.88 (m, 1 H), 1.79-1.71 (m, 1 H), 1.62-

[0347] 1 .49 ppm (m, 2H).

[0348] Synthesis of compound 47

[0349] Following general procedure D with 47. i (0.35eq.) as the carboxylic acid

[0350] 1H NMR (400 MHz, D2O) 5 8.97-7.03 (m, 5H), 4.64-4.27 (m, 1 H), 4.15-2.83 (m, 23.2H), 2.21 -1 .32 ppm (m, 4H).

[0351] Synthesis of compound 47.i

[0352] Following general procedure B using ethyl isonipecotate and quinoxaline-2-carbonyl chloride

[0353] 1 H NMR (400 MHz, CDCI3) 5 9.15 (s, 1 H), 8.23-8.05 (m, 2H), 7.93-7.78 (m, 2H), 4.46-4.38 (m, 1 H), 3.84-3.76 (m, 1 H), 3.28-3.20 (m, 1 H), 3.12-3.03 (m, 1 H), 2.65-2.58 (m, 1 H), 2.04-1.94 (m, 1 H), 1 .87-1 .79 (m, 1 H), 1 .71 -1 .53 ppm (m, 2H). Synthesis of compound 48

[0354] Following general procedure D with 48. i (0.35 eq.) as the carboxylic acid

[0355] 1H NMR (400 MHz, D2O) 5 8.57-7.58 (m, 6H), 4.56-4.38 (m, 1 H), 4.0-2.9 (m, 21 ,6H), 2.1 -1 .2 ppm (m, 4H).

[0356] Synthesis of compound 48.i

[0357] Following general procedure B using ethyl isonipecotate and quinoline-2-carbonyl chloride

[0358] 1H NMR (400 MHz, MeOD) 5 8.39-8.34 (m, 1 H), 8.02-7.82 (m, 2H), 7.76-7.69 (m, 1 H), 7.64-7.48 (m, 2H), 4.48 (dtd, J = 13.1 , 4.0, 1 .6 Hz, 1 H), 3.63 (dtd, J = 13.6, 4.0, 1 .6 Hz, 1 H), 3.14-2.96 (m, 2H), 2.43-2.36 (m, 1 H), 2.01 -1 .93 (m, 1 H), 1 .83-1 .55 ppm (m, 3H).

[0359] Synthesis of compound 49

[0360] Following general procedure D with 48. i (0.42 eq.) as the carboxylic acid

[0361] 1H NMR (400 MHz, D2O) 5 8.70-7.55 (m, 6H), 4.59-4.38 (m, 1 H), 4.11 -2.44 (m, 16.2H), 2.13-1 .30 ppm (m, 4H). Synthesis of compound 50

[0362] Following general procedure D with 50. i (0.35eq.) as the carboxylic acid

[0363] 1 H NMR (400 MHz, D2O) 5 8.60-8.56 (m, 1 H), 8.43-8.36 (m, 1 H), 8.03-7.70 (m, 2H), 7.44-7.38 (m, 1 H), 4.52-4.47 (m, 1 H), 4.22-4.17 (m, 1 H), 4.02-2.76 (m, 23.7H), 2.11 -1 .52 ppm (m, 4H).

[0364] Synthesis of compound 5O.i

[0365] Following general procedure B using ethyl isonipecotate and imidazo[1 ,2-a]pyridine-2-carbonyl chloride

[0366] 1H NMR (400 MHz, CDCI3) 6 8.19 (dt, J = 6.8, 1 .2 Hz, 1 H), 8.08 (s, 1 H), 7.74 (d, J = 9.2 Hz, 1 H), 7.32 (ddd, J = 9.1 , 6.8, 1.3 Hz, 1 H), 6.93 (td, J = 6.8, 1.1 Hz, 1 H), 4.82-4.72 (m, 1 H), 4.34-4.23 (m, 1 H), 3.38-3.26 (m, 1 H), 3.01 -2.89 (m, 1 H), 2.28-2.18 (m, 1 H), 1.86-1.72 (m, 2H), 1.58-1.46 ppm (m, 2H).

[0367] Synthesis of compound 51

[0368] Following general procedure D using 51 .i (0.35 eq.) as the carboxylic acid

[0369] 1 H NMR (400 MHz, D2O) 5 7.476-7.02 (m, 4H), 4.63-2.65 (m, 23.5H), 2.21 -1 .30 ppm (m, 4H). Synthesis of compound 51.i

[0370] Following general procedure C using ethyl isonipecotate and Benzimidazole-2-carboxylic acid

[0371] 1 H NMR (400 MHz, DMSO-d6) 5 13.09 (s, 1 H), 7.82-7.48 (m, 2H), 7.36-7.22 (m, 2H), 5.33-5.24 (m, 1 H), 4.46-4.37 (m, 1 H), 3.55-3.43 (8 m, 1 H), 3.1 1 -2.99 (m, 1 H), 2.67-2.57 (m, 1 H), 2.02-1 .92 (m, 2H), 1.68-1.49 ppm (m, 2H).

[0372] Synthesis of compound 52

[0373] Following general procedure D with 52. i (0.35 eq.) as the carboxylic acid

[0374] 1H NMR (400 MHz, D2O) 5 7.33-6.64 (m, 4H), 4.42-4.24 (m, 1 H), 4.09-0.37 ppm (m, 29.81 H).

[0375] Synthesis of compound 52.i

[0376] Following general procedure B using 4-Piperidine acetic acid methyl ester and 4-methoxybenzoyl chloride

[0377] 1H NMR (400 MHz, DMSO-d6) 5 12.12 (s, 1 H), 7.38-7.30 (m, 2H), 7.02-6.94 (m, 2H), 4.51 -4.24 (m, 1 H), 3.80 (s, 3H), 3.73-3.55 (m, 1 H), 3.06-2.74 (m, 2H), 2.24-2.16 (m, 2H), 1 .98-1 .87 (m, 1 H), 1.77-1.61 (m, 2H), 1.21 -1.02 ppm (m, 2H).

[0378] Synthesis of compound 53 Following general procedure D with 52. i (0.41 eq.) as the carboxylic acid

[0379] 1H NMR (400 MHz, D2O) 5 7.18-6.79 (m, 4H), 4.52-0.55 ppm (m, 26.18H).

[0380] Synthesis of compound 54

[0381] Following general procedure D with 1 .i (0.08eq.) as the carboxylic acid

[0382] 1H NMR (400 MHz, D2O) 5 7.37-7.28 (m, 2H), 7.02-6.95 (m, 2H), 4.58-4.31 (m, 1 H), 4.05-2.78 (m, 97H), 2.14-1 .42 ppm (m, 4H).

[0383] Synthesis of compound 55

[0384] Following general procedure D with 1 .i (0.31 eq.) as the carboxylic acid

[0385] 1H NMR (400 MHz, D2O) 5 7.39-6.65 (m, 4H), 4.52-4.31 (m, 1 H), 4.22 - 2.52 (m, 23.09H), 1 .93- 1 .28 ppm (m, 4H).

[0386] Synthesis of compound 56

[0387] Following general procedure D with 1 .i (0.19 eq.) as the carboxylic acid

[0388] 1H NMR (400 MHz, D2O) 5 7.36-6.77 (m, 4H), 4.51 -4.35 (m, 1 H), 4.32-2.74 (m, 32.93H), 1.96- 1 .43 ppm (m, 4H). Synthesis of compound 57

[0389] Following general procedure D using 1 .i (0.29 eq.) as the carboxylic acid

[0390] 1 H NMR (400 MHz, D2O) 5 7.62-6.67 (m, 4H), 4.51 -4.35 (m, 1 H), 4.17-2.67 (m, 23.7H), 2.29- 1 .08 ppm (m, 4H).

[0391] Synthesis of compound 58

[0392] Following general procedure D with 51 .i (0.42 eq.) as the carboxylic acid

[0393] 1H NMR (400 MHz, D2O) 5 7.37-6.60 (m, 4H), 4.55-2.46 (m, 21 .76H), 1 .95-1 .32 ppm (m, 4H).

[0394] Synthesis of compound 59

[0395] Following general procedure D with 1 .i (0.3 eq.) as the carboxylic acid

[0396] 1H NMR (400 MHz, D2O) 5 7.39-6.69 (m, 4H), 4.57-2.54 (m, 27.61 H), 1 .98-1 .39 ppm (m, 4H).

[0397] Example 3. Syntheses of comparative compounds of formulas 2-11, 17, 20 and 26.

[0398] The inventors also carried out some comparative data using the following comparative compounds of formulas 2-1 1 , 17, 20 and 26 as disclosed in Table 2 below. Table 2. Structures of comparative compounds.

[0399] Synthesis of comparative compound 2

[0400] Following general procedure D with 2.i (0.4 eq.) as the carboxylic acid

[0401] 1H NMR (400 MHz, D2O) 6 7.71 -7.36 (m, 2H), 6.97-6.54 (m, 2H), 4.04-0.76 ppm (m, 22.75H).

[0402] Synthesis of comparative compound 2.i

[0403] Following general procedure B using 4-Aminobutyric acid methyl ester, hydrochloride and 4- methoxybenzoyl chloride

[0404] 1H NMR (400 MHz, DMSO-d6) 5 9.61 (t, J = 4.6 Hz, 1 H), 7.91 -7.83 (m, 2H), 7.00-6.92 (m, 2H), 3.80 (s, 3H), 3.24-3.19 (m, 2H), 2.1 1 -2.02 (m, 2H), 1 .72-1 .68 ppm (m, 2H). Synthesis of comparative compound 3

[0405] Following general procedure D with 2.i (0.35 eq.) as the carboxylic acid

[0406] 1H NMR (400 MHz, D2O) 6 7.72-7.40 (m, 2H), 7.02-6.57 (m, 2H), 4.25-1 .27 ppm (m, 26.09H).

[0407] Synthesis of comparative compound 4

[0408] Following general procedure D with 4.i (0.3 eq.) as the carboxylic acid

[0409] 1H NMR (400 MHz, D2O) 5 7.56-7.1 1 (m, 2H), 7.1 1 -6.71 (m, 2H), 4.22-2.69 (m, 31 ,7H), 2.63-1 .46 ppm (m, 4H).

[0410] Synthesis of comparative compound 4.i

[0411] Following general procedure B using methyl 4-(methylamino)butanoate and 4-methoxybenzoyl chloride

[0412] 1H NMR (400 MHz, MeOD) 5 7.38-7.22 (m, 2H), 6.91 -6.85 (m, 2H), 3.73 (s, 3H), 3.56-3.52 (m, 1 H), 3.35-3.23 (m, 1 H), 3.03-2.90 (m, 3H), 2.34-2.19 (m, 1 H), 2.08-1 .95 (m, 1 H), 1 .92-1 .79 ppm (m, 2H). Synthesis of comparative compound 5

[0413] Following general procedure D with 5.i (0.35 eq.) as the carboxylic acid

[0414] 1H NMR (400 MHz, D2O) 6 7.42-7.17 (m, 2H), 7.07-6.73 (m, 2H), 4.44-4.15 (m, 1 H), 4.06 - 2.66 (m, 27H), 2.13-1.19 ppm (m, 4H).

[0415] Synthesis of comparative compound 5.i

[0416] Following general procedure B using ethyl nipecotate and 4-methoxybenzoyl chloride

[0417] 1H NMR (400 MHz, MeOD) 5 7.32-7.24 (m, 4H), 6.93-6.84 (m, 4H), 4.48-4.24 (m, 1 H), 4.14-3.96 (m, 1 H), 3.73 (s, 3H), 3.18-3.01 (m, 2H), 2.51 -2.37 (m, 1 H), 2.05-1 .96 (m, 1 H), 1.75-1.58 (m, 2H), 1.53-1.374 ppm (m, 1 H).

[0418] Synthesis of comparative compound 6

[0419] Following general procedure D with 6.i (0.35 eq.) as the carboxylic acid

[0420] 1 H NMR (400 MHz, D2O) 5 7.62-6.61 (m, 4H), 4.63-2.50 ppm (m, 34.19H).

[0421] Synthesis of comparative compound 6.i

[0422] Following general procedure B using methyl azetidine-3-carboxylate hydrochloride and 4- methoxybenzoyl chloride1H NMR (400 MHz, DMSO-d6) 5 7.63-7.55 (m, 2H), 7.02-6.94 (m, 2H), 4.35-4.22 (m, 2H), 4.09- 3.95 (m, 2H), 3.80 (s, 3H), 2.98-2.90 ppm (tt, J = 8.9, 6.1 Hz, 1 H).

[0423] Synthesis of comparative compound 7

[0424] Following general procedure D with 7.i (0.35 eq.) as the carboxylic acid

[0425] 1H NMR (400 MHz, D2O) 6 7.92-6.35 (m, 4H), 4.33-0.88 ppm (m, 31.28H).

[0426] Synthesis of comparative compound 7.i

[0427] Following general procedure B using methyl pyrrolidine-3-carboxylate hydrochloride and 4- methoxybenzoyl chloride

[0428] 1H NMR (400 MHz, DMSO-d6) 5 7.53-7.44 (m, 2H), 6.99-6.92 (m, 2H), 3.79 (s, 3H), 3.69-3.32 (m, 4H), 2.71 -2.55 (m, 1 H), 2.09-1 .83 ppm (m, 2H).

[0429] Synthesis of comparative compound 8

[0430] Following general procedure D with 8.i (0.35 eq.) as the carboxylic acid

[0431] 1H NMR (400 MHz, D2O) 6 7.90-6.41 (m, 1 H), 3.98-1 .21 ppm (m, 37.32H). Synthesis of comparative compound 8.i

[0432] Following general procedure B using methyl azepane-4-carboxylate hydrochloride and 4- methoxybenzoyl chloride

[0433] 1H NMR (400 MHz, DMSO-d6) 5 7.37-7.29 (m, 2H), 7.01 -6.93 (m, 2H), 3.79 (s, 3H), 3.76-3.20 (m, 4H), 2.48-2.38 (m, 1 H), 2.08-1 .46 ppm (m, 6H).

[0434] Synthesis of comparative compound 9

[0435] Following general procedure D with 6.i (0.55eq.) as the carboxylic acid

[0436] 1H NMR (400 MHz, D2O) 5 8.10-5.87 (m, 4H), 4.8-3.2 ppm (m, 18.16H).

[0437] Synthesis of comparative compound 10

[0438] Following general procedure D with 7.i (0.51 eq.) as the carboxylic acid

[0439] 1H NMR (400 MHz, D2O) 5 7.51 -6.57 (m, 4H), 4.12-1.13 ppm (m, 23.04H).

[0440] Following general procedure D with 8.i (0.53 eq.) as the carboxylic acid1H NMR (400 MHz, D2O) 6 7.37-6.62 (m, 4H), 4.18-0.46 ppm (m, 27.2H).

[0441] Synthesis of comparative compound 17

[0442] Following general procedure D with 17.i (0.4 eq.) as the carboxylic acid

[0443] 1H NMR (400 MHz, D2O) 6 7.48-6.70 (m, 4H), 4.57-0.53 ppm (m, 20.8H).

[0444] Synthesis of comparative compound 17.i

[0445] Following general procedure A using ethyl isonipecotate and 4-(trifluoromethoxy)benzoyl chloride

[0446] 1H NMR (400 MHz, DMSO-d6) 5 12.32 (s, 1 H), 7.61 -7.49 (m, 2H), 7.49-7.37 (m, 2H), 4.39-4.23 (m, 1 H), 3.58-3.44 (m, 1 H), 3.21 -2.86 (m, 2H), 2.63-2.45 (m, 1 H), 2.01 -1.70 (m, 2H), 1.63-1.41 ppm (m, 2H).

[0447] Synthesis of comparative compound 20

[0448] Following general procedure D with the commercially available 1 -acetylpiperidine-4-carboxylic acid (0.3 eq.) as the carboxylic acid

[0449] 1H NMR (400 MHz, D2O) 54.39-4.30 (m, 1 H), 4.07-3.09 (m, 23.4H), 3.00-2.86 (m, 1 H), 2.78-2.67 (m, 1 H), 2.08-2.00 (m, 3H), 1.91 -1.34 ppm (m, 4H). Synthesis of comparative compound 26

[0450] Following general procedure D with 26. i (0.4 eq.) as the carboxylic acid

[0451] 1H NMR (400 MHz, D2O) 6 7.74-6.95 (m, 4H), 4.54-0.89 ppm (m, 21 H).

[0452] Synthesis of comparative compound 26.i

[0453] Following general procedure A using ethyl isonipecotate and 4-(trifluoromethyl)benzoyl chloride

[0454] 1H NMR (400 MHz, DMSO-d6) 5 12.33 (s, 1 H), 7.81 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8 Hz, 2H), 4.39-4.28 (m, 1 H), 3.51 -3.40 (m, 1 H), 3.17-2.92 (m, 2H), 2.64-2.51 (m, 1 H), 2.04-1.69 (m, 2H), 1.62-1.44 ppm (m, 2H).

[0455] Example 4. Lentivirus production

[0456] Lentivirus expressing the GFP reporter gene was produced in HEK-293T cells grown in suspension in supplemented Freestyle F17 media. Cells were seeded and cultured before being co-transfected by 4 plasmids (as described in the Material and Methods).

[0457] Figure 2 shows the efficiency of different transfection reagents on lentivirus production. Each reagent was used according to the recommendations of the supplier. According to the results, a significant range of lentivirus titer, determined 72 hours post-transfection, is from 5.0E+07 Tll / mL to at least 2.0E+8 TU / mL, obtained with the product jetPRIME® commercialized by Polyplus Transfection.

[0458] One of the objectives of the invention was to develop a novel family of transfection reagents which exhibits specific properties to produce viral vectors in HEK293 cells.

[0459] The inventors previously demonstrated how the introduction of a hydrophobic moiety in cationic polymers (mainly PEI) can specifically improve the in vitro nucleic acid delivery into cells. In particular, unsaturated nitrogen-containing heterocycles such as triazoles (WO2021 / 023798) or benzimidazoles (WO2021 / 023796) have shown specific affinities with plasmids, inducing high productions of viral vectors (LV, AAV, adenoviruses, ...). The heterocycle alone is not capable to promote the DNA delivery. The linker between the cationic polymer and the heterocycle, and the groups attached directly to this heterocycle are critical to induce a high transfection efficiency. In the examples shown in Figure 2, compounds 1 .42 as described in the patent application WO2021 / 023796 and 2.22 as described in the patent application WO2021 / 023798, the hydrophobic moieties are linked to the polymer by butyramide or propionamide respectively. Both derivatives have shown excellent efficiency.

[0460] 1 . 6-membered ring linker

[0461] Herein the inventors demonstrated that the presence of sterically hindered linker could help to improve the production of therapeutic viral vector.

[0462] The inventors compared 3 different compounds: (i) compound 1 of the invention (2.0E+08 TU / mL, ratio [DNA / TR]: 1 / 1.5; 3.8E+08 TU / mL, ratio [DNA / TR]: 1 / 1 ) with a piperidine-4-carboxamide linker, (ii) comparative compound 3 (7.8E+07 TU / mL, ratio [DNA / TR]: 1 / 1.5) with a 4- aminobutanamide linker, and (iii) comparative compound 4 (4.6E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ) with a 4-(methylamino)butanamide linker. The introduction of a piperidine ring permitted to increase the LV production.

[0463] Furthermore, the size of the ring was critical to maintain a high LV productivity. Piperidine (see compound 1 of the invention: 2.0E+08 Tll / mL, ratio [DNA / TR]: 1 / 1.5) ring was preferential in comparison with azetidine (see comparative compound 6: 7.4E+07 Tll / mL, ratio [DNA / TR]: 1 / 1 .5), pyrrolidine (see comparative compound 7:: 8.3E+07 TU / mL, ratio [DNA / TR]: 1 / 1.5) or azepane (see comparative compound 8: 7.1 E+07 TU / mL, ratio [DNA / TR]: 1 / 1 .5). The inventors next investigated the Nitrogen-position in the piperidine ring. Piperidine-4- carboxamide 1 of the invention (1.5E+08 Tll / mL, ratio [DNA / TR]: 1 / 1 ) was more efficient than piperidine-3-carboxamide 5 (comparative compound 5, 3.3E+07, ratio [DNA / TR]: 1 / 1 ), showing the geometry of the hydrophobic moiety was crucial to improve the transfection efficiency.

[0464] In conclusion, the 4-piperidinyl moiety on a cationic polymer was directly responsive of an increase in a viral vector production.

[0465] 2. Aromatic ring

[0466] If the choice of a 4-piperidinyl moiety was essential, an aromatic ring attached to this linker was required to obtain a high transfection efficiency.

[0467] For example, an order of magnitude was observed in LV production between 4-methoxybenzoyl derivative (see compound 55 of the invention, 2.5E+08 Tll / mL, ratio [DNA / TR]: 1 / 1 ) and acetyl derivative 20 (see comparative compound 20, 3.9E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ).

[0468] Moreover, the nature of the aromatic ring had been explored by the inventors with various modifications. Indeed, the inventors investigated the introduction of a nitrogen-containing heteroaromatic rings, fused ring, alkaryl rings. The inventors also studied the influence of electrondonating, electron-withdrawing groups.

[0469] The heteroaromatic rings could be a 6-membered (such as pyridine, pyrazine, pyrimidine or pyridazine) or a fused (such quinoline, quinaxoline, imidazopyridine or benzimidazole) unsaturated ring. The nitrogen position in the ring or the number of nitrogens were both compatible with a high LV production. 2-pyridine (see compound 41 of the invention, 4.6E+07 Tll / rnL, ratio [DNA / TR]: 1 / 2) and 3-pyridine (see compound 42 of the invention, 4.6E+07 Tll / rnL, ratio [DNA / TR]: 1 / 2) exhibited the same efficiency. The LV production was maintained with 2-nitrogen heterocyclic rings since 2- pyrazine (see compound 43 of the invention, 2.3E+07 Tll / rnL, ratio [DNA / TR]: 1 / 2), 2-pyrimidine (see compound 45 of the invention, 2.2E+07 Tll / rnL, ratio [DNA / TR]: 1 / 2), 2-pyrazine (see compound 46 of the invention, 5.9E+07 Tll / rnL, ratio [DNA / TR]: 1 / 2) showed high efficacy in LV production.

[0470] In the same manner, the addition of fused heterocycles to the cationic polymer such as quinoline see compound 48 of the invention, 8.0E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ), quinaxoline (see compound 47 of the invention, 9.2E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ), imidazopyridine (see compound 50 of the invention, 4.3E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ) or benzimidazole (see compound 51 of the invention, 1.9E+08 TU / mL, ratio [DNA / TR]: 1 / 1 ) gave high LV production. Finally, as shown with the examples cited above, the grafting of heteroaromatic rings, 6-membered or fused, to a cationic polymer permitted to improve the LV production compared to a polymer without any substitution to its skeleton.

[0471] The aromatic ring could also be an alkaryl and the distance between the piperidine and this ring didn’t modify the transfection efficiency of the polymers. Indeed, polyethyleneimine (PEI22k) with a phenol (see compound 12 of the invention, 1.8E+08 TU / mL, ratio [DNA / TR]: 1 / 1 ), a 4- hydroxybenzyl (see compound 13 of the invention, 8.6E+07 TU / mL, ratio [DNA / TR]: 1 / 1 ), a 4- hydroxyphenethyl (see compound 14 of the invention, 1.4E+08 TU / mL, ratio [DNA / TR]: 1 / 1 ) or 4- hydroxyohenylpropyl group (see compound 15 of the invention, 1 .3E+08 TU / mL, ratio [DNA / TR]: 1 / 1 ).

[0472] The inventors next investigated the influence of adding electron-donating or electron-withdrawing groups (EDG or EWG) to the aromatic ring.

[0473] 3-methoxy- (compound 16), 4-methoxy- (compound 1), 4-hydroxy- (compound 12), 3,4- dimethoxy- (compound 18), 3,4-methylenedioxy- (compound 19), or 4-trifluoromethoxy- (compound 17) were synthetized to demonstrate the impact of EDG. 4-bromo- (compound 34), 4-chloro- (compound 33), 4-fluoro-(compound 24), 4-trifluoromethyl- (compound 26), 3-fluoro- (compound 25), 3,4-dichloro- (compound 35), 3,4-difluoro- (compound 29), 2,4-difluoro(compound 30), 3,5-difluoro-(compound 28), 2,5-difluoro (compound 31), 2,3- difluoro- (compound 32), 2,6-dif luoro (compound 27), 3,5-dimethyl- (compound 36), 3,4,5-trifluoro- (compound 37), 2,3,4,5-tetrafluoro (compound 38) or 2,3,4,5,6-pentafluoro- (compound 39) were evaluated to determinate the impact of EWG.

[0474] Both functional groups were tolerated and gave a LV production between 5.0E+07 and 3.0E+08 TU / mL.

[0475] In conclusion, various investigations were explored by the inventors around the introduction of a hydrophobic moiety directly grafted to cationic polymers. Even if the molecule was more sterically hindered compared to previous grafting compounds described in the patent applications WO2021 / 023796 and WO2021 / 023798, a piperidine group afforded high efficiency in viral vector production.

[0476] Figures 4.a to 4.c show the production of LV with different compounds described in the invention.

[0477] 3. Cell line

[0478] Figure 6 shows the production of LV from suspension HEK293F cells. All the selected compounds showed an improvement of the LV titer compared to the reference PEIpro. These results showed the great potential of the compounds in a different cell line than the HEK-293 T cells.

[0479] 4. Adherent cells

[0480] Figure 5., 5.b and 5.c show the production of LV from adherent HEK-293T cells. The results were expressed in TU / mL. Most of the compounds (18, 30, 31 , 32 and 1) showed promising LV production yields compared to the reference PEIpro® and improvements when compared to the references TransITO-Lenti and jetPRIME® (Figure 5a) Repeated experiments (Figure 5b and 5c) show improvements of LV titers when compared to the reference PEIpro® with a great potential when the quantity of DNA was decreased to 0.75 pg DNA / million cells. At the ratio DNA:TR of 1 :1 , all the selected compounds outperform against the reference PEIpro® at the same ratio.

[0481] 5. Complexation buffer or media

[0482] Figure 7 shows the versatility of the compounds in different complexation buffers to prepare the transfection solution between DNA and transfection reagent. Lentiviruses were produced with different complexation buffer or media and compound 59. The complexation buffers or media used are commercially available and include PBS, DMEM High glucose (5 g / L), 150 mM NaCL, Opti- MEM™ ThermoFisher), and Freestyle™ F17 (ThermoFisher). The results show that all conditions tested were able to generate lentiviral particles. Depending on the choice of the buffer or medium to form the transfection complexes, it is possible to manage the volume and / or the time of complexation and to find optimal conditions providing efficient virus production. The transfection protocol is flexible and can be adapted to different working conditions such as viral production at small scale or large scale.

[0483] 6. Complexation

[0484] Figure 8 shows the reproducibility of the results obtained with the compound 1 using different complexation volumes, from 1 to 10% of the total cell culture volume) at the ratio [1 :1]. On the contrary, for the reference jetPRIME® commercialized by Polyplus Transfection, the titers varied according to the complexation volumes. In addition, the results obtained with compound 1 at low volume of complexation is an advantage to work at large scale providing a better control for the transfer of the transfection solution in a well-defined time window into large scale bioreactors.

[0485] 7. DNA amount

[0486] Figure 9 shows the production of lentivirus using compound 55 of the invention at different ratio DNA:transfection reagent (from 1 .5 to 2 pL reagent / pg DNA), and different DNA quantity (from 0.5 to 1 pg / million cells) or with PEIpro using recommended transfection protocol (ratio 1 :1 , and 1 pg / million cells).

[0487] The use of compound 55 of the invention as transfection reagent allows to use less DNA quantity while optimizing titers to improve process economics.

[0488] Example 5. AAV production

[0489] The production of AAV particles from suspension HEK-293T cells in Freestyle F17 medium with selected compounds of the invention was performed using AAV-2 and AVV-9 expressing the GFP reporter gene. Cells were seeded and cultured before being co-transfected by 3 plasmids (pAAV- RC vector expressing Rep and Cap, pHelper vector, and pAAV-GFP control vector expressing the GFP under the control of a CMV promoter) as described in the Material and Methods. AAV titers (viral genome titer, VG / mL) were determined 72 hours post-transfection.

[0490] Figure 3.a and Figure 3.b show the production of AAV serotype 2 and AAV serotype 9 from suspension HEK-293T cells. Figure 3.a shows the production of AAV-2 from suspension HEK-293T cells. Cells were seeded and cultured for 24 hours before being transfected by 3 plasmids (pAAV-RC2 vector expressing Rep and Cap-2, pHelper vector, and pAAV-GFP) with the reference FectoVIR-AAV® (Polyplus Transfection) or various compounds. Genome titers (VG / mL) were determined 72 hours posttransfection.

[0491] Figure 3b shows the production of AAV-9 from suspension HEK-293T cells. Cells were seeded and cultured for 24 hours before being transfected by 3 plasmids (pAAV-RC2-9 vector expressing Rep and Cap-9, pHelper vector, and pAAV-GFP) with the reference FectoVIR-AAV® (Polyplus Transfection) or various compounds. Genome titers (VG / mL) were determined 72 hours posttransfection.

[0492] Most compounds performed similarly in AAV productivity than the reference FectoVIR-AAV® commercialized by Polyplus Transfection.

[0493] Example 6. Residual test

[0494] The method consists of inducing an acidic hydrolysis of biological products (viruses) without affecting the chemical stability of the transfection reagent. After purification and removal of the degraded biological material by ultrafiltration, the residual transfection reagent is collected and analyzed using HPLC or LIPLC conditions.

[0495] The following protocol is given for a final concentration of 0.1 % HCI during the hydrolysis step of the sample. To a solution of viral vector (LV or AAV, 200 pL) was added a solution of HCI (0.37%, 135 pL), a volume of pure water (65 pL) and a solution of transfection reagent (concentration: X ppm, 100 pL). The tube was sealed, then heated at 60°C for 2 hours. After cooling to room temperature, the mixture was diluted in 3.5 mL pure water then filtered by centrifugation using an Amicon Ultra 4 for 30 minutes at 5.000g to afford a solution between 250-300 pL. The solution was completed to 4 mL and filtered at 5.000g for 60 minutes. The same procedure was repeated 2 more times. The final retentate was diluted with pure water to give a solution of 500 pL and analyzed by UHPLC.

[0496] The HCI concentration (0.1 % and 1 %) and heating temperature (60°C, 80°C, and 1 10°C) as well as the heating time (from 2 hours to 24 hours) were evaluated as these parameters are critical for acidic hydrolysis. Finally, the optimized conditions were fixed to an acidic hydrolysis in a HCI solution 0.1 % at 60°C for 2 hours as no noticeable degradation of the transfection compounds (many examples are presented in Table 3 below) was observed. Figure 10 shows the analysis of compound 55 of the invention without HCI 0.1 % treatment, with HCI 0.1% treatment for 2 hours at 60°C and a control sample with HCI 0.1 % treatment for 2 hours at 60°C following with a spike of compound 55 of the invention before analysis. The recovery of compound 55 of the invention without HCI 0.1 % treatment, and with HCI 0.1% treatment for 2 hours at 60°C was >90% when compared to the control sample, showing the stability of this compound in acidic hydrolysis conditions.

[0497] The linearity of the method is shown on the Figure 11 with a LCD = 0.4 and LOQ = 1 .4 determined for the compound 55 of the invention used in this example after HCI 0.1 % treatment for 2 hours at 60°C.

[0498] Stability of compounds 16, 18, 19, 30, 44, 55, 50, 51, and 52 according to the invention and comparative compound 5 under acidic hydrolysis conditions

[0499] The stability of compounds 16, 18, 19, 30, 44, 55, 50, 51, and 52 of the invention and comparative compound 5 was studied under the acidic hydrolysis as described herein. The inventors carried out the following experiment: a transfection reagent (200 ppm) in a solution of HCI (1 mL, 0.1 %) was heated at 60°C for 2 hours. After cooling, 100 pL of the solution was diluted in 900 pL of water and analyzed by UHPLC. Results are reported in Table 3 below. Most of the molecules were fully stable under the acidic conditions.

[0500] Table 3. Examples of polymers

[0501] Example 7. DNA or mRNA transfection

[0502] The inventors tested the compounds 1 , 13, 15, 16, 23, 27, 33, 47, 50, 51 , 52, 53, 55, 58 and 59 according to the invention as previously described, and the comparative compounds 2, 4 and 20 as previously described, for DNA and / or mRNA transfection. Materials and Methods

[0503] - HEK293T cells

[0504] Human embryonic kidney 293 cells containing the SV40 T-antigen (HEK-293T) were grown on cell flask and cultured in DMEM glucose 4.5 g / L supplemented with Fetal bovine serum (FBS, 10 %), L-Glutamine (1 %), and Penicillin-Streptomycin (2 %).

[0505] GFP pDNA transfection

[0506] For transfection experiments, HEK-293T cells were seeded at 10 000 cells per well of 96-well plates in complete medium one day before transfection. Transfection efficiency was assessed 48 hours post-transfection by flow cytometry.

[0507] • With PEIpro® (Polyplus Transfection) as a commercial reference

[0508] On the day of transfection, the reference PEIpro® (Polyplus Transfection) / GFP DNA complexes were prepared according to the manufacturers’ recommendations. Briefly, transfection with the reference PEIpro® was performed as described: 150 ng of GFP-encoding pDNA (per well of 96- well plate) and 0.3 pL of the reference PEIpro® were first diluted in DMEM high glucose separately, diluted PEIpro® was then added to diluted DNA and mixed by vortex. Following an incubation of 10 minutes at room temperature, 12.5 pL of the reference PEIpro® / DNA complexes were simply added dropwise to cells in their complete growth medium.

[0509] • With compounds 1 , 13, 15, 16, 23, 27, 33, 47, 50, 51, 52, 53, 55 and 58 according to the invention, or comparative compounds 2, 4 and 20

[0510] On the day of transfection, 150 ng of GFP-encoding pDNA (per well of 96-well plate) were first diluted in DMEM high glucose, followed by the mixing-in of 0.3 pL of the compound 1, 13, 15, 16, 23, 27, 33, 47, 50, 51, 52, 53, 55 or 58 of the invention, or the comparative compound 2, 4 or 20 (ratio 1 :2 pg DNA / pL of the compound). Following an incubation of 10 minutes at room temperature, 12.5 pL of the compound / DNA complexes were simply added dropwise to cells in their complete growth medium.

[0511] GFP mRNA transfection

[0512] For transfection experiments, HEK-293T cells were seeded at 10 000 cells per well of 96-well plates in complete medium 1 day before transfection. Transfection efficiency was assessed 24 hours post-transfection by flow cytometry.

[0513] • With jetPRIME® (Polyplus Transfection) as a commercial reference

[0514] On the day of transfection, the reference jetPRIME® (Polyplus Transfection) / GFP mRNA complexes were prepared according to the manufacturers’ recommendations. Briefly, transfection with the reference jetPRIME® was performed as described: 100 ng of GFP-encoding mRNA (per well of 96-well plate) were first diluted in jetPRIME® buffer, followed by the addition of the reference jetPRIME® (N / P 5) and mix by pipetting up and down. Following an incubation of 10 minutes at room temperature, 12.5 pL of the reference jetPRIME® / mRNA complexes were simply added dropwise to cells in their complete growth medium.

[0515] • With compounds 13, 16, 23, 33, 51 , 52, 55 and 59 according to the invention, or comparative compounds 2, 4 and 20

[0516] On the day of transfection, 100 ng of GFP-encoding mRNA (per well of 96-well plate) were first diluted in DMEM high glucose, followed by the mixing-in of 0.2 pL of the compound 13, 16, 23, 33, 51 , 52, 55 or 59 of the invention, or the comparative compound 2, 4 or 20 (ratio 1 :2 pg mRNA / pL of the compound). Following an incubation of 10 minutes at room temperature, 12.5 pL of the compound / mRNA complexes were simply added dropwise to cells in their complete growth medium.

[0517] Results

[0518] • DNA transfection

[0519] HEK-293T cells were transfected with GFP pDNA with PEIpro® (Polyplus Transfection) as a commercial reference, or compounds 1 , 13, 15, 16, 23, 27, 33, 50, 51, 53, 55 and 58 according to the invention, or comparative compounds 2, 4 and 20 (Figure 12), or compounds 47 and 52 (Figure 13). Transfection efficiency was assessed 48 hours post-transfection by GFP fluorescence. High transfection efficiency (> 85 % of GFP expression) was reached with the reference PEIpro® and all the compounds tested except with the comparative compound 20 for which no transfection efficiency was observed (< 10 % of GFP expression).

[0520] • mRNA transfection

[0521] HEK-293T cells were transfected with GFP mRNA with jetPRIME® (Polyplus Transfection) as a commercial reference, or compounds 13, 16, 23, 33, 51, 52, 55 and 59 according to the invention or comparative compounds 2, 4 and 20 (Figure 14). Transfection efficiency was assessed 24 hours post-transfection by GFP fluorescence. Good transfection efficiency (> 30 % of GFP expression) was observed with jetPRIME® and all the compounds except with comparative compounds 2, 4 and 20 for which low transfection or no transfection efficiency was observed (< 20 % of GFP expression). References

[0522] Kulkarni, J. A., Witzigmann, D., Thomson, S.B. et al. The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 16, 630-643 (2021 ). https: / / doi.org / 10.1038 / s41565-021 -00898- 0

[0523] Terrence Dobrowsky, Davide Gianni, John Pieracci, Junghae Suh, AAV manufacturing for clinical use: Insights on current challenges from the upstream process perspective, Current Opinion in Biomedical Engineering, Volume 20, 2021, 100353, ISSN 2468-4511, https: / / doi.Org / 10.1016 / j.cobme.2021.100353.

[0524] Bulcha, J.T., Wang, Y., Ma, H. et al. Viral vector platforms within the gene therapy landscape. Sig Transduct Target TherG, 53 (2021 ). https: / / doi.org / 10.1038 / s41392-021 -00487-6

[0525] Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr JP. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A. 1995 Aug 1 ;92(16):7297-301 . doi: 10.1073 / pnas.92.16.7297. PMID: 7638184; PMCID: PMC41326.

[0526] Akinc, A., Thomas, M., Klibanov, A.M. and Langer, R. (2005), Exploring polyethylenimine- mediated DNA transfection and the proton sponge hypothesis. J. Gene Med., 7: 657-663.

[0527] Sonawane ND, Szoka FC Jr, Verkman AS. Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes. J Biol Chem. 2003 Nov 7;278(45):44826- 31. doi: 10.1074 / jbc.M308643200. Epub 2003 Aug 27. PMID: 12944394.

[0528] Perry C, Rayat ACME. Lentiviral Vector Bioprocessing. Viruses. 2021 Feb 9;13(2):268. doi: 10.3390 / v13020268. PMID: 33572347; PMCID: PMC7916122.

Claims

CLAIMS1 . A transfection composition suitable for the production of a biological product, wherein the composition comprises (i) at least one compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, and (ii) and acceptable excipient, buffering agent, cell culture medium, or transfection medium:wherein: - R is selected from the group consisting of the compounds having the following formulasR1-R54:, wherein Wi, W2, W3, W4and W5may be identical or different, and represent H, a linear or branched, saturated or insaturated (C1-C10) alkyl, or a linear or branched, saturated or insaturated 0-(Ci-Cio) alkyl, and m represents an integrer between 1 and 10, preferably between 1 and 3;- Z represents: -C=O-; or -(CH2)n-C(O)-, with n representing an integer between 1 and 3;- P+represents a graft cationic polymer selected from the group consisting of a linear or branched polyethyleneimine (PEI) and PEI dendrimers.

2. The composition according to claim 1 , which further comprises at least one nucleic acid molecule to be transfected in a cell, for the production of the biological product, preferably at least one nucleic acid molecule selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA / RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, a selfamplifying RNA (saRNA), a synthetic DNA, more preferably a DNA or a mRNA, and an expression vector encoding said nucleic acid molecule, in particular a plasmid encoding said nucleic acid molecule or a plasmid expressing said nucleic acid molecule.

3. The composition according to claim 2, which comprises multiple nucleic acid molecules, preferably multiple DNA molecules.

4. The composition according to any one of claims 1 to 3, wherein the biological product is selected from the group consisting of:(i) a biological product encoded by the nucleic acid from the composition, which is selected from a protein, a peptide, an antibody and a fragment thereof,(ii) a virus, in particular a recombinant virus, preferably a recombinant virus selected from the group consisting of an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus (Ad), an oncolytic virus, and a baculovirus, more preferably AAV or LV, even more preferably LV, and(iii) a biological product which comprises virus-like particles.

5. The composition according to any one of claims 1 to 4, wherein the graft cationic polymer is a linear or branched PEI, preferably a linear PEI.

6. The composition according to any one of claims 1 to 5, wherein:(i) the graft cationic polymer P+has a grafting ratio ranging from 5% to 50%, preferably from 20% to 35%, more preferably is 25%; and / or(ii) the graft cationic polymer P+has an average molecular weight (Mw) ranging from 1 kDa to 50 kDa, preferably from 5 kDa to 30 kDa or from 10 kDa to 25 kDa, in particular the graft cationic polymer P+has an Mw of 8, 10, 15, 22, 25 or 30 kDa, preferably of 10 or 22 kDa, more preferably 22 kDa.

7. The composition according to any one of claims 1 to 6, wherein:- R represents R7; and / or- Z represents -C=O; and / or- P+represents a linear or branched PEI, preferably a linear PEI; and / or P+has a grafting ratio of 25%; and / or P+has an Mw of 10 or 22 kDa, preferably 22 kDa.

8. The composition according to any one of claims 1 to 7, wherein the at least one compound of general formula (I) is selected from the group consisting of the following compounds:

9. The composition according to claim 8, wherein the at least one compound of general formula (I) is selected from the group consisting of the compounds 1 , 14, 18, 30, 44, 52 and 55, more preferably is compound 55.

10. A method comprising the in vitro or ex vivo use of the composition according to any one of claims 2 to 9, wherein the method is for the production of (i) a biological product encoded by the nucleic acid from the composition, which is selected from a protein, a peptide, an antibody and a fragment thereof; or (ii) a biological product, which is a virus, in particular a recombinant virus, such as adeno-associated virus (AAV), lentivirus (LV), adenovirus, oncolytic virus, or baculovirus, wherein said composition comprises multiple nucleic acid molecules for cotransfection; or (iii) a biological product which comprises virus-like particles, wherein said composition comprises multiple nucleic acid molecules for co-transfection.1 1 . The method according to claim 10, for the production of recombinant AAV, said composition comprising (i) at least one compound selected from the group consisting of compounds 1 , 14, 18, 30, 44, 52 and 55, preferably compound 55, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, and (iii) optionally a booster.

12. The method according to claim 10, for the production of recombinant LV, said composition comprising (i) at least one compound selected from the group of compounds 1 , 14,18, 30, 44, 52 and 55, preferably compound 55, and (ii) an acceptable excipient, buffering agent, cell culture medium, or transfection medium, and (iii) optionally a booster.

13. The method according to any one of claims 10 to 12, for the production of recombinant virus, said composition comprising a plurality of expression vectors such as plasmid vectors to transfect in eukaryotic cells, in particular in mammalian cells or in insect cells, preferably mammalian cells, more preferably adherent mammalian cells or suspension mammalian cells, even more preferably suspension and adherent HEK293T cells, wherein said vectors, in particular plasmids, are construct expressing viral structural sequences and transfer vector genome for virus or virus-like production and optionally expressing molecules of interest encoded by the transfer vector genome.

14. A method for in vitro or ex vivo transfection of live cells comprising introducing in the cells the transfection composition according to any one of claims 1 to 9.

15. In vitro or ex vivo use of the at least one compound of general formula (I) as defined in any one of claims 1 and 5-9 to transfect at least one nucleic acid molecule in a cell, in particular a eukaryotic cell, wherein the at least one nucleic acid molecule is preferably selected from the group consisting of a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), a DNA / RNA hybrid, a short interfering RNA (siRNA), a microRNA (miRNA), a short hairpin RNA (shRNA), a messenger RNA (mRNA), a CRISPR guide RNA, a self-amplifying RNA (saRNA), a synthetic DNA, more preferably a DNA or a mRNA, and an expression vector encoding said nucleic acid molecule, in particular a plasmid encoding said nucleic acid molecule or a plasmid expressing said nucleic acid molecule.

16. The in vitro or ex vivo use according to claim 15, wherein the at least one compound of general formula (I) is selected from the group consisting of compounds 1 , 13, 15, 16, 23, 27, 33, 47, 50, 51, 52, 53, 55 and 58, preferably compound 55, and the at least one nucleic acid molecule is DNA.

17. The in vitro or ex vivo use according to claim 15, wherein the at least one compound of general formula (I) is selected from the group consisting of compounds 13, 16, 23, 33, 51 , 52, 55 and 59, preferably from the group consisting of compounds 16, 33, 51 , 55 and 59, more preferably compound 55, and the at least one nucleic acid molecule is mRNA.

18. A method for purifying, detecting and / or quantifying the at least one compound of general formula (I) as defined in any one of claims 1 and 5-9, wherein the at least one compound of general formula (I) is comprised in a liquid mixture comprising a biological matrix, wherein the biological matrix comprises a recombinant virus or virus-like particles produced using the at least one compound of general formula (I), wherein the method comprises the step of:(a) performing an acidic hydrolysis of the liquid mixture by incubating the liquid mixture comprising the biological matrix in an aqueous solution comprising from 0.1% to 10% (v / v) hydrochloric acid (HCI) at a temperature ranging from 60°C to 1 10°C for a time period ranging from 2 hours to 24 hours, preferably an aqueous solution comprising 0.1 % (v / v) HCI at a temperature of 1 10°C for 2 hours or in an aqueous solution comprising 1 % (v / v) HCI at a temperature ranging from 60°C to 80°C for 2 hours, wherein said acidic hydrolysis does not degrade the at least one compound of general formula (I),(b) purifying the reaction mixture obtained in step (a) in order to obtain a purified compound of general formula (I),(c) detecting and / or quantifying the purified compound of general formula (I) obtained in step (b).

19. The method according to claim 18, wherein the recombinant virus is selected from the group consisting of an adeno-associated virus (AAV), a lentivirus (LV), an adenovirus, an oncolytic virus and a baculovirus, preferably is an AAV or a LV, more preferably is a LV.

Images