Cationic poloxamers and their uses in transduction

Abstract

To provide enhancement of transduction of target cells with viral vectors.SOLUTION: The invention relates to a method for enhancing the transduction of a target cell with a viral vector using a cationic block-copolymer introduced as an additive alone or formulated with nanoparticles. The method comprises a step of bringing a target cell into contact with a virus and a cationic block co-polymer. The structure of this additive comprises both hydrophilic and hydrophobic regions which represent different areas in the backbone of the polymer. This polymeric construction is ended by cationic chemical functions which contribute to further enhancement of the viral transduction. Also disclosed are new cationic poloxamers that can be used in the disclosed method. Furthermore, another embodiment is the colloidal stabilization of iron-based nanoparticles using these polymers and their use in increasing transduction efficiency.SELECTED DRAWING: None

Description

[Technical field]

[0001] The methods described in this document relate to the enhancement of transduction of target cells by viral vectors using cationic block copolymers, introduced as an additive alone or formulated with nanoparticles (also referred to herein as chemical adjuvants). The methods include contacting target cells with a virus and a cationic block copolymer. The structure of the additive incorporates distinct regions in the polymer backbone, both hydrophilic and hydrophobic regions. The polymer architecture is terminated by cationic chemical functions that contribute to further enhancement of viral transduction. The invention also relates to novel cationic poloxamers that can be used in the methods of the invention. Furthermore, another embodiment of the invention relates to the use of these polymers in colloidal stabilization of iron-based nanoparticles and their increased transduction efficiency. [Background technology]

[0002] In gene and cellular applications, the use of viruses is the method of choice for the genetic modification of most cells, especially primary cells. This class of vectors, especially retroviruses, allows stable gene expression in infected cells and is quickly becoming an important cornerstone for both research and therapeutic applications.

[0003] In recent years, lentivirus (LV) vectors have been successfully used to infect human or other mammalian cells, whether they are growing or not. In most cases, the success of these procedures requires the use of pseudotyped LVs with glycoproteins. Thus, such vectors can stably integrate into the chromosomes of target cells. In the same way, adenoviruses and adeno-associated viruses have been widely used for genetic modification or engineering of various cells and tissues as well.

[0004] Currently, there is an increasing interest in finding highly efficient transduction conditions involving lentiviruses or adenoviruses or adeno-associated viruses, or more conventional viruses, to deliver genes to therapeutic cells, including, but not limited to, lymphocytes, immune cells, lymphoid cell lines, stem cells and primary hematopoietic stem cells. Recent clinical advances are based on introducing genetic material into primary lymphocytes, hematopoietic tumor cells or pluripotent stem cells, which are known to be difficult to achieve. Studies on such cells often imply the use of highly concentrated and highly pure grade virus preparations to be sufficiently efficient. As a result, large-scale transduction of parental cells requires upscaling of virus production and elaborate processing to obtain high virus titers and highly concentrated virus stocks. Such optimization therefore implies significant financial costs.

[0005] In order to reduce the required viral production and, consequently, the cost of clinical trials, it is of paramount importance to improve viral transduction efficiency. Furthermore, studies with lower multiplicities of infection (MOI) will reduce the mutagenic risk for insertion of multiple viral copies per parental cell.

[0006] In this context, the expression "such as" should be understood as non-limiting and as "such as an example," implying that the elements described thereafter are merely examples cited among others not mentioned that may fit fully within the scope of the present invention.

[0007] Several strategies have been used to improve the efficiency of virus-related transduction procedures.

[0008] The first improvement point depends on the vector itself. The purity and quality of the virus preparation, the multiplicity of infection (MOI) (ratio of the number of infectious agents - e.g. infectious virus particles - to the number of hosts - e.g. cells) and the virus assembly are important parameters to control to achieve optimal efficiency of transduction. In fact, some studies have reported that the presence of endogenous inhibitors in the virus may explain the reduced efficiency in the transduction procedure. The use of highly concentrated and purified virus preparations, which may help to circumvent these concerns, has been obtained so far by ultracentrifugation or ultrafiltration strategies. An efficient transduction procedure also requires optimal cell density before virus application, limited cell passage number and careful control of cell culture medium composition.

[0009] In addition to issues related to the purity and quality of viral vectors, there are several fundamental biophysical constraints that limit viral gene transfer efficiency. In fact, several groups have demonstrated that viral diffusion over time and rapid inactivation are the main causes of the relatively low observed efficiency. With viral particles capable of diffusing for only a few microns before losing biological activity, the majority of initially active viral particles are inactivated before they can interact with target cells, fundamentally limiting the achievable level of transduction efficiency.

[0010] Mechanical or physical approaches have made it possible to increase the number of viral particles that reach the target cell and are still infectious. Among others, the centrifugation (or spinoculation) technique (AB Bahnson et al.; J. Virol. Methods 1995, 54: 131-143), flow-through transduction (S. Chuck et al.; Hum. Gene Ther. 1996, 7: 743-750) or the use of magnetic nanoparticles (MNPs) (Sloutskin A. et al.; J Virol Methods. 2014, 206: 128-32) can be cited as techniques that can increase the concentration of active viral particles on the cell surface. These techniques have in common the addition of a convective element to the viral mass transport, thereby increasing the probability and frequency of active virus-cell interaction. Magnetofection™ technology, which principally uses cationic magnetic nanoparticles, can promote, enhance and simultaneously induce both viral infection and viral adsorption in a wide variety of in vitro, ex vivo and in vivo biological models (C. Plank, et al.; Adv Drug Del Rev 2011, 63:1300-1331). Although proven to be effective, their cost and difficult scale-up somehow limit their potential as components in large-scale in vivo clinical settings.

[0011] Another strategy relied on the use of chemical adjuvants. A wide range of chemical adjuvants have been used over the past years to promote transduction, such as recombinant polypeptides, polycationic compounds, or neutral compounds such as poloxamers. Among all the molecules tested, polycations such as cationic liposomes or cationic polymers such as polyethylenimine (PEI), DEAE-dextran plotamine sulfate, or poly-L-lysine have been extensively studied.

[0012] However, the most common cationic additive that can be found in the described transduction procedures is called polybrene. It is an aprotic cationic linear polymer that has been demonstrated to significantly improve gene transduction rates in a wide range of target cells. These interesting features have ensured polybrene's leading position in the field of adjuvant-assisted viral gene delivery methods. Unfortunately, polybrene, together with many polycationic molecules used for this purpose, shares negative features that have limited its impact in clinical trials. The first and most important polycations with high positive charge density have been found to be somehow toxic to the target cells, which does not allow their use on sensitive cell types such as primary parental cells. As a result, polybrene can only be used for short-term applications and has limited toxicity to the target cells at low concentrations (below 10 μg / mL). This aspect is clearly a major drawback for the use of polybrene in clinical applications, especially when the target is a highly sensitive cell such as primary hematopoietic stem cells.

[0013] In addition to polycations, another class of compounds used as transduction enhancers are the linear or X-shaped non-ionic poloxamers (AV Kabanov et al.; J. Control. Release, 2002, 82:189-212; A. Rey-Rico et al.; Int. J. Mol. Sci. 2018, 19:775). Poloxamers represent a well-known class of non-ionic triblock copolymers consisting of a central hydrophobic chain of polyoxypropylene (PPO) surrounded by two hydrophilic chains [PEO-PPO-PEO] belonging to the polyethylene oxide (PEO) family. These poloxamers can be linear or X-shaped, the latest of which is known as poloxamine. Meroxapol, also known in the literature as "reverse poloxamer", is also included in this family, in which a hydrophilic block based on polyethylene oxide is present, surrounded by two hydrophobic blocks based on polypropylene oxide polymers. By extension, we contemplate these X-shaped variations, "reverse poloxamines" in the present invention. All these polymers (poloxamers, poloxamines, reverse poloxamines) will be referred to as poloxamers in the remainder of this document.

[0014] Poloxamers can be called according to their trade names and may be "Pluronic" or "Symperonic" depending on the manufacturer. These molecules are characterized by different hydrophilic / lipophilic (e.g. hydrophobic) balances (HLB) and critical micelle concentrations (CMC). These structural features have been found to play an important role in their biological properties (A; Kabanov A. et al.; Adv. Gen. 2005, 53: 231-61). In addition to their well-described self-assembly and micellization behavior, poloxamers are widely used in cosmetics, herbal medicines and a wide variety of pharmaceutical preparations. They are also used in non-viral gene delivery procedures for in vitro and / or in vivo applications, as well as electroporation methods due to their sealing properties. They have also been used in combination with cationic polymers to generate polyplexes with DNA.

[0015] Poloxamers have been found to act as polymer excipients during virus-mediated gene delivery procedures (AV. Kabanov et al.; Adv Drug Del Rev 2002,54:223-3). The first example of such use was reported by March et al., who used poloxamer 407 (also known as F127) as a transduction enhancer such as rate and frequency of change in vascular smooth muscle cells using recombinant adenovirus vectors (KL. March, et al.; Hum. Gene Ther,1995,6:41-53). This study highlighted the modulation of the pharmacokinetics of adenovirus-mediated gene delivery by poloxamers. Although the mechanism underlying this beneficial effect was not explicitly understood in the report, the authors hypothesized that poloxamer 407 formed a gel under the experimental conditions of the study, acting as a "delivery reservoir" for the virus that enhanced gene delivery by maintaining a high concentration of vector around the cells.

[0016] After this pioneering work, several other studies have highlighted the beneficial effects of poloxamers during transduction experiments. Among others, we can cite the use of poloxamer 407 in the transduction of rat arteries with adenovirus as vector (LJ.Feldman, et al.; Gene Ther. 1997, 4, 189-198) or the use of poloxamer 407 as a gel for percutaneous adenovirus-mediated gene transfer with stenting of rabbit iliac arteries (Van Belle et al. Hum. Gene Ther. 1998, 9, 1013-1024). The enhancing behavior of poloxamers in transduction was also demonstrated using lentiviruses as vectors in several studies with, among others, endothelial or smooth muscle cells as targets (KL.Dishart, et al. J.Mol.Cell.Cardiol.2003,35:739-748). In this case, AAV serotype and vesicular stomatitis virus glycoprotein-pseudotype lentivirus systems were placed together with similar results, enhanced by the presence of poloxamer 407 as an adjuvant. Another very interesting study involved the use of AAV associated with linear (Pluronic F68) or X-shaped (poloxamine Tetronic T908) poloxamer micelles. In this case, micellar encapsulation with poloxamers allowed both an increase in stability and bioactivity of AAV, resulting in higher levels of transgene expression without adverse effects on cells both in vitro and in experimental osteochondral defects.

[0017] Since then, enthusiasm for using poloxamers as adjuvants in virus-mediated gene delivery procedures has proven consistent. These recent developments involving novel gene therapy clinical trials involving lentiviruses have increased and expanded this interest, generating several patent applications involving poloxamers in the context of virus-mediated gene delivery directed to both in vitro and in vivo applications. Among several others, one can cite the combined use of polybrene and poloxamer 338 in retrovirus-mediated gene transfer strategies (WO 2013 / 127964 (A1), WO 2017 / 139576 (A1)), or the induction of primary T cells containing CARs (chimeric antigenic receptors) using lentiviruses and a wide range of different poloxamer-containing adjuvants (WO 2015 / 162211 (A1), WO 2018 / 148502 (A1)).

[0018] However, the efficiency of poloxamers is highly dependent on the cell type targeted, the poloxamer structure used, and in most cases requires the addition of cationic polymers such as polybrene. Moreover, their non-ionic properties are not favorable for these combinations, nor for formulations containing nanoparticles, such as iron-based magnetic nanoparticles. In fact, magnetic nanoparticles have been shown to improve and promote viral transduction efficiency in various models. As a result, it could be imagined that the addition of magnetic nanoparticles and poloxamers would lead to a strong synergistic effect. Unfortunately, the non-ionic properties of poloxamers lead to nanoparticle destabilization or precipitation, which hinders their use as nanoparticle coatings or formulations.

[0019] Due to these interesting properties, several studies have focused on the chemical modification of poloxamers, with the aim of adding novel physicochemical properties to the existing inherent characteristics of these polymers in order to circumvent certain limitations.

[0020] For example, in non-viral strategies, some groups have used the hydroxyl functional groups of poloxamers as attachment points to covalently bond with cationic polymers of different natures, allowing poloxamers to interact and condense with naked plasmid DNA. For example, the behavior of block copolymers consisting of Pluronic 123 coupled with branched polyethyleneimine with a molecular weight of about 2000 Da as non-viral vectors was described. The same concept was also applied to the development of novel non-viral vectors based on poloxamer 407 covalently linked with polylysine, or acrylate-modified poloxamers condensed with polyethyleneimine for efficient gene delivery. In the majority of these cases, the modification occurred at the hydroxyl end groups (Gyulain G.et al eXPRESS Polymer Letters 2016,10:216-26). The reactions involved in these procedures often rely on polyethylene glycol chemistry. Summary of the Invention

[0021] However, such modifications with cationic groups or polymers attached at the termini of the poloxamers have not been realized or attempted in the context of enhancing viral transduction to avoid the need to use additional cationic polymers such as polybrene to increase their efficiency in some cell types as previously mentioned. Similarly, the potential synergistic effects of modified poloxamers with magnetic nanoparticles have not been sensed and monitored. The present invention aims to solve the technical problems associated with poloxamers used to enhance viral transduction, alone or in combination with magnetic nanoparticles as described herein above. The present invention also aims to create alternative and / or improved means and methods for transducing cells with viruses.

[0022] Furthermore, the present invention also relates to the modification of the poloxamer end groups, which allows further tuning of the properties and behavior of the chemical adjuvant. More specifically, the present invention describes the systematic introduction of cationic organic functional groups at the end of the poloxamer polymeric backbone, so as to create in one entity, instead of two separate entities (non-ionic and ionic), synergistic properties that promote viral transduction and to be formulated with magnetic nanoparticles as transduction enhancers. The added value of the invention described in this document is the synergistic combination, in the same polymeric entity, of the surfactant properties of poloxamers with cationic moieties that are able to interact with both viruses and cells. Although blends of poloxamers and cationic polymers have been described previously, this is the first time, to the applicant's knowledge, that a single polymer exhibiting both amphiphilic properties and cationic functional groups is used in connection with viruses.

[0023] More specifically, the present invention describes the systematic introduction of cationic organic functional groups at the termini of the polymeric backbone of poloxamers.

[0024] The present invention describes for the first time a method of using cationic poloxamers to enhance viral transduction of target cells. [Brief description of the drawings]

[0025] [Figure 1] Disclosed are preferred structures of compounds of formula I using the general linear cationic poloxamer structure, where the values ​​of "a", "b" and "a-1" are as defined herein. [Diagram 2] Detailed procedures are disclosed to obtain compound Ib, an intermediate of polymer I. Condition i): TsCl, DMAP, DCM / pyridine at room temperature; Condition ii): NH3(MeOH), 80°C. [Diagram 3] The synthesis of compound 1e, an intermediate of polymer I, is disclosed; condition i) NaOH, MeOH; condition ii): H2, Raney Nickel EtOH; condition iii): Boc2O, NaOH, THF / H2O. [Figure 4]The synthesis of polymers from 1b and 1e is disclosed. Conditions i): DIC, HOBt, Et3N, DCM / DMF; Conditions ii): TFA, DCM. [Diagram 5] Detailed procedures to obtain compound II are disclosed. Condition i): TsCl, DMAP, DCM / pyridine at room temperature; Condition ii): NH3(MeOH), 80° C. Condition iii): MeI, DIPEA, DMF at room temperature. [Figure 6] Detailed procedures are disclosed to obtain compound III: Condition i): CBr4, PPh3, DCM, room temperature; Condition ii): PPh3, DMF; 120°C. [Figure 7] Detailed procedures for obtaining compound IV are disclosed: Condition i): NaN3, DMF, 90°C; Condition ii): Propargylamine, CuSO4, Ascorbic acid, PPh3, H2O / DMSO room temperature; Condition iii): MeI, DIPEA, DMF room temperature. [Figure 8] Detailed procedures are disclosed to obtain compound V. Condition i): TsCl, DMAP, DCM / pyridine room temperature; Condition ii): NH3(MeOH), 80°C. Condition iii): acetaldehyde, TosMIC, K2CO3 DMF, room temperature. Condition iv): dimethyl sulfate, toluene, room temperature. [Figure 9] Detailed procedures are disclosed to obtain compound VI. Condition i): Boc3R, DIC, HOBt, Et3N, DCM / DMF at room temperature; Condition ii): Trifluoroacetic acid, DCM at room temperature. [Figure 10] Detailed procedures are disclosed to obtain compound VII. Condition i): Boc2Hist, DIC, HOBt, Et3N, DCM / DMF at room temperature; Condition ii): Trifluoroacetic acid, DCM at room temperature. [Figure 11] We disclose the results of the evaluation of cationic poloxamers in transduction procedures with lentiviral vectors: NIH-3T3 cell lines were infected with lentivirus (MOI 1) in the presence or absence of different doses of cationic poloxamer. After 72 h of incubation, the % of GFP-positive cells (A) and the mean intensity of transduced cells (B) were assessed by flow cytometry. [Figure 12]We present a second round of cationic poloxamer evaluation in transduction procedures with lentiviral vectors: NIH-3T3 cell lines were infected with lentivirus (MOI 1) in the presence or absence of various doses of cationic poloxamer. After 72 h of incubation, the % of GFP-positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 13] Figures 13A-F disclose the differential effect of cationic poloxamer-induced infection. HEK293T cell lines were infected with lentivirus encoding GFP protein at an MOI of 1 in the presence or absence of cationic poloxamer. 72 hours after infection, the % and mean intensity of GFP positive cells were analyzed by flow cytometry. [Figure 14] The results of the assay are disclosed in which BV2 and HEK-293 cell lines were infected with lentivirus encoding GFP at the indicated MOI in the presence or absence of F108+polybrene, compounds 2b and II. After 72 h of incubation, the % of GFP positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 15] The results of the assay are shown in which Jurkat T cells were infected with lentivirus (MOI 0.75) in the presence or absence of natural or modified cationic poloxamers. After 72 h of incubation, the % of GFP positive cells (A) and the mean intensity of transduced cells (B) were assessed by flow cytometry. [Figure 16] The results of the assay are disclosed in which the KG1a cell line was infected with lentivirus (MOI 2) in the presence or absence of a wide range of doses of cationic poloxamer. After 72 h of incubation, the % of GFP positive cells (A) and the mean intensity of transduced cells (B) were assessed by flow cytometry. [Figure 17]Figures 17A and B disclose the results of an assay in which BV2, HeLa, Jurkat, C6, CLU-500 and KG1a cell lines were infected with lentivirus encoding GFP at the indicated MOI in the presence or absence of cationic poloxamer 2b. After 72 h of incubation, the % of GFP positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 18] Figures 18A and B disclose the results of an assay in which primary human CD34+ stem cells were infected with lentivirus encoding GFP in the presence or absence of a wide range of doses of modified cationic poloxamer at an MOI of 5. After 72 hours of incubation, the % of GFP positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 19] Figures 19A and B disclose the results of an assay in which C6 and HEK-293 cell lines were infected with adenovirus encoding GFP (AdGFP) in the presence or absence of F108 and 2b at an MOI of 5. After 72 h of incubation, the % of GFP positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 20] Serum effect on cationic poloxamer-induced transduction. NIH-3T3 cell lines were infected with adenovirus encoding GFP protein at an MOI of 4 in the presence or absence of two concentrations of cationic poloxamer (final concentrations 6.25% or 12.5%) in medium with or without serum. After 72 hours of incubation, the % of GFP-positive cells was analyzed by flow cytometry and the fold change in the % of transduced cells. [Figure 21] Figures 21A and B disclose the results of an assay in which a HEK-293 cell line was transduced with an adenovirus encoding GFP (AAV-GFP) at 100,000 genome copies per cell in the presence or absence of II. After 72 hours of incubation, the % of GFP-positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. [Figure 22]Cationic poloxamers differentially enhance AAV infection and transduction. HEK293 cell lines were infected with AAV encoding GFP protein at 100,000 genome copies (GC) per cell in the presence or absence of cationic poloxamers at a final concentration of 7.5% or 15%. 72 hours after infection, the percentage and average intensity of GFP-positive cells were analyzed by flow cytometry. [Figure 23] Figures 23A and B disclose the results of an assay in which the KG1a suspension cell line was transduced with lentivirus encoding GFP in combination with ViroMag magnetic nanoparticles (VM), cationic poloxamer 2b or ViroMag coated with a 5% (weight / volume) solution of 2b (VM+2b) with an MOI of 5. After 72 hours of incubation, the % of G GFP positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The invention described in this document relates to a method for enhancing transduction of target cells by viral vectors using cationic poloxamers (which may otherwise be referred to herein as "chemical adjuvants" or "additives") introduced as the sole additive or formulated with nanoparticles.

[0027] According to the invention, the method involves contacting target cells with a virus and a cationic poloxamer. The structure of this additive incorporates distinct regions in the polymer backbone, both hydrophilic and hydrophobic regions. The polymeric architecture is terminated by a cationic chemical functionality that contributes to further enhancing viral transduction.

[0028] Additionally, another embodiment of the present invention relates to the use of these cationic poloxamers in colloidal stabilization and enhancing the transduction efficiency of iron-based nanoparticles.

[0029] The applicant has focused on modifying some poloxamers into cationic poloxamers, which, in combination with viruses, contact with target cells to dramatically improve the efficiency of transduction without any kind of toxicity. The details of the invention are set out in the claims below, while the technical advantages will be discussed in the embodiments related thereto.

[0030] According to this, a first object of the invention relates to a method for transducing target cells with a viral vector using a cationic poloxamer.

[0031] In the present text, the term "transduction" refers to its common and accepted meaning in the scientific community. Transduction refers to the method of introducing natural, wild-type or recombinant genetic material into a target cell by a virus and, if necessary, its integration into the genome. This method is often also called "infection"; both terms are used interchangeably in this document. In contrast, transfection refers to the method of delivering genetic material into a cell using a synthetic or non-viral delivery system. The nature of the genetic material introduced and the mechanism involved in this step depend on the type of virus used in the transduction procedure. For example, the genetic material introduced by the virus can code for a wide variety of genes of interest, such as reporter genes, enzymes, endonucleases, nucleases or recombinases for genome editing, short hairpin RNAs, antisense RNAs for gene silencing, mRNAs, tRNAs, receptors such as engineered antigen receptors (T cell receptors, chimeric antigen receptors, chimeric cytokine receptors...), growth factors, hormones, cell surface proteins, secreted proteins, signaling proteins or any polypeptide or protein or nucleic acid for therapeutic purposes.

[0032] In particular, the present invention relates to a transduction method comprising at least one step of contacting a target cell with a viral vector and a cationic poloxamer.

[0033] According to the present invention, the claimed transfection methods can be carried out using known cationic poloxamers as well as novel cationic poloxamers, such as those newly synthesized by the applicant and claimed herein below.

[0034] According to the present invention, any known virus that can be used as a viral vector (natural, recombinant, pseudotyped or wild type) and that can be used in transduction can be used in the method according to the present invention.

[0035] By way of example, and without limitation, viruses (viral vectors) for use in the practice of the present invention include: a) double-stranded DNA viruses, such as adenovirus, adeno-associated virus, canine hepatitis virus, papillomavirus, polyomavirus, herpes simplex virus (HSV), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, smallpox virus, vaccinia virus, and hepatitis B virus; b) Single-stranded DNA, e.g., parvoviruses, anelloviruses; c) double-stranded RNA, e.g., reovirus, rotavirus; d) single-stranded RNA such as, for example, picornaviruses, coxsackieviruses, caliciviruses, togaviruses, alphaviruses, arenaviruses, flaviviruses, pestiviruses, West Nile virus, orthomyxoviruses, influenza viruses, enteroviruses, polioviruses, paramyxoviruses, measles virus, Newcastle disease virus, bunyaviruses, rhabdoviruses, vesicular stomatitis virus (VSV), filoviruses, coronaviruses, astroviruses, bornaviruses, arteriviruses, and hepeviruses; e) reverse transcribing viruses, such as retroviruses, lentiviruses, hepatitis B viruses, and f) other viruses, e.g. arboviruses, bacteriophages, etc. Examples include:

[0036] Preferably, the viral gene delivery vehicle can be any gene therapy delivery vehicle known in the art for use in gene therapy, including, but not limited to, adenoviruses, adeno-associated viruses (AAV), lentiviruses, retroviruses, gammaretroviruses, herpes viruses and other viruses.

[0037] A brief description of the main viruses relevant to the present invention is given below:

[0038] -Retroviruses are the mainstay of current gene therapy approaches, since most of them can stably integrate into the host genome. To do this, retroviruses use retrotranscriptases to convert their RNA into DNA, which is then integrated into the host genome by integrases. Replication-defective retroviruses are the most common choice in most studies, since the modification of their genomes further allows them to infect target cells and deliver their viral payload, but blocks the subsequent pathway that leads to cell lysis and death. This family includes gammaretroviruses, which are popular in gene therapy due to their advantages over other retroviral vectors, especially since their genomes are very simple and easy to use.

[0039] -Despite their tremendous potential as vectors, retroviruses suffer from several drawbacks, such as their inability to infect cells that are not actively dividing. Furthermore, a significant number of viral particles can lead to insertional mutations that may be the cause of cancer or leukemia. Retroviruses encompass a number of different virus species that can be used in gene therapy-based clinical trials, the major species being lentiviruses and gammaretroviruses. These two subclasses have been the subject of more than 300 clinical trials.

[0040] -Lentiviruses are a subclass of retroviruses and have the ability to integrate their genome into host cells. Moreover, lentiviruses show the unique advantage of being equally effective on dividing or non-dividing cells. Lentiviruses used in gene therapy are mostly non-replicating for safety reasons. This means that lentiviruses are still infectious but cannot enter any replication cycle and lack helper proteins that provide the missing viral proteins for new virion production. Almost always, the genes necessary for replication are replaced by an expression cassette that contains a target sequence for introduction into the host genome. This sequence may, for example, contain genes encoding therapeutically useful proteins or markers, or control sequences for RNA interference or miRNA expression.

[0041] - Adenoviruses have a linear dsDNA genome and can replicate in the cell nucleus using the host replication machinery. In contrast to lentiviruses, adenoviral DNA does not integrate into the genome and is not replicated during cell division. Their main applications are gene therapy and vaccination. Humans usually come into contact with adenoviruses, resulting in respiratory, gastrointestinal and eye infections, and the majority of patients already have neutralizing antibodies that can inactivate the virus before it can reach the target cells. To overcome this problem, science is currently studying adenoviruses that infect different species to which humans have no immunity.

[0042] - Adeno-associated viruses (AAV) are small viruses that infect humans and other primate species. AAV is currently known not to cause disease and only elicits a very mild immune response. AAV can infect both dividing and non-dividing cells and can integrate its genome into the host cell. Furthermore, AAV mostly resides as an episome (replicates without integration into chromosomes); long-term and stable expression. These characteristics make AAV a very attractive candidate for creating viral vectors for gene therapy. However, AAV grows to a maximum size of less than 5 kb, which is quite small compared to the original capacity of AAV.

[0043] Furthermore, for its potential use as a gene therapy vector, researchers have created a modified AAV called self-complementary adeno-associated virus (scAAV). AAV packages a single strand of DNA and requires the process of second-strand synthesis; scAAV packages both strands that anneal together to form double-stranded DNA. Skipping second-strand synthesis allows scAAV to rapidly express in cells. Otherwise, scAAV retains many of the characteristics of its AAV counterpart.

[0044] In the context of the present invention, the cationic poloxamer and the viral vector must be in contact with the target cells for transduction to occur. The contact with the different elements for efficient transduction has been shown many times in the literature and is highly dependent on the properties of the selected viral vector and the target cells. Some cells are more difficult to infect than others, and these may require specific management before contact, such as transformation in a specific culture medium or extended contact time with both the virus and chemical adjuvants.

[0045] According to the invention, the cationic poloxamer and the viral particles can be added to the cells simultaneously, either as a mixture or sequentially. However, it is of utmost importance to allow contact between the virus, the cationic poloxamer and the cells for a sufficiently long time to ensure an efficient transduction procedure. Contact or incubation times within the scope of the invention are in the range of 5 seconds to 3 months. More preferably, the contact time should be comprised between 10 minutes and 2 weeks, preferably between 20 minutes and 1 week. More preferably, it should be comprised between 0.5 hours and 120 hours.

[0046] Exemplary conditions can be found in the "Examples" section of this document. The cationic poloxamer and the viral particles can be added to the cells simultaneously, either as a mixture or sequentially. Optimal conditions for transduction according to the guidelines of the present invention have been described many times for different viral vectors such as lentiviruses (Arnoult et al Nature 2017,549:548-552;), AAV (Yelamarthi, T., Thesis Dissertation, 2012. University of Houston) or adenoviruses (Sapet et al Pharm Res. 2012,29:1203-18).

[0047] Preferably, the correct amounts of virus and cationic poloxamer will be mixed before depositing them on the cells, and the resulting mixture will require gentle mixing before proceeding.

[0048] According to the present invention, various multiplicities of infection (MOI) or viral titers may be used depending on the target cells, the selected viral vector, and the transduction needs. Some parameters can be modified as necessary to improve the efficiency of the transduction procedure. The skilled person will easily define the exact MOI in function of the viral vector used. As an example, a preferred MOI in the range of 0.001 to 5,000,000 may be considered.

[0049] According to the method of the present invention, the cationic poloxamer can be provided at a stock concentration of 0.01 to 500 mg / mL, preferably 0.05 to 300 mg / mL, more preferably 1 to 200 mg / mL, and even more preferably 5 to 150 mg / mL.

[0050] According to the method of the present invention, the cationic poloxamer is used at a final concentration obtained by diluting the above stock solution 1-100,000-fold, preferably 2-10,000-fold, more preferably 2-5,000-fold.

[0051] Optionally, according to a variant of the invention, the entire culture medium may be replaced with some fresh pre-warmed medium after the incubation time of the virus, the cationic poloxamer formulated or not with nanoparticles and the target cells, which may be modified according to procedures commonly performed in cell biology (Harrisson MA et al in General Techniques of Cell Culture Ed: Cambridge University Press 1993, ISBN-10:052157496X).

[0052] According to another variant of the invention, a centrifugation step can be performed after administration of the viral vector / cationic poloxamer, formulated or not, with the nanoparticle mixture. Indeed, an increase in the adjuvanted transduction efficiency can be observed by centrifuging the plates immediately after administration of the vector (AB Bahnson et al.; J. Virol. Methods 1995, 54: 131-143).

[0053] According to the invention, any kind of cell culture material or cell culture method can be used on a two-dimensional (2D) or three-dimensional (3D) carrier, respectively, for transducing said "target cells". 2D devices allow cells to be cultured in an adherent manner (cells adhere at the bottom of the well), in a suspension manner (cells remain suspended in the culture medium), or in treated or untreated cell culture plates and dishes (1536-well to 4-well cell culture plates, 35 mm, 60 mm, 90 mm, 100 mm Petri dishes) or cell flasks (25 cm 2 , 75cm 2 Or 150cm 2 ...) in co-cultures (two or more different cell types without separation or intercalation). The invention may be applied to 3D matrices such as hydrogels or solid 3D scaffolds of various compositions such as collagen, atelocollagen, glycosan, polystyrene, hyaluronic acid, polycaprolactone, polyethylene glycol, etc.

[0054] According to yet another variation of the invention, magnetic nanoparticles may be added to the mixture of target cells, cationic poloxamer and virus.

[0055] When cationic poloxamers are combined with magnetic nanoparticles, the method may include an optional additional step in which a continuous magnetic field or an oscillating magnetic field or an electronic magnetic field may be applied to the experiment after contacting the cationic poloxamer, formulated or unformulated, with the virus, cells and nanoparticles. This step may last from 10 seconds to 96 hours, preferably from 1 minute to 48 hours. More preferably, it will last from 5 minutes to 4 hours.

[0056] According to the invention, said "target cells" can also be infected and / or transduced on a magnetic cell separation device, with or without the use of columns supporting positive or negative cell selection. The term "cell selection" refers to an immunomagnetic cell separation technique used to isolate specific cell populations (hematopoietic stem cells, T lymphocytes, B lymphocytes, natural killer, PBMCs, splenocytes...) from different species (human, mouse, rat, non-human primate, rabbit, cow...) with the aid of a magnetic field. Thus, the invention may be used to infect or transduce cells either retained on the separation device or immediately after the selection process; furthermore, cationic poloxamers formulated with magnetic nanoparticles can also be used with viruses to retain viral particles on the magnetic separation device and further directly infect the cells on the same device. The present invention is particularly interesting for infecting various cell types ranging from cell lines to primary cells such as Jurkat T cells, K562, cord blood primary CD34+ cells, bone marrow cells or human mesenchymal stem cells (Sanchez-Antequera Y., Blood. 2011 Apr 21; 117(16): e171-81). When adding cationic poloxamer, formulated or not with magnetic nanoparticles, onto the separation device, the contact time with the separation device within the scope of the present invention ranges from 15 seconds to 1 week. More preferably, the contact time should be comprised between 1 minute and 6 hours. More preferably, it should be comprised between 1 minute and 1 hour.

[0057] According to the present invention, the "target cell" may be any cell that can be targeted for transduction with a viral vector. As used in the context of the present invention, the term "cell" may refer to a single and / or isolated cell or a cell that is part of a multicellular entity such as a tissue, an organism or a cell culture. That is, the method may be performed in vivo, ex vivo or in vitro. The cell may be a primary cell (as used herein, the term "primary cell" is known in the art and refers to a cell that has been isolated from a tissue and established for growth in vitro) or an established cell line or a stem or progenitor cell. The cells may be eukaryotic cells such as endothelial cells, epithelial cells, fibroblasts, stem cells, hematopoietic cells (lymphocytes, monocytes, macrophages natural killer dendritic cells, etc.), muscle cells, neural cells (plasmatic cells, hippocampal cells, sensory neurons, motor neurons, dorsal root ganglia, oligodendrocytes, cerebellar granule cells, neural stem cells and all types of neurons such as basket cells, Betz cells, medium spiny neurons, Purkinje cells, pyramidal cells, Renshaw cells, granule cells or anterior horn cells, glial cells, astrocytes, etc.), stem cells, embryonic stem cells, hematopoietic stem cells, germ cells, tumor cells, lymphoid cell lineages, and somatic cells, to name a few. The cells may be prokaryotic cells (bacteria), plant cells, insect cells, yeast cells, or parasitic cells. They may be differentiated or totipotent or totipotent or multipotent or oligopotent or unipotent or pluripotent. These may be embryonic stem cells or induced pluripotent stem cells (iPS) or may be differentiated from embryonic or iPS. The cells to be transduced may be non-permissive cells, cells that are difficult to infect, or cells that are susceptible to infection.Tumor cells and cell lines known in the art can be, for example, but are not limited to, lymphoma cell lines (e.g., Jurkat, CEM, H9, Daudi, SUP-M2, KARPAS-299, etc.), dendritic cell lines (DC2.4, Mutu, etc.), natural killer cell lines (NK-92, KHYG-1, etc.), epithelial cell lines (NIH-3T3, COS, CHO, HEK293, etc.), pancreatic tumor cells (PANC-1, etc.), stem cell lines (KG1a), breast cancer cells (MCF-7, T74D, MDA-MB361), neuroblastomas (SH-SY5Y, N2a, etc.), fibrosarcomas (HT1080, etc.), astrocytomas (1321N1, etc.), endothelial cells (HUVEC, HMEC, HCAEC, etc.). As used herein, a eukaryotic cell refers to any cell of a multicellular eukaryotic organism, including cells from animals such as vertebrates.

[0058] Preferably, said target cell may be a mammalian cell. As used herein, the term "mammalian cell" is well known in the art and refers to any cell belonging to or derived from an animal grouped under the mammalian classification.

[0059] In the method of the present invention, the improved transduction of the virus using cationic poloxamers can target the aforementioned tissues or organs. The tissues can be, for example, but not limited to, muscle tissue, connective tissue, epithelial tissue or nervous tissue. Methods for obtaining samples from various tissues and establishing primary and immortalized cell lines are well known in the art (Freshney, RICulture of Animal Cells: A Manual of Basic Technique, Fifth Edition, 2005 Ed: John Wiley & Sons, Inc.).

[0060] According to the invention, the cationic poloxamer will be based on a block copolymer comprising at least three blocks. A block means a polymeric construct having either hydrophilic or hydrophobic properties. Preferably, the hydrophilic block represents a poly(ethylene) oxide polymer chain, whereas the hydrophobic block preferably represents a poly(propylene) oxide polymer backbone. The order and sequence of these different blocks defines the general structure of the polymers used as transduction adjuvants in the present invention.

[0061] If the cationic poloxamer is based on the regular and linear poloxamer family, the first central block, identified as the hydrophobic / lipophilic block, will preferably be composed of a poly(propylene) oxide polymer chain. Said hydrophobic / lipophilic block will preferably be surrounded by two similar or different more hydrophilic blocks, based on poly(ethylene) oxide. If the cationic poloxamer is based on the "reverse poloxamer" (or meroxapol) family, the central block will be composed of a poly(ethylene) oxide polymer surrounded by two hydrophobic blocks, identified as poly(propylene) oxide polymer chains.

[0062] Such cationic and reverse cationic poloxamers can be linear or X-shaped, the latest of which are known as poloxamines or reverse poloxamines according to the same definition. All these families of polymers are described under the general name of cationic poloxamers in the remainder of this document.

[0063] Due to their synthetic methods involving anionic polymerization reactions, cationic poloxamers usually display two hydroxyl (or alcohol) functional groups at the termini of their backbones, what polymer chemists generally refer to as "polymer end groups."

[0064] In the present invention, these end groups have been modified to introduce cationic moieties at both ends of the polymeric molecule. A complete description of these modifications is detailed further in this document, including the characterization of the complete synthetic procedure and biological evaluation.

[0065] Thus, in this document, the term cationic poloxamer refers to a triblock copolymer composed of hydrophobic chains of polypropylene oxide and hydrophilic chains of polyethylene oxide originally terminated with hydroxyl functional groups and subsequently modified using well-known organic chemistry procedures to introduce cationic organic functional groups at both ends of the polymer chains.

[0066] The present invention also relates to novel cationic poloxamers that can be used in the methods of the present invention as well as cationic poloxamers known in the prior art at the date of the present invention, such as the cationic poloxamers described in G. Gyulain et al in eXPRESS Polym Letters, 2016, 10:216:26 or Slobodkin et al. US Patent Publication No. 2010 / 0004313 A1. Thus, the present invention also relates to novel linear or branched cationic triblock copolymers which can be represented by the following formula I or formula II:

[0067] [ka]

[0068] [ka]

[0069] According to formulas I and II, P can represent the backbone of a triblock copolymer that can include hydrophobic chains of polypropylene oxide and hydrophilic chains of polyethylene oxide. According to formula I, in the case of linear cationic poloxamers, P can be described according to the following formula IIIa or b:

[0070] [ka]

[0071] where Formula III covers the cases of regular cationic poloxamers, where a hydrophobic poly(propylene) oxide block is surrounded by two hydrophilic poly(ethylene) oxide blocks (Formula IIIa), and "reverse cationic poloxamers / meroxapols," where a hydrophilic poly(ethylene) oxide block is surrounded by two hydrophobic poly(propylene) oxide blocks (Formula IIIb).

[0072] According to formula II, which covers the case of modified X-shaped cationic poloxamers known as poloxamines, P can be described according to the following formula IVa or b:

[0073] [ka]

[0074] According to formula I; II, IIIa, IIIb, IVa and IVb: "a" represents the number of hydrophilic units repeated in the polymer backbone and is an integer which may range from 2 to 10000, preferably from 5 to 1000, more preferably from 20 to 200; - "a-1" can represent the number obtained by subtracting 1 unit from the above "a". It can therefore range from 2 to 10,000, preferably from 5 to 1,000, more preferably from 20 to 200; - "b" represents the number of hydrophobic units repeated along the polymer backbone and is an integer which may range from 2 to 1000, preferably from 5 to 500, more preferably from 15 to 80; - "b-1" can represent the number obtained by subtracting 1 unit from the above "b". It can therefore range from 2 to 1000, preferably from 5 to 500, more preferably from 15 to 80; - X1, X2, X3 and X4 may represent heteroatoms and are preferably selected from nitrogen, phosphorus, silicon, sulfur and oxygen, more preferably nitrogen, phosphorus, silicon or sulfur, even more preferably nitrogen, phosphorus or sulfur. X1, X2, X3 and X4 may be the same or different. X1, X2, X3 and X4 are covalently bonded to R1, R2, R3 and R4, respectively; - n is an integer comprised between 1 and 20, more preferably between 2 and 6; - R1, R2, R3 and R4 can be selected such that the non-polymeric entities "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" can be cationic. "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" can contain one or several positive charges in an aqueous environment at a pH ranging from 4 to 9, preferably at a physiological pH ranging from 6 to 8. This can include positive charges localized on heteroatoms such as nitrogen, phosphorus, sulfur, silicon or oxygen, preferably nitrogen, phosphorus, silicon or sulfur. "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" may share the same structure or may be different according to the invention.

[0075] According to this, R1, R2, R3 and R4 may or may not be present simultaneously. 1 to 6 hydrogen atoms, preferably 2 to 4 hydrogen atoms. 1 to 8 heteroatoms, preferably 2 to 4 heteroatoms selected from, for example, nitrogen, phosphorus, silicon, sulfur and oxygen, preferably nitrogen, phosphorus, silicon or sulfur, more preferably nitrogen, phosphorus or sulfur. 1 to 24, preferably 1 to 12, more preferably 1 to 6 straight chain, branched chain and / or cyclic saturated or unsaturated hydrocarbon groups containing 1 to 24 carbon atoms, with or without one or more heteroatoms such as oxygen, nitrogen, silicon, sulfur, phosphorus. · 1 to 6, more preferably 1 to 3, amino acid residues, natural or unnatural. Any combination of these definitions As a result, and in accordance with the present invention, "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" are: Primary, secondary or tertiary amine cationic moieties. Amines such as guanidine, hydrazine, guanidinium, hydrazinium can be preferred entities according to the invention; o Organic quaternary phosphonium moieties such as, for example, substituted tri-n-butylphosphonium, substituted triphenylphosphonium, substituted triethylphosphonium, etc.; o Quaternary ammonium salts based on a nitrogen atom covalently bonded to a four-carbon moiety, such as, for example, substituted quaternary trimethylammonium, substituted quaternary tri-n-butylammonium, substituted quaternary tri-n-octylammonium; o Organic tertiary sulfonium salts based on sulfur covalently bonded to a three carbon moiety, e.g., substituted dimethylsulfonium, substituted di-n-butylsulfonium, substituted di-n-octylsulfonium, etc.; ○ Organic heterocycles having a net positive charge, delocalized or not, on the ring. Heterocycle means an organic carbocyclic structure of 3-20, preferably 3-8 carbon atoms incorporating at least 1-6 heteroatoms, similar or different, such as oxygen, nitrogen, sulfur, phosphorus, etc. These heterocycles may or may not contain at least one unsaturation that can provide aromaticity, such as, for example, pyridine and its cationic counterpart pyridinium; imidazole and its cationic counterpart imidazolium; triazole and its cationic counterpart triazolium; piperidine and its cationic counterpart piperidinium; morpholine and its cationic counterpart morpholinium; o Basic amino acid residues as the source of cationic charge, e.g. natural or unnatural lysine, arginine, histidine, ornithine, tryptophan; o It may be, for example, a naturally occurring non-polymeric polyamine, such as a spermine, spermidine or thermospermine derivative that is not polymeric; o Furthermore, according to the present invention, any combination of the described chemical structures for "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" attached to a single cationic poloxamer end group is included within the scope of the present invention. Thus, according to the present invention, the "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4" structures can contain one or several carbon-based chains, linear or branched chains, heteroatoms with or without heteroatoms, carbocyclic rings, heterocyclic rings. As an extension of this point, and as illustrated in the text, figures and examples of the present desire, the defined chemical structures for "-X1-R1", "-X2-R2", "-X3-R3", and "-X4-R4" exclude the possibility that these entities are polymeric, meaning that "-X1-R1", "-X2-R2", "-X3-R3", and "-X4-R4" cannot be composed of the same chemical pattern repeated an undefined number of times. A1, A2, A3 and A4 may represent the same or different counterions, which may be chosen, for example, from one or several organic groups, such as: o halogen anions, such as iodide, bromide, chloride or fluoride; o Organic groups having a negative charge, whether or not centered on a carbon atom, such as, for example, methanesulfonate, trifluoromethanesulfonate, trifluoroacetate, acetate, formate, paratoluenesulfonate, carbonate, hydrogen carbonate, etc.; o Inorganic anions, e.g., sulfate, phosphate, nitrate, hydrogen sulfate, hydrogen phosphate, etc.; o Inorganic non-coordinating anions, defined as anions that do not interact or only slightly interact with their associated cations. We can mention, for example, tetrafluoroborate, hexafluorophosphate or perchlorate, carba-closo-dodecaborate, etc.; ○ It can be a boron-centered organic anion based on the tetrakis[3,5-bis(trifluoromethyl)phenyl]borate framework; and - F108 skeleton, a is an integer in the range of 130 to 135, b is an integer in the range of 48 to 52, a-1 is an integer in the range of 129 to 134, X1R1=X2R2=NH2, or X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - F127 skeleton, a is an integer in the range of 98 to 103, b is an integer in the range of 52 to 58, a-1 is an integer in the range of 97 to 102, X1R1 = X2R2 = NH2, or X1R1 = X2R2 = -OC(O)-NH-(CH2)2-NH2, or X1R1 = X2R2 = -OC(O)-NH-(CH2)2-NH-C 19 -H 18 -N7-O5, or X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2, or X1R1=X2R2=-OC(O)-CH(NH2)-CH-(CH3)2, or X1R1=X2R2=-OC(O)-NH-(CH2)2-NH-(CH2)2-NH-(CH2)2-NH2, or X1R1=X2R2=-OC(O)-CH(NH2)-CH2-SH; - F68 skeleton, a is an integer in the range of 72 to 78, b is an integer in the range of 25 to 32, a-1 is an integer in the range of 71 to 77, X1R1=X2R2=NH2, and X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - P123 skeleton, a is an integer in the range of 17 to 23, b is an integer in the range of 67 to 73, a-1 is an integer in the range of 16 to 22, and X1R1=X2R2=-OC(O)-NH-(CH2)2-NH2; - L121 skeleton, a is an integer ranging from 8 to 12, b is an integer ranging from 64 to 72, a-1 is an integer ranging from 7 to 11, X1R1=X2R2=-CH=N-(CH2)2-NH2 or X1R1=X2R2=-CH=N-(CH2)4-NH2 or X1R1=X2R2=-CH=N-(CH2)2-O-(CH2)2-O-(CH2)2-NH2; - P85 skeleton, a is an integer in the range of 24 to 28, b is an integer in the range of 38 to 42, a-1 is an integer in the range of 23 to 27, and X1R1=X2R2=-OC(O)-NH-(CH2)3-NH2; - P105 skeleton, a is an integer in the range of 34 to 39, b is an integer in the range of 52 to 60, a-1 is an integer in the range of 33 to 38, X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - F88 skeleton, a is an integer in the range of 94 to 100, b is an integer in the range of 34 to 42, a-1 is an integer in the range of 93 to 99, and X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - P124 skeleton, a is an integer in the range of 8 to 14, b is an integer in the range of 16 to 24, a-1 is an integer in the range of 7 to 13, and X1R1=X2R2=-OC(O)-CH2-C 12 H 20 N3O8, or X1R1=X2R2=N((CH2)3-NH2)-(CH2)4)-NH2; - P104 skeleton, a is an integer in the range of 24 to 30, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 23 to 29, X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2; - P103 skeleton, a is an integer in the range of 14 to 20, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 13 to 19, X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2; - L64 skeleton, a is an integer in the range of 10 to 16, b is an integer in the range of 26 to 34, a-1 is an integer in the range of 9 to 15, X1R1=N3 and X2R2=-C 17 H 15 N2O2; - T908 skeleton, a is an integer in the range of 116 to 122, b is an integer in the range of 15 to 20, and a-1 is an integer in the range of 115 to 121, and may exclude X1R1 = X2R2 = X3R3 = X4R4 = NH2;

[0076] According to the present invention, A1, A2, A3 and A4 can maintain the neutrality of the whole polymer as a cationic moiety is incorporated, since the terminal groups of the cationic poloxamer chains imply the presence of an anionic counterpart to preserve the neutrality of the whole molecular assembly. As a result, the number of negative charges contained in A1, A2, A3 and A4 will be directly related to the number of positive charges present in "-X1-R1", "-X2-R2", "-X3-R3" and "-X4-R4". Furthermore, the nature of "A" may depend on the reactants used during the modification of the cationic poloxamer. If it is not possible to use reactants that combine the chemical properties required for the polymer modification and the exact nature of "A", a salt metathesis reaction will be set up using commonly described experimental conditions (Gutowski KE et al J.Am.Chem.Soc.,2003,125:6632-6633).

[0077] According to the present invention, the selection of "a" and "b" will adjust the structural composition of the described cationic poloxamer and therefore modify its physicochemical properties. Among these, the properties can be highlighted as HLB (hydrophilic lipophilic balance), a well-known semi-empirical value, which reflects the hydrophilic or lipophilic behavior of commonly used surfactants. According to the present invention, the Davies definition of HLB (Davies JT, Proceedings of the International Congress of Surface Activity 1957, 426-438) was used, which was extended to the neutral poloxamer definition by Guo et al. (Guo X et al. Journal of Colloid and Interface Science 2006 298: 441-450).

[0078] It should be noted that in accordance with the present invention, both "regular" poloxamines (wherein a polypropylene oxide block can be linked with a central amine functionality) and "reverse poloxamines" (wherein a polyethylene oxide block can be linked with a central amine functionality) are also contemplated in the present invention.

[0079] Additionally, all molecules depicted in formulas I and II, regardless of their linear or X-shaped or inverted character, are referred to in this document as cationic poloxamers.

[0080] According to the present invention, the described cationic poloxamers can be constructed with values ​​of "a" and "b" selected such that the HLB value of the resulting polymer before cationic modification can be between 1 and 100, more preferably between 10 and 40, more preferably between 15 and 30. This goal can be achieved by careful selection of poloxamer starting materials, which are commercially available from different suppliers such as BASF or CRODA.

[0081] Polymer synthesis, particularly by anionic polymerization, as in the present invention, cannot lead to perfectly controlled molecules, including the exact number of repeating units, and in most cases, the quality of the purification methods that allow the manufacturer to obtain a "pure" form of such a polymer at the end of the synthesis can be conditioned. Therefore, it is very difficult, if not impossible, for a person skilled in the art to obtain the exact values ​​of the parameters related to the molecular weight of the described cationic poloxamer. As outlined herein, the synthesis of cationic poloxamers can result in a mixture of polymers with different molecular weights. Therefore, the term "average" in relation to the molecular weight, or HLB, is a consequence of the technology not being able to produce cationic poloxamers that all have the same composition.

[0082] The cationic poloxamer used may therefore exist as a mixture of cationic poloxamers, each of which exhibits variation with respect to their molecular weight and composition. Parameters such as molecular weight, HLB, and the values ​​"a" and "b" in formulae IIIa, IIIb, IVa and IVb reflect this variation and must be considered as characteristic of the mixture of cationic poloxamers used in this study.

[0083] According to the present invention, a preferred structure of the compound of formula I can be that disclosed in formula I using the general linear cationic poloxamer structure:

[0084] [ka] [ka] [ka] [ka] [ka]

[0085] wherein the values ​​of "a", "b" and "a-1" and "b-1" can be as defined earlier in the text.

[0086] As mentioned above, the following molecules are excluded from the present invention as defined by formula I and II, but are included in the present invention in relation to the transduction method as cationic poloxamers: - F108 skeleton, a is an integer in the range of 130 to 135, b is an integer in the range of 48 to 52, a-1 is an integer in the range of 129 to 134, X1R1=X2R2=NH2, or X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - F127 skeleton, a is an integer in the range of 98 to 103, b is an integer in the range of 52 to 58, a-1 is an integer in the range of 97 to 102, X1R1 = X2R2 = NH2, or X1R1 = X2R2 = -OC(O)-NH-(CH2)2-NH2, or X1R1 = X2R2 = -OC(O)-NH-(CH2)2-NH-C 19 -H 18 -N7-O5, or X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2, or X1R1=X2R2=-OC(O)-CH(NH2)-CH-(CH3)2, or X1R1=X2R2=-OC(O)-NH-(CH2)2-NH-(CH2)2-NH-(CH2)2-NH2, or X1R1=X2R2=-OC(O)-CH(NH2)-CH2-SH; - F68 skeleton, a is an integer in the range of 72 to 78, b is an integer in the range of 25 to 32, a-1 is an integer in the range of 71 to 77, X1R1=X2R2=NH2, and X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - P123 skeleton, a is an integer in the range of 17 to 23, b is an integer in the range of 67 to 73, a-1 is an integer in the range of 16 to 22, and X1R1=X2R2=-OC(O)-NH-(CH2)2-NH2; - L121 skeleton, a is an integer ranging from 8 to 12, b is an integer ranging from 64 to 72, a-1 is an integer ranging from 7 to 11, X1R1=X2R2=-CH=N-(CH2)2-NH2 or X1R1=X2R2=-CH=N-(CH2)4-NH2 or X1R1=X2R2=-CH=N-(CH2)2-O-(CH2)2-O-(CH2)2-NH2; - P85 skeleton, a is an integer in the range of 24 to 28, b is an integer in the range of 38 to 42, a-1 is an integer in the range of 23 to 27, and X1R1=X2R2=-OC(O)-NH-(CH2)3-NH2; - P105 skeleton, a is an integer in the range of 34 to 39, b is an integer in the range of 52 to 60, a-1 is an integer in the range of 33 to 38, X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - F88 skeleton, a is an integer in the range of 94 to 100, b is an integer in the range of 34 to 42, a-1 is an integer in the range of 93 to 99, and X1R1=X2R2=-OC(O)-NH-(CH2)2-SS-C5H4N, or X1R1=X2R2=-OC(O)-NH-NH2; - P124 skeleton, a is an integer in the range of 8 to 14, b is an integer in the range of 16 to 24, a-1 is an integer in the range of 7 to 13, and X1R1=X2R2=-OC(O)-CH2-C 12 H 20 N3O8, or X1R1=X2R2=N((CH2)3-NH2)-(CH2)4)-NH2; - P104 skeleton, a is an integer in the range of 24 to 30, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 23 to 29, X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2; - P103 skeleton, a is an integer in the range of 14 to 20, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 13 to 19, X1R1=X2R2=-OC(O)-(CH2)2-NH-(CH2)3-NH-(CH2)4-NH-(CH2)3-NH2; - L64 skeleton, a is an integer in the range of 10 to 16, b is an integer in the range of 26 to 34, a-1 is an integer in the range of 9 to 15, X1R1=N3 and X2R2=-C 17 H 15 N2O2; - T908 skeleton, a is an integer in the range of 116 to 122, b is an integer in the range of 15 to 20, a-1 is an integer in the range of 115 to 121, and X1R1 = X2R2 = X3R3 = X4R4 = NH2.

[0087] According to the present invention, the cationic poloxamers described herein as well as any other cationic poloxamers can be used as chemical adjuvants in transduction experiments. They can be combined with viral vectors before contacting target cells. The concepts of contact, viral vector and target cells are explained in this document.

[0088] Cationic poloxamers have the property of forming gels at high concentrations. The cationic poloxamers described in this invention can be provided as liquids or gels.

[0089] The cationic poloxamers can be dissolved in water or any suitable aqueous buffer routinely used in cell biology at a given range of concentrations. Examples of such buffers can be: HEPES, DPBS (with or without calcium and / or magnesium), Tris, carbonate, acetate, citrate. The selection of dissolution concentration and dissolution medium can be made to maintain an efficient and reproducible transduction efficiency and to preserve the formulation according to the invention in a liquid physical state. The cationic poloxamers can also be associated with different components such as surfactants capable of forming emulsions, gels, nanoemulsions or micelles.

[0090] The novel cationic poloxamers, as well as the cationic poloxamers known from the prior art at the date of the present invention, can be associated or formulated with different types of chemical molecules, such as adjuvants capable of specifically targeting or binding determinants on the cell surface and / or inside, and can be covalently or non-covalently linked to the compounds corresponding to formula I, II or to other molecules contained in the compositions comprising the compounds of formula I, II, such as ligands of receptors expressed on the surface of the target cells, such as sugars, folates, transferrin, insulin, hormones, prostaglandins, peptides, reductases, metabolites, vitamins or other molecules capable of recognizing extracellular receptors, or elements of intracellular vectorization for targeting specific compartments, such as the nucleus or cytoplasm, for example, the mitochondrion, the nucleus or the cytoplasm, or mitochondrial localization signals, such as sugars, peptides, proteins, antibodies, antibody fragments, ligands or ligand fragments.Activators or inhibitors of protein kinase C or cellular metabolic pathways (AMPK signaling, insulin receptor signaling, glutamine metabolic pathway, etc.), such as activators or inhibitors of TLR-pathways (Toll-like receptors), PRR-pathways (pattern recognition receptors), PAMP-pathways (pathogen-associated molecular patterns), DAMP-pathways (damage-associated molecular patterns), AhR-ligands (aryl hydrocarbon receptor ligands), NOD-like receptors (NLRs), RIG-I-like receptors (RLRs), cytosolic DNA sensors (CDSs), C-type lectin receptors (CLRs), activators or inhibitors of inflammasomes or inflammation or autophagy pathways, activators or inhibitors of the JAK / STAT pathway, activators or inhibitors of the ubiquitin and ubiquitin-like / proteasome pathways, PI3K / A Signaling molecules such as activators or inhibitors of the KT pathway, activators or inhibitors of tyrosine kinases, activators or inhibitors of the MAP kinase pathway, activators or inhibitors of GPCR, calcium, cAMP signaling pathways, activators or inhibitors of cell cycle, checkpoint control and DNA repair mechanisms, activators or inhibitors of apoptosis pathways, regulators of reactive oxygen species (ROS, NOS...), regulators of protein synthesis and RNA degradation processes, regulators of key elements with a role at the chromatin or DNA level (i.e. protein acetylation, histone lysine methylation, DNA methylation, nuclear receptor signaling...), regulators of microtubule and actin dynamics pathways, and molecules that may be associated with signaling pathways inside the target cell, may also be considered in the present invention.

[0091] Cationic poloxamers can also be conjugated to molecules known to affect the transduction procedure, including a wide range of molecules such as recombinant or non-recombinant polypeptides, liposomes, cationic lipids, cationic polymers such as polyethyleneimine, protamine sulfate, polybrene, poly-L-lysine, DEAE dextran, polylactic-co-glycolic acid, chitosan, neutral polymers such as unmodified poloxamers, polyethylene glycol and derivatives, anionic polymers such as starch and hyaluronic acid, etc. These molecules can be added to the cells 1 week before to 4 weeks after the addition of the present invention, preferably 1 day before to 7 days after the addition of the present invention.

[0092] The main objective of the present invention is to design a novel and efficient adjuvant for transduction procedures. Magnetic nanoparticles were used to enhance, synchronize and promote transduction. Therefore, cationic poloxamers were tested in combination with magnetic nanoparticles, and in the present invention, the cationic poloxamers described herein can also act as stabilizers and synergists for magnetic nanoparticles.

[0093] In fact, the use of unmodified poloxamers on the surface of magnetic colloidal suspensions of beads is well known. Due to their extreme biological compatibility, mainly due to the presence of ethylene oxide chains, some examples associate them with iron-based magnetism, thus enhancing the properties of these colloidal suspensions intended to be used in biological environments, especially in in vivo procedures. However, in any case, the unmodified poloxamers need to be combined with another type of molecule, polymeric or not, to provide the nanoparticles with a zeta potential, or surface potential. In fact, it is paramount that electrostatic repulsive interactions occur in the colloidal suspension, so that the magnetic core does not aggregate, resulting in micrometer material, which may have disastrous consequences for the safety of the host organism. By extension, magnetic nanoparticles coated only with unmodified poloxamers remained stable for a long time due to the lack of stabilizing charges. The positive charges added to the molecules of the present invention, as well as other cationic poloxamers, introduce electrostatic interactions, a missing factor necessary for stabilization by the magnetic iron core. According to the present invention, the particles produced by the method are able to retain the nanometer size range for many weeks, whereas their counterparts coated with unmodified poloxamers are unable to achieve colloidal stability for many hours. This novel and interesting feature has allowed the applicant to develop a new class of magnetic iron nanoparticles that show improved transduction efficiency when combined with viral vectors.

[0094] The present invention also relates to a composition, preferably a therapeutic or cosmetic or life science composition, comprising at least one compound of formula I or II as defined above.

[0095] In another aspect, the invention relates to a composition comprising at least one compound of formula I or II and a virus.

[0096] In yet another aspect, the present invention relates to a composition comprising at least one compound of formula I or II and a virus for ultimately transducing a target cell.

[0097] The present invention also relates to a kit comprising the cationic poloxamer, alone or formulated as described above, finally further comprising a virus, and optionally instructions for use.

[0098] The present invention relates to transduced cells obtained by the method according to claims 1 to 11. Said cells can be used in cell therapy.

[0099] Other features and advantages of the present invention will be seen in the following illustrative and non-exhaustive examples and the accompanying drawings which illustrate the results of tests carried out with compounds of the invention. EXAMPLES

[0100] a) Material Most of the solvents and reagents were obtained from VWR Prolabo (Briare, France), Sigma-Aldrich SA (Saint-Quentin-Fallavier, France), TCI Europe NV (Antwerp, Belgium) and Bachem Biochimie SARL (Boisins-le-Bretonneux, France). Poloxamers used as starting materials were obtained from BASF Performance polymers Europe (Rudolstadt, Germany) and CRODA Industrial specialties Europe (Gouda, The Netherlands). All reagents purchased at reagent grade were used without further purification unless otherwise specified. All amino acids used in this study were derived from natural amino acids (i.e., L-isomers) unless otherwise specified.

[0101] b) Method Thin-layer chromatography (TLC) was performed on aluminum plates coated with silica gel 60 Fasa (Merck). Compounds were developed with iodine under UV light (254 nm) by immersion in ninhydrin staining solution (0.2% in butanol) and then heated at 150 °C. For compounds with primary amine functions, immersion in cerium / enriched molybdate (HO / HSO4) / (NH4)MoO·4H2O / Ce(SO)·3HO developing solution: 90 / 10 / 15 / 1) and then heated at 110 °C for sulfur-containing compounds.

[0102] The synthesis products are purified on a silica chromatography column: flash chromatography separation is performed on silica gel 60 (230-400 mesh ASTM) (Merck).

[0103] Unless otherwise stated, polymer purification was performed by dialysis using membranes of different cut-offs sold by Sigma Aldrich or Spectrum Labs Europe BV (Breda, The Netherlands). Dialysis was performed against 4 liters of double distilled water. The medium was changed after 2, 8, 24, 36 and 48 hours.

[0104] Freeze drying was performed using a Heto PowerDry3000 freeze dryer with samples kept frozen at -80°C for 12 hours.

[0105] Size Exclusion Chromatography (SEC) The SEC experiments were carried out according to the gel permeation chromatography technique using an Agilent HPLC1260 Chain (Agilent, Les Ulis, France). The equipment is equipped with: a manual injection valve, a refractive index detector RID Agilent1260 Infinity, a multi-wavelength Agilent infinity 1260 UV detector, and a thermostatic column oven Agilent 1260 infinity. The data were collected and processed using the Agilent Open Lab LC Chemstation software and its GPC extension. The stationary phase used in these experiments is an Agilent Biosec 3 column (100 Å, 7.8 × 300 mm) thermostatically controlled at 40 °C. The mobile phase is composed of 0.5 M acetate buffer, the pH of which has been corrected to 4.75. The mobile phase was degassed multiple times by ultrasound and filtered twice before use. The pressure was approximately 105 bar (1.05 × 10 7 The flow rate was adjusted to 1 mL / min to reach a working pressure in the range of 100 psi (1 Pa).

[0106] For the analysis of the molecular mass characteristics of the polymers, a molecular weight calibration curve was prepared using polyethylene oxide standards (Mw ranging from 20400 to 136 Da) provided by Agilent. The samples to be analyzed were dissolved in double distilled water at a concentration of 1 mg / mL and filtered before injection (syringe filtration, 0.22 μm, Merck). The dilution water used to dissolve the samples contained tetrahydrofuran (THF, 10 μL / 100 mL of water), which was used as an elution marker for all measurements.

[0107] Particle size and zeta potential measurement Mean hydrodynamic particle size and charge measurements of cationic poloxamers were performed in grade 2 water using dynamic light scattering (DLS) and laser Doppler velocimetry (LDV), respectively, using a Malvern Nano ZS instrument and DTS software (Malvern Instruments, UK). Different amounts of cationic poloxamer were diluted in 100 μl water and mixed with the same amount of the same water containing DNA. Measurements were performed in automatic mode and results were presented as mean ± SEM, n = 3. Each average is the average of 30 measurements.

[0108] Synthetic molecule examples A large collection of modified cationic poloxamers was synthesized according to the method described in this invention. All molecules obtained in these examples were derived from commercially available unmodified "hydrophilic" linear and regular poloxamers and have various HLB values, preferably greater than or close to 20 (see above for full definition). The main structural features of the commercially available polymers used as starting materials for this study are summarized in Table 1 below.

[0109] [Table 1] Table 1: Physical properties of commercially available unmodified poloxamers used as templates in this study. Molecular weights (MW) are given in Daltons (Da) and were provided by the manufacturer. "a" and "b" are defined further on in the text and were calculated according to MW. HLB was calculated by the manufacturer.

[0110] A large panel of cationic modifications is presented in the following examples, where different strategies were applied to different commercially available unmodified poloxamers mentioned above, resulting in cationic polymers belonging to the present invention. Each of these polymers is named hereafter after a number (tag) as shown in Table 2.

[0111] [Table 2] Table 2: Numbers (tags) belonging to the cationic poloxamers synthesized for this study: each polymer is identified by the modification of its end groups with cationic chemical functional groups in combination with the unmodified poloxamer backbone from a commercial source.

[0112] In order to keep the examples as simple as possible, only one example of each synthetic strategy is described in full, however, all of these synthetic procedures were applied with similar efficiency to obtain the compounds listed in Table 2.

[0113] Therefore, each synthetic step is fully described for the selected cationic poloxamer, but results with other polymer backbones are summarized in the tables each time following the main steps.

[0114] Example 1: Synthesis of bispermine cationic poloxamer derivatives Example 1 deals with the introduction of spermine derivatives as terminal groups on different hydrophilic backbones of unmodified poloxamers. The chemical strategy would result in compounds with the following general structure:

[0115] [ka]

[0116] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 3 below.

[0117] [Table 3] Table 3: Main physicochemical characteristics of the spermine-grafted cationic poloxamer derivatives described in this study, Sd: spermine derivative; Tfa: trifluoroacetate; XR-A: cationic end group (XR)-counter anion (A); Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as defined above. HLB stands for hydrophilic / lipophilic balance as previously described.

[0118] To illustrate the synthetic strategy, a detailed procedure for obtaining compound I is developed below, starting with the synthesis of compound 1b depicted in Scheme II:

[0119] Step 1: Synthesis of bistosylated F87 1a. Before synthesis, 10 g of F87 (Croda) was dissolved in 100 mL of milli-Q double distilled water and placed in a 500 mL flask. The mixture was then frozen overnight at -80°C and then lyophilized.

[0120] 3.36 g (0.436 mmol) is then dissolved in 86 mL of a mixture of anhydrous dichloromethane with pyridine (73 / 13 volume ratio dichloromethane / pyridine) under argon. After vigorously stirring the mixture, catalytic amounts of DMAP (TCI) and paratoluenesulfonic acid chloride (Sigma-Aldrich, 4.36 mmol, 891 mg) are added in portions over 45 min. The reaction is then stirred at room temperature for 48 h. The entire mixture is then concentrated under vacuum and the resulting yellowish gum is redissolved in DCM (50 mL). The organic extract is then washed twice with saturated aqueous NH4Cl (2 x 100 mL) and allowed to stand. The combined washings are then extracted multiple times with dichloromethane (5 x 50 mL), after which the combined organic extract is washed twice with saturated aqueous NH4Cl with Milli-Q water and once with brine (150 mL each). The resulting organic phase was then dried over magnesium sulfate, filtered and concentrated in vacuo to give the title compound which was used without further purification (3.17g).

[0121] Table 4 below shows the numbers and amounts of the starting materials and the names and amounts of the bistosylated products, where m indicates the mass of the compound and n indicates the number of moles.

[0122] [Table 4]

[0123] Step 2: Synthesis of bisamino F87 1b. Compound 1b (1.66 g, 0.208 mmol) is dissolved in 30 mL of methanol solution containing 2 M ammonia (TCl) under vigorous stirring. The solution is degassed with argon and then heated to reflux for 24 h. Ammonia in methanol is then added (15 mL) and the mixture is refluxed for another 6 h before being concentrated under vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept overnight at 4° C. and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double distilled water with five medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (1.48 g). This compound is cationic. It therefore belongs to the present invention.

[0124] The following Table 5 shows the process evaluation for tosyl substitution with ammonia: the table gives the number and amount of starting material and the name and amount of the bisamino product, m gives the mass of the compound, and n gives the number of moles.

[0125] [Table 5]

[0126] To obtain the title compound, the synthesis of the ornithine derivative 1e is then required.

[0127] The synthesis of compound 1e, an intermediate of I, is summarized in FIG. 3: Step 3: Synthesis of 2,5-bis(2-cyanoethylamino)pentanoic acid 1c Sodium hydroxide (1.78 g, 44.5 mmol, VWR) is ground in a mortar and suspended in 150 mL of methanol in a round-bottom flask. L-ornithine monohydrochloride (5 g, 29.7 mmol, TCI) is then introduced in portions and stirred vigorously for 1 h 30 min. The suspension is then filtered through a short pad of Celite (Sigma-Aldrich). The remaining clear filtrate is reintroduced into the flask and, after stirring, 4.29 mL of acrylonitrile is added dropwise over 1 h. After complete addition, the mixture is stirred in the dark at room temperature for 36 h until TLC indicates completion. After stirring, 12 mL of 37% HCl (VWR) is carefully added to the mixture, which produces a cloudy precipitate while stirring. The solid is collected by filtration and dried in vacuum (S1, 2.51 g). A few drops of 30% concentrated aqueous sodium hydroxide (VWR) was then added, resulting in the immediate formation of a solid which was collected and similarly dried (S2, 1.13 g). Analysis by TLC showed that the remaining filtrate contained no target material, whereas S1 and S2 were pure target product. The overall yield was 46% (3.64 g, 13.7 mmol).

[0128] Step 4: 5-amino-2-(3-aminopropylamino)pentanoic acid 1d 2,5-Bis(2-cyanoethylamino)pentanoic acid 1c (2.51 g, 9.44 mmol) is suspended in 40 mL of absolute ethanol. The pH of the mixture is increased slightly with sodium hydroxide to aid dissolution and then transferred to a 50 mL steel laboratory autoclave (BuchiGlass; Uster, Switzerland). A catalytic amount of Raney Nickel (Sigma Aldrich) is introduced, the autoclave is closed and pressurized at 8 bar (8 × 10 5 The mixture is stirred in this manner for 12 hours. It is then evacuated and the mixture is filtered through a short pad of Celite. After removal of the ethanol, the oil obtained is used in the next step without further purification.

[0129] Step 5: 2,5-bis[tert-butoxycarbonyl-[3-(tert-butoxycarbonylamino)propyl]amino]pentanoic acid 1e The crude product from step 4 is placed in a three-necked round-bottom flask containing 15 mL of a solution of tetrahydrofuran and water (5 / 1 by volume). The mixture is then cooled to 0° C. using an ice / water bath. Under stirring, 4 portions of Boc2O (11.4 g, 57 mmol) dissolved in 4 M aqueous NaOH (9.12 mL) and THF (12 mL) are added, alternating with each other. The entire addition process takes 30 minutes to complete, and the reaction is then stirred at room temperature for 12 hours. The reaction is then concentrated under vacuum, redispersed in 100 mL of 6 M HCl, and extracted with diethyl ether (6×35 mL). The organic extract is then dried over magnesium sulfate, filtered, and concentrated. The product is purified on silica gel using DCM / MeOH (2% to 12% MeOH gradient in DCM) as the mobile phase. After concentration, the desired compound is isolated in 48% yield (2 steps, 2.91 g, 4.53 mmol).

[0130] This compound was synthesized in bulk and used in combination with each bisaminopoloxamer according to the strategy depicted in FIG.

[0131] Step 6: Coupling of 1b and 1e Compound 1e (32mg, 4.8×10 -2 Dissolve 10 mmol of DIC (9 μL, 5.6 × 10 -2 mmol) and HOBt (8 mg, 5.6 × 10 -2 The mixture is successively added with 10 mmol of 1b (1 mmol) and stirred for 2 h 30 min. Poloxamer 1b (127 mg) is then added directly to the flask with stirring and the reaction is followed for 24 h at room temperature before being concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept overnight at 4° C. and introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is carried out against 4 liters of double distilled water with five medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (123 mg).

[0132] The following Table 6 shows the process summary for coupling with compound 1e: the table gives the number and amount of starting material and the name and amount of the bis-protected product, m gives the mass of the compound and n gives the number of moles.

[0133] [Table 6]

[0134] Step 7: Synthesis of bis-substituted F87 derivative I. Compound 1f (123 mg) is dissolved in 4 mL of dichloromethane solution. TFA is then introduced dropwise (VWR, 2 mL). The solution is degassed with argon and then stirred for 2 h. The mixture is then concentrated in vacuum, coevaporated twice with DCM and three times with diethyl ether, and dried overnight under vacuum. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double-distilled water with five medium changes (2 h, 8 h, 24 h, 36 h, and 48 h). The final product is then recovered after lyophilization (124 mg).

[0135] The following Table 7 shows the process summary for Boc deprotection: the table shows the number and amount of starting material and the name and amount of bisspermine product, m shows the mass of the compound, and n shows the number of moles.

[0136] [Table 7]

[0137] Example 2: Synthesis of bistrimethylammonium cationic poloxamer derivatives Example 2 deals with the introduction of trimethylammonium moieties as terminal groups of different hydrophilic unmodified poloxamers. The chemical strategy will result in compounds with the general structure shown below:

[0138] [ka]

[0139] Structure of trimethylammonium grafted cationic poloxamer derivatives

[0140] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 8 below.

[0141] [Table 8] Table 8: Main physicochemical characteristics of the trimethylammonium grafted cationic poloxamer derivatives described in this study. XR: cationic end group (XR); counter anion (A) as previously described; Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as previously defined. HLB stands for hydrophilic / lipophilic balance as previously described.

[0142] The synthetic strategy, detailed procedure to obtain compound II, is developed in FIG.

[0143] Step 1: Synthesis of bistosylated F108 2a. Before synthesis, 20 g of F108 (Croda) was dissolved in 200 mL of milli-Q double distilled water and placed in a 1000 mL flask. The mixture was then frozen overnight at -80°C and then lyophilized.

[0144] Compound 2a is obtained by starting from 16.8 g of F108 and following the strategy and ratios used in the synthesis of compound 1a, 15.8 g of 2a is obtained. See Table 4 for detailed results.

[0145] Step 2: Synthesis of bisamino F108 2b. Compound 2b is obtained by starting from 4.65 g of 2a and following the strategy and ratios used in the synthesis of compound 1b, 3.38 g of 2b is obtained. This compound is cationic. Therefore, it belongs to the present invention and is further evaluated in this document.

[0146] See Table 5 for detailed results.

[0147] Step 3: Synthesis of bistrimethylammonium F108 derivative II. 300 mg (2.06 × 10) of water was dissolved in 8 mL of anhydrous DMF in a round-bottom flask under argon. -2 After complete dissolution within a few minutes, DIPEA (Sigma-Aldrich, 60 μL, 3.44 × 10 mmol) was added to the reaction mixture. -1 100 mmol) and methyl iodide (Sigma-Aldrich, 75 μL, 1.21 mmol) are added and the resulting mixture is stirred at room temperature for 48 h. After removal of the solvent, the remaining residue is resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is carried out against 4 liters of double distilled water with 5 medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (290 mg).

[0148] The following Table 9 shows the trimethylation process evaluation: The table gives the number and amount of starting material and the name and amount of the bistrimethylated product, where m is the mass of the compound and n is the number of moles.

[0149] [Table 9]

[0150] Example 3: Synthesis of triphenylphosphonium cationic poloxamer derivatives Example 3 deals with the introduction of a triphenylphosphonium moiety as the terminal group of different hydrophilic unmodified poloxamers. The chemical strategy will result in compounds with the general structure shown below:

[0151] [ka]

[0152] General structure of triphenylphosphonium-grafted cationic poloxamer derivatives

[0153] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 10 below.

[0154] [Table 10] Table 10: Main physicochemical characteristics of the triphenylphosphonium grafted cationic poloxamer derivatives described in this study (T; b: triphenylphosphonium; bromide); XR; A: the cationic end group (XR); the counter anion (A); Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as defined above. HLB stands for hydrophilic / lipophilic balance as previously mentioned.

[0155] To illustrate the synthetic strategy, a detailed procedure to obtain compound III is laid out in FIG.

[0156] Step 1: Synthesis of bisbrominated F127 3a. Before synthesis, 20 g of F127 (Croda) was dissolved in 200 mL of milli-Q double distilled water and placed in a 1000 mL flask. The mixture was then frozen overnight at -80°C and then lyophilized.

[0157] In a 100 mL round-bottom flask, 5.45 g of F127 (0.432 mmol) are dissolved under argon atmosphere. Carbon tetrabromide (Sigma, 2.46 g, 7.42 mmol) and triphenylphosphine (Sigma, 2.13 g, 8.14 mmol) are added simultaneously in three portions over 1 h. The mixture is stirred vigorously at room temperature for 48 h, after which the dichloromethane is removed under vacuum. The resulting solid is then resuspended in 50 mL of Milli-Q water and kept at 4° C. for 12 h. After removing the solid by centrifugation, the title product is isolated without further purification by lyophilization (1.62 g).

[0158] The following Table 11 shows the bromination process evaluation: The table gives the number and amount of starting material and the name and amount of the bis-brominated product, where m is the mass of the compound and n is the number of moles.

[0159] [Table 11]

[0160] Step 2: Synthesis of bisphosphonium F127 derivatives III. 1 g (0.078 mmol) of compound 3a is dissolved in 30 mL of anhydrous DMF. PPh3 (1.6 mmol, 420 mg) is then added under argon and the reaction is heated to 120° C. in an oil bath while maintaining an argon atmosphere. After 36 h of stirring, the reaction is cooled to room temperature and concentrated under vacuum. The resulting syrup is washed several times with some hexane to obtain a solid, which is then dried under high vacuum for 12 h. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double-distilled water with five medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (654 mg).

[0161] The following Table 12 shows the phosphonium incorporation process evaluation: The table gives the number and amount of starting material and the name and amount of the bisphosphonium product, where m is the mass of the compound and n is the number of moles.

[0162] [Table 12] Example 4: Synthesis of bis-1-methyl-5-[(trimethyl-λ5-azanyl)methyl]-1λ5,2,3-triazacyclopenta-1,4-diene (MTAMTD) - a cationic poloxamer derivative

[0163] Example 4 deals with the introduction of 1-methyl-5-[(trimethyl-λ5-azanyl)methyl]-1λ5,2,3-triazacyclopenta-1,4-diene (MTAMTD) moieties as terminal groups on different hydrophilic unmodified poloxamers. The chemical strategy will result in compounds with the following general structure:

[0164] [ka]

[0165] General structure of MTAMTD-grafted cationic poloxamer derivatives

[0166] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 13 below.

[0167] [Table 13] Table 13: Main physicochemical characteristics of the MTAMTD grafted cationic poloxamer derivatives described in this study. XR: cationic end group (XR); counter anion (A) as previously described; Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as previously defined. HLB stands for hydrophilic / lipophilic balance as previously described.

[0168] To illustrate the synthetic strategy, a detailed procedure to obtain compound IV is laid out in FIG.

[0169] Step 2: Synthesis of bis(azide) F108 4a. Compound 2a (7.44 g, 0.5 mmol), described further on in this document, is dissolved in 30 mL of anhydrous DMF under argon. Sodium azide (6 mmol, 390 mg) is then introduced and the mixture is stirred at 90° C. for 3 days. After cooling, the DMF is removed using a rotary evaporator. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is carried out against 4 liters of double-distilled water with 5 medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (6.18 g).

[0170] The following Table 14 shows the azide incorporation process evaluation: the table shows the number and amount of starting material and the name and amount of the bisazide product, m shows the mass of the compound, and n shows the number of moles.

[0171] [Table 14]

[0172] Step 2: Synthesis of bis-1H-triazol-4-ylmethanamine F108 4b. Copper sulfate heptahydrate (VWR, 0.012 mmol, 3 mg), ascorbic acid (Sigma, 0.06 mmol, 12 mg) and triphenylphosphine (0.012 mmol, 3 mg) are mixed in 1 mL of anhydrous DMSO under stirring. Compound 4a (2 g, 0.136 mmol) is dissolved in 4 mL of a 3 / 1 mixture of water / DMSO while stirring this solution (15 min). After complete dissolution of the polymer, the copper-based yellowish solution is added to the reaction flask and stirred with the starting material for 12 h at room temperature. The reaction is then diluted in 10 mL of water and extracted with 4 x 20 mL of DCM. The organic extracts are then combined, dried over magnesium sulfate, filtered and concentrated under vacuum. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). The medium is changed 5 times (2 hours, 8 hours, 24 hours, 36 hours and 48 hours) and dialyzed against 4 liters of double distilled water. The final product is then recovered after lyophilization (1.72 g). This compound is cationic. Therefore, it belongs to the present invention.

[0173] The following Table 15 shows the Huisgen Click reaction process evaluation: This table shows the number and amount of the starting material and the name and amount of the product having a triazole ring, m shows the mass of the compound, and n shows the number of moles.

[0174] [Table 15]

[0175] Step 3: Synthesis of bis-1-methyl-5-[(trimethyl-λ5-azanyl)methyl]-1λ5,2,3-triazacyclopenta-1,4-diene F108 derivative IV

[0176] 600 mg (4.05 × 10) of DMF in 18 mL of anhydrous DMF in a round-bottom flask under argon -2 After complete dissolution within a few minutes, DIPEA (Sigma-Aldrich, 120 μL, 6.88 × 10 mmol) was added to the reaction mixture. -1 100 μL, 2.42 mmol) and methyl iodide (Sigma-Aldrich, 150 μL, 2.42 mmol) are added and the resulting mixture is stirred at room temperature for 48 h. After removal of the solvent, the remaining residue is resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is carried out against 4 liters of double distilled water with 5 medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (501 mg).

[0177] The following Table 16 shows the methylation reaction process evaluation: the table shows the number and amount of the starting material and the name and amount of the product having a methylated triazolium ring, m shows the mass of the compound, and n shows the number of moles.

[0178] [Table 16]

[0179] Example 5: Synthesis of bis-3,4-dimethyl-1H-imidazolium cationic poloxamer derivatives Example 5 deals with the introduction of 3,4-dimethyl-1H-imidazolium moieties as terminal groups of different hydrophilic unmodified poloxamers. The chemical strategy will lead to compounds with the general structure shown below:

[0180] [ka]

[0181] General structure of 3,4-dimethyl-1H-imidazolium-grafted cationic poloxamer derivatives

[0182] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 17 below.

[0183] [Table 17] Table 17: Main physicochemical characteristics of the 3,4-dimethyl-1H-imidazolium grafted cationic poloxamer derivatives described in this study. XR; A: cationic end group (XR); counter anion (A) as previously described. Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as previously defined. HLB stands for hydrophilic / lipophilic balance as previously described. 3,4-dm1Hiz: 3,4-dimethyl-1H-imidazolium; ms: methyl sulfate.

[0184] To illustrate the synthetic strategy, a detailed procedure to obtain compound V is laid out in FIG.

[0185] Step 1: Synthesis of bistosylated F127 5a. Before synthesis, 20 g of F127 (Croda) was dissolved in 200 mL of milli-Q double distilled water and placed in a 1000 mL flask. The mixture was then frozen overnight at -80°C and then lyophilized.

[0186] Compound 5a is obtained starting from 11.7 g of F127 and following the strategy and proportions used in the synthesis of compound 1a, 11.3 g of 5a is obtained.

[0187] See Table 4 for detailed results.

[0188] Step 2: Synthesis of bisamino F127 5b. Compound 5b is obtained by starting from 3g of 5a and following the strategy and ratio used in the synthesis of compound 1b, 2.2g of 5b is obtained. This compound is cationic. Therefore, it belongs to the present invention.

[0189] See Table 5 for detailed results.

[0190] Step 3: Synthesis of bis-methylimidazole F127 5c. Compound 5b (1g, 7.93×10 -2 1000 mmol) is dissolved in 10 mL of anhydrous DMF under argon atmosphere. After complete dissolution, acetaldehyde (TCI, 1.6 mmol, 100 μL) is introduced into the mixture, which is then stirred at room temperature for 3 hours. Potassium carbonate (VWR, 0.47 mmol, 65 mg) and para-toluenesulfonic isocyanide (Sigma, 0.47 mmol, 91 mg) are added in succession and the reaction is kept under stirring at room temperature for 24 hours. After the reaction is over, the solvent is evaporated under vacuum and then coevaporated several times with ethyl acetate. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is carried out against 4 liters of double-distilled water with 5 medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product is then recovered after lyophilization (831 mg). The compound is cationic. Therefore, it belongs to the present invention.

[0191] The following Table 18 shows the Van Reusen cyclization reaction process evaluation: This table shows the number and amount of the starting material and the name and amount of the product having an imidazole ring, m indicates the mass of the compound, and n indicates the number of moles.

[0192] [Table 18]

[0193] Step 4: Synthesis of bismethylimidazolium salt F127 V. Compound 5c (412 mg) is dissolved in 20 mL of anhydrous toluene under argon. The flask is then cooled to 0° C. using an ice bath. Dimethyl sulfate (Sigma, 6.3 mmol, 600 μL) is introduced carefully into the mixture under vigorous stirring so that the temperature inside the flask does not rise above 40° C. After the addition is complete, the reaction is stirred for another 2 days at room temperature, concentrated under vacuum, and then coevaporated several times with diethyl ether and ethyl acetate. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double-distilled water with five medium changes (2 h, 8 h, 24 h, 36 h, and 48 h). The final product is then recovered after lyophilization (274 mg).

[0194] The following Table 19 shows the imidazole methylation process evaluation: the table shows the number and amount of the starting material and the name and amount of the product having a methylated imidazolium ring, m shows the mass of the compound, and n shows the number of moles.

[0195] [Table 19]

[0196] Example 6: Synthesis of bis-arginine-based cationic poloxamer derivatives Example 6 deals with the introduction of an arginine moiety as the terminal group of different hydrophilic unmodified poloxamers. The chemical strategy will result in compounds with the general structure shown below:

[0197] [ka]

[0198] General structure of cationic poloxamers containing arginine moieties

[0199] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 20 below.

[0200] [Table 20] Table 20: Main physicochemical characteristics of the arginine grafted cationic poloxamer derivatives described in this study (A; TFA: arginine / trifluoroacetate); XR; A: the cationic end group (XR); the counter anion (A); Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as defined above. HLB stands for hydrophilic / lipophilic balance as previously described.

[0201] To illustrate the synthetic strategy, a detailed procedure to obtain compound VI is laid out in Scheme IX.

[0202] Step 1: Coupling of 5b and Boc3Arg Boc3Arg(Bachem, 30mg, 6.4×10 -2 Dissolve 15 μL of DIC (9.6 × 10 mmol) in 2 mL of anhydrous DMF. -2 mmol) and HOBt (17 mg, 13 × 10 -1 The mixture is successively added with stirring for 2 hours and 30 minutes. Then, cationic poloxamer 5b (200 mg) is added with stirring to triethylamine (TCI, 4 × 10 -2 The reaction is allowed to proceed for 24 h at room temperature and then concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept overnight at 4° C. and transferred to a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is performed against 4 liters of double distilled water with five medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product 6a is then recovered after lyophilization (198 mg).

[0203] The following Table 21 shows the coupling process evaluation with protected arginine: the table shows the number and amount of starting material and the name and amount of the product having a protected arginine moiety, m shows the mass of the compound, and n shows the number of moles.

[0204] [Table 21]

[0205] Step 2: Synthesis of bis-arginine substituted F127 derivative VI. Compound 6a (198 mg) is dissolved in 4 mL of dichloromethane solution. TFA is then introduced dropwise (VWR, 1 mL). The solution is degassed with argon and then stirred for 2 h. The mixture is then concentrated in vacuum, coevaporated twice with DCM and three times with diethyl ether, and dried overnight under vacuum. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double-distilled water with five medium changes (2 h, 8 h, 24 h, 36 h, and 48 h). The final product is then recovered after lyophilization (194 mg).

[0206] The following Table 22 shows the deprotection step evaluation with protected arginine: The table shows the number and amount of starting material and the name and amount of the product having the deprotected arginine moiety, where m is the mass of the compound and n is the number of moles.

[0207] [Table 22]

[0208] Example 7: Synthesis of bis-histidine-based cationic poloxamer derivatives Example 7 deals with the introduction of a histidine moiety as the terminal group of different hydrophilic unmodified poloxamers. The chemical strategy will result in compounds with the general structure shown below:

[0209] [ka]

[0210] General structure of histidine-based cationic poloxamer derivatives

[0211] The physicochemical characteristics of the related compounds depend on the polymer backbone chosen and are summarized in Table 23 below.

[0212] [Table 23] Table 23: Main physicochemical characteristics of the histidine grafted cationic poloxamer derivatives described in this study. XR; A: cationic end group (XR); counter anion (A) as above. Tag: number belonging to the obtained polymer; a; b: number of PEO (a) and PPO (b) blocks as defined above. HLB stands for hydrophilic / lipophilic balance as previously described.

[0213] To illustrate the synthetic strategy, a detailed procedure to obtain compound VII is laid out in FIG.

[0214] Step 1: Coupling of 5b and Boc2Hist Di-Boc-histidine dicyclohexylammonium salt (Sigma, 34 mg, 6.4 × 10 -2 Dissolve 15 μL of DIC (9.6 × 10 mmol) in 2 mL of anhydrous DMF. -2 mmol) and HOBt (17 mg, 13 × 10 -1 The mixture is successively added with stirring for 2 hours and 30 minutes. Then, cationic poloxamer 5b (200 mg) is added with stirring to triethylamine (TCI, 4 × 10 -2 The reaction is then stirred at room temperature for 24 hours and then concentrated under high vacuum. The resulting syrup is then dissolved in 20 mL of double distilled water, kept at 4° C. overnight and then introduced into a dialysis bag with a 3.5 kDa cut-off (Spectrum Labs Europe). Dialysis is performed against 4 liters of double distilled water with five medium changes (2 h, 8 h, 24 h, 36 h and 48 h). The final product 6a is then recovered after lyophilization (198 mg).

[0215] The following Table 24 shows the coupling step evaluation with protected histidine: the table shows the number and amount of starting material and the name and amount of the product having a protected histidine moiety, m shows the mass of the compound, and n shows the number of moles.

[0216] [Table 24]

[0217] Step 2: Synthesis of bis-histidine substituted F127 derivative VII. Compound 7a (159 mg) is dissolved in 4 mL of dichloromethane solution. TFA is then introduced dropwise (VWR, 1 mL). The solution is degassed with argon and then stirred for 2 h. The mixture is then concentrated in vacuum, coevaporated twice with DCM and three times with diethyl ether, and dried overnight under vacuum. The remaining residue is then resuspended in Milli-Q water and introduced into a dialysis bag with a 3.5 kDa cutoff (Spectrum Labs Europe). Dialysis is performed against 4 liters of double-distilled water with five medium changes (2 h, 8 h, 24 h, 36 h, and 48 h). The final product is then recovered after lyophilization (160 mg).

[0218] The following Table 25 shows the histidine deprotection process evaluation: the table shows the number and amount of starting material and the name and amount of the product having the deprotected histidine moiety, m shows the mass of the compound, and n shows the number of moles.

[0219] [Table 25]

[0220] Example 8: Synthesis of cationic poloxamer coated magnetic nanoparticles. Some of the cationic poloxamer derivatives described in this invention were used to coat negatively or positively charged iron-based magnetic nanoparticles. Thus, after the initial NP synthesis, several coating strategies were considered. All of these involved the resuspension of the ferrofluid (aqueous or ethyl suspension of NPs) in an aqueous polymer suspension. During the initial trials, the inventors considered three main factors: -Cationic end group selection -Properties of the polymer backbone - Concentration of cationic poloxamer solutions Depending on the concentration, large differences in stabilizing behavior between the cationic poloxamers were observed.

[0221] Based on these observations, several experimental strategies were tried simultaneously to find out what is the best way to obtain colloidally stable magnetic NPs coated with cationic poloxamers. So, after the synthesis of the magnetic cores by a co-precipitation strategy well described and known in the art (other synthesis procedures can also be considered), the polymer coating was introduced using different routes depending on the concentration of cationic poloxamer used. We can classify these routes into four categories as "dilute", "moderately concentrated", "highly concentrated" and "very concentrated", corresponding to aqueous solutions of cationic poloxamer coating equal to 0.25, 1, 5 and 10% (weight / volume), respectively, while the iron content was fixed at 0.5 mg of iron per mL of aqueous polymer solution each time.

[0222] Step 1: Synthesis of hydrophobic coated iron-based magnetic cores. Iron-based magnetic cores are obtained by a conventional co-precipitation strategy (Lu, AHet al. Angew. Chem. Int. Ed. 2007, 46:1222-44; Huber, DLSmall 2005, 1:482-501). Iron(III) chloride hexahydrate (Sigma) and iron(II) chloride tetrahydrate (Sigma) are used, both dissolved in Milli-Q water. After filtration, precipitation is carried out under argon atmosphere with mechanical stirring. Iron oxide is precipitated using NH4OH (TCI). Then, a hydrophobic pre-coating is introduced at a temperature of 80°C, while maintaining stirring for 2 hours. Then, after cooling to room temperature, the reaction is transferred to an Erlenmeyer and pseudo-decanted for 12 hours. The supernatant is removed and the remaining solid is washed multiple times with absolute ethanol (4 times 30 mL) and used directly in the next step, suspended in 20 mL of ethanol, as solution A.

[0223] Step 2: Coating of the magnetic cores with a dilute aqueous solution of cationic poloxamer (0.25% (w / v)). Aqueous solutions of the cationic poloxamer of interest are prepared by suspending the polymer in Milli-Q water at a concentration of 100 mg / mL (10% (wt / vol)). Once suspended, the polymer is kept at 4°C for 12 hours with regular stirring to facilitate dissolution and prevent gelation. After complete dissolution, the solution is filtered through a 0.22 μm membrane to obtain what is called the mother liquor.

[0224] Step 3.1: Coating of the magnetic cores with a dilute aqueous solution of cationic poloxamer (0.25% (wt / vol)). To test using the "dilute" condition, 1 mL of the mother liquor is diluted with 37 mL of Milli-Q water to obtain a solution labeled B. For the coating procedure, solution A is titrated using a standard colorimetric method (Jiang C et al.; J Magn Magn Mater. 2017, 439: 126-134) to obtain the total iron concentration of the oleic acid-coated magnetic cores. A volume of solution A containing 0.5 mg of iron is then magnetically decanted onto a magnetic rack (OZ Biosciences, Marseille, France) for 12 h. After removal of the supernatant, the remaining solids are then resuspended in 1 mL of solution B and vortexed vigorously for 5 min. The resulting suspension is then characterized by DLS after sonication (Branson apparatus, power 3, 70% duty factor, 10 min sonication).

[0225] Step 3.2: Coating of the magnetic cores with a moderately concentrated aqueous solution (1% (wt / vol)) of cationic poloxamer. The previous steps 1 and 2 are repeated and solution A is titrated as described above. Meanwhile, 100 μL of the polymer aqueous mother liquor is diluted in 900 μL of Milli-Q water to obtain solution C (cationic poloxamer concentration is 1% (w / v)). A volume of solution A containing 0.5 mg of iron is then magnetically decanted on a magnetic rack (OZ Biosciences, Marseille, France) for 12 hours. Then, after removal of the supernatant, the remaining solid is resuspended in 1 mL of solution C and vortexed vigorously for 5 minutes. The resulting suspension is then sonicated (Branson apparatus, power 3, 70% duty factor, 10 minutes sonication) and then characterized by DLS.

[0226] Step 3.3: Coating of the magnetic cores with a highly concentrated aqueous solution of cationic poloxamer (5% (wt / vol)). The previous steps 1, 2 and 3.2 are repeated and solution A is titrated as described above. Meanwhile, 500 μL of the polymer aqueous mother liquor is diluted in 500 μL of Milli-Q water to obtain solution D (the cationic poloxamer concentration is 5% (w / v)). The NPs resuspended in 1% (w / v) cationic poloxamer (see step 3.2) are then magnetically decanted again for 12 h on a magnetic rack. Then, after removal of the supernatant, the remaining solids are resuspended in 1 mL of solution D and vortexed vigorously for 5 min. The resulting suspension is then sonicated (Branson apparatus, power 4, 80% duty factor, 10 min sonication). After cooling to room temperature, the NPs are characterized by DLS after a second sonication cycle.

[0227] Step 3.4: Coating of the magnetic cores with a highly concentrated aqueous solution of cationic poloxamer (10% (w / v)). The previous steps 1, 2, 3.2 and 3.3 are repeated and solution A is titrated as described above. The NPs resuspended in 5% (wt / vol) cationic poloxamer (see step 3.3) are then magnetically decanted again on a magnetic rack for 12 h. After removal of the supernatant, the remaining solids are then resuspended in 1 mL of cationic poloxamer mother liquor and vortexed vigorously for 5 min. The resulting suspension is then sonicated (Branson apparatus, power 4, 80% duty factor, 10 min sonication). After cooling to room temperature, a second sonication cycle is performed, then a third, after which the NPs are characterized by DLS.

[0228] Biological evaluation of modified cationic poloxamers Materials and methods for transduction experiments Cationic poloxamer formulation in water An aqueous solution of the cationic poloxamer of interest is prepared by suspending the polymer in Milli-Q water at a concentration of 100 mg / mL (10% (w / v)). Once suspended, the polymer is kept at 4° C. for 12 hours with regular stirring to facilitate dissolution and prevent gelation. After complete dissolution, the solution is filtered through a 0.22 μm membrane and is ready for immediate use in transduction.

[0229] For adenovirus or AAV mediated experiments, a 250 mg / mL (25% weight / volume) cationic poloxamer solution is prepared following the same general guidelines for storing liquid mixtures.

[0230] Cells and viruses Human cervical carcinoma (HeLa), human lung cancer (HEK-293T), adult mouse hypothalamic cell line (CLU-500), mouse fibroblast (NIH-3T3), immortalized mouse microglial cell line (BV2), and rat glioma (C6) cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBD, St. Louis, MO, USA), 2 mM final L-glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin (Lonza, Walkersville, MD, USA). Human immortalized Jurkat T cells were cultured in Roswell Park Memorial Institute medium (RPMI, Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBD, St. Louis, MO, USA), 2 mM final L-glutamine, 100 units / ml penicillin and 100 μg / ml streptomycin (Lonza, Walkersville, MD, USA). CD34+ KG-1a cell lines were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Lonza, Walkersville, MD, USA) supplemented with 20% fetal bovine serum. Primary CD34+ stem cells were isolated from umbilical cord blood and cultured in P6-well plates, and all cell lines were cultured in a humidified incubator (Sanyo Electric Co., Ltd., Tokyo, Japan) at 37°C with a 5% CO2 atmosphere at 75 cm 2Cells were cultured in flasks (Sarstedt AG & Co., Germany) and trypsinized at 80% confluence to maintain exponential division rates prior to transduction assays. Transduction experiments were performed in 24-well plates (Sarstedt AG & Co., Germany).

[0231] Preparation of lentivirus / cationic poloxamer solution for infection HIV-1 vectors encoding a green fluorescent protein (GFP) reporter gene under the control of SFFV (HIV-SFFV-GFP) were produced at the SFR Biosciences Facility (UMS3444 / CNRS, US8 / INSERM, ENS de Lyon, UCBL, France). Viral suspensions were collected from cell supernatants, aliquoted without a concentration step, and stored at -150°C (Sanyo Electric Co., Tokyo, Japan). Prior to each experiment, viral stocks were thawed and diluted at the desired multiplicity of infection (MOI) in 50 μL of complete medium. After addition of cationic poloxamer, the suspension was mixed by inverting the tube and added dropwise directly onto the cells. The cells were then incubated under conventional conditions for 72 h until experimental evaluation.

[0232] Adenovirus and adeno-associated virus infections Type 5 replication-deficient adenovirus encoding GFP (Ad-GFP) and AAV serotype A virus, both under the control of the cytomegalovirus (CMV) promoter, were purchased from Vector Biolabs. 11 1x10 corresponding to VP / ml 10 Ad-GFP aliquots of IFU / ml virus stock were extemporaneously diluted in medium without nutrient supplements to achieve the desired MOI. AAV-1 was diluted in medium without nutrient supplements for a final MOI of 100,000 genome copies per cell. If necessary, cationic poloxamer was mixed with the virus particles and added dropwise onto the cells. The cells were then incubated under conventional conditions for 72 hours until experimental evaluation.

[0233] Flow cytometry. Cells were washed twice with calcium- and magnesium-free phosphate-buffered saline (PBS, Lonza, Walkersville, MD, USA), and adherent cells were detached with trypsin / EDTA 0.2% solution (Lonza, Walkersville, MD, USA) and then fixed in 200 μL of 4% PFA (Sigma Aldrich). Cells were then analyzed for % GFP-positive cells and fluorescence intensity by flow cytometry using a CytoFlex flow cytometer (Beckman Coulter, Miami, FL, USA).

[0234] Evaluation of the use of modified cationic poloxamers for lentivirus-based chemical transduction in transduction experiments As described herein, the main objective of the present invention is to develop novel methods using chemical agents that can efficiently and reliably improve the infectious behavior of viruses. The first part of the results focuses on the use of lentiviruses as viral vectors, as they are described as strong candidates for cell therapy clinical trials. For this evaluation, the inventors used a mouse fibroblast cell line, NIH-3T3; the cationic poloxamers described in the examples were combined with an HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene. This first evaluation described modified cationic poloxamers derived from F87, F127 and F108.

[0235] NIH-3T3 cell lines were infected with lentivirus (MOI 1) in the presence or absence of various doses of cationic poloxamer. After 72 hours of incubation, the percentage of GFP-positive cells (A) and the mean intensity of transduced cells (B) were assessed by flow cytometry. The results are depicted in FIG. 11.

[0236] The biological evaluation of cationic poloxamers as transduction enhancers has highlighted their favorable properties in such experiments. Compounds I, 2b, II and VI show a net increase in the number of infected cells compared to the viral vector used without adjuvant at the same MOI, up to a 6-fold increase when using 10 μL of compound II. This increase can also be seen to a lesser extent when comparing the average intensity of different experiments.

[0237] For the compounds showing a favorable effect, a clear dose-dependence is noteworthy, with a dramatic increase in the number of infected cells associated with increasing amounts of cationic poloxamer. This dose effect is less visible when considering the average potency, with clear increases only being observed when compounds 2b and VI are selected.

[0238] On the other hand, compound III did not show a favorable effect on transduction when used under the same experimental conditions. The number of infected cells remains at the same level as lentivirus alone, regardless of the dose of cationic poloxamer used. However, it is important to note that compound III does not inhibit the behavior of the virus or affect its infection technique.

[0239] The difference in behavior between compounds III and VI, both based on the F127 scaffold, is the first evidence of the tremendous importance of the nature of the end group on transduction efficiency.

[0240] A second set of potent adjuvants was evaluated following the same experimental design as above, and the results are summarized in FIG.

[0241] This evaluation of different cationic poloxamer candidates gives similar results to the previous one: in fact, some of the modifications clearly have a favorable impact on transduction efficiency, whereas others do not seem to bring about any improvement.

[0242] In the case of the tetrazole-based compounds 4b and IV, IV alone leads to an improvement in transduction efficiency in terms of both the number of infected cells and the average GFP intensity. A dose response can also be clearly seen, highlighting the favorable influence of the adjuvant. At the same time, the use of unsubstituted tetrazole groups to modify the cationic poloxamer did not lead to the same level of enhancement. In fact, when 4b is used as a transduction enhancer, the number of transduced cells remains indeed close to the level achieved with lentivirus alone.

[0243] Both imidazole-based compounds 5c and V show favorable effects in transduction in combination with lentivirus. In any case, the number of cells infected with the virus depends on the amount of modified cationic poloxamer used. However, the improvement is much more pronounced with compound V, showing about 4 times more transduced cells than the virus alone. In this case, the methylation of the heteroatom ring leads to better behavior as an adjuvant, with compound V being more efficient than its counterpart 5c.

[0244] The screen was finally completed by a final round of transduction experiments, this time involving the use of lentivirus to infect HEK-293 and NIH-3T3. Here again the results demonstrated differences in behavior among the different poloxamers, as shown in Table 26.

[0245] [Table 26] Table 26: Differential transduction efficacy of cationic poloxamers depending on MOI and cell type. NIH-3T3 and HEK293 cell lines were infected with lentivirus encoding GFP protein at MOI of 0.5 and 1 in the presence or absence of 5 or 10 μL of cationic poloxamer. 72 hours after infection, the % of GFP positive cells was analyzed by flow cytometry.

[0246] For example, in the case of NIH-3T3, all cationic poloxamers shown in this table induce a favorable effect on the amount of transduced cells. Compound XXIX, based on the F87 backbone, is clearly efficient and is able to increase the number of infected cells by a factor of 6 at a lentiviral MOI of 0.5 (13.32% of infected cells vs. 2.5% for lentivirus used alone), while at an MOI of 1, the number of infected cells increases by a factor of more than 10 when compared to the lentiviral control. Moreover, a clear dose effect can be seen using these conditions, since when doubling the amount of poloxamer used with the same amount of virus, the number of transduced cells goes from 12.7% to more than 50%.

[0247] This poloxamer behaves similarly on HEK-293 cells, even though no effect can be seen due to the greater permissiveness of these cells to lentiviral infection. At an MOI of 0.5, compound XXIX allows the observation of a number of infected cells that goes from 23% to 37% when 10 μL of adjuvant is added in the experiment. An increase in the amount of virus does not change the favorable trend brought about by the use of this compound. At an MOI of 1, the addition of compound XXIX brings the number of infected cells to 42-50%. These results are a clear illustration of one of the main advantages of the present invention, which is to use less virus to obtain a similar amount of favorably transduced cells.

[0248] Compounds 5b, VI and IX, all based on the F127 backbone, were also included in the present experiment, and three of them show favorable results for both cell lines when combined with various amounts of lentivirus. However, these efficiencies are lower than those observed with compound XXIX used simultaneously. In fact, the greatest enhancement is observed when using compound VI for NIH-3T3 cells, and a 5-fold enhancement is observed at an MOI of 1 when using 10 μL of poloxamer for virus alone. For HEK-293, the three compounds provide similar enhancement factors, providing about 10% to 15% efficacy in transducing small cells compared to lentivirus used alone, whatever the MOI used.

[0249] An interesting observation can be made when comparing the results observed with compounds XXIX and IX, which are based on the F87 and F127 scaffolds, respectively, although both are modified to contain a quaternary trimethylammonium salt as a terminal group.

[0250] In each of these experiments, compound XXIX provides transduction with enhanced behavior that is almost always better than that observed with compound IX, and when NIH-3T3 is used as the cell, this difference is greatest with lentivirus used at an MOI of 0.5 (3-fold enhancement with compound IX vs. 6-fold enhancement with compound XXIX). This observation, which correlates with other results observed in the current request, fully emphasizes the importance of finding a good balance between the selection of the polymer backbone and the selection of the cationic end group to achieve optimal efficiency. The observation of these data is clear, as it is not possible to adopt predictable behavior, and it is not possible to select either the best backbone or the best end group to achieve a perfect balance.

[0251] Figures 13A-F highlight this point by showing the results of several transduction experiments combining the use of lentivirus as the viral vector for HEK-293T cells with a broad range of modified poloxamers as transduction adjuvants.

[0252] The poloxamers used in this diagram are categorized according to the poloxamer backbone used for modification.

[0253] As a result, Figures 13A and 13B show the results of experiments using lentiviral infection on HEK-293T cells containing F87-based cationic poloxamers, i.e. compounds 1b and XXIX, which contain a primary amine and a quaternary trimethylammonium as terminal groups, respectively, both of which show a favorable effect on the number of infected cells, achieving a 10% effect when compared to conditions using virus alone.

[0254] Figures 13C and 13D highlight the behavior of poloxamers based on the F108 backbone, in this case comparing a wide range of modifications. All compounds tested induced a favorable effect on the infection rate, with compounds 2b and VIII obtaining the best results. In these conditions, almost 100% of the cells were transduced, compared to only 75% of the infected cells when using lentivirus alone. Compounds II, XX and XIV also showed a globally favorable effect. When considering the average fluorescence intensity, the trend is clear, with compounds 2b, II and VIII providing the best infection enhancement. Compound 2b allows a fluorescence intensity that reaches 150% of the value observed with the virus alone, while in the case of II, the effect is 120%. On the other hand, poloxamers XX and XIV, which have arginine and histidine, respectively, as terminal groups, did not reach high levels of fluorescence intensity, which would be expected when considering the number of infected cells. This is further evidence of the tremendous importance of the choice of cationic modification and how several read-outs must be considered at the same time to effectively evaluate the efficacy of a cationic modification in enhancing the transduction procedure.

[0255] Figures 13E and 13F feature the same experiment with poloxamers based on the F127 backbone with different cationic modifications. In Figure 13E, the number of transduced cells obtained with each poloxamer used as an adjuvant is compared to that obtained with the lentiviral vector alone. Here again, the results are favorable, with compounds 5b, XV, IX, and VII showing clear effects when used to enhance infection rates. Compound XV, which contains a spermine residue as a terminal group, appears to be a more efficient candidate, allowing the ratio of transduced cells to reach 90% at the end of the experiment. A similar beneficial effect is observed with compound VII, which has a cationic histidine.

[0256] Observing the average fluorescence intensity for this experiment allowed us to easily identify the polymers being evaluated. Compounds XV and VII, which are the most promising adjuvants when looking at the number of infected cells when combined with lentivirus, remained the most effective enhancers, since both allowed us to observe a higher average intensity than that observed with the virus alone. It is clear that compound XV is the most efficient overall when dealing with the number of infected cells, since both provided similar enhancements, but its association with the viral vector gives a 40% increase in intensity compared to only 3% for compound VII. Furthermore, we can observe that compounds 5b and IX, which are promising in terms of the number of infected cells, are not efficient when we look at the average fluorescence intensity.

[0257] This entire set of experiments provides interesting insights into the selection of the best cationic poloxamer to efficiently enhance transduction experiments. - First, the choice of the polymer scaffold that will serve as the modification platform is obviously important, since different poloxamers provide different responses in transduction, but these effects are unpredictable, since no studies provide a comparison of all the poloxamers available as transduction enhancers. - Secondly, the entire experiment should be viewed with several reads to closely screen all possible candidates. - First, cationic modifications show a key role in the overall efficiency of the experiment. However, Figure 13 clearly highlights the fact that a particular cationic functional group will not give the same results in transduction when combined with different poloxamer backbones, preventing scientists from predicting the enhancement capacity of the polymers of the invention simply based on the choice of cationic end group. This can be seen when comparing adjuvants 1b, 2b, 5b, based on poloxamers F87, F108 and F127, respectively, all bearing primary amine functional groups as end groups. From Figure 13, it is clear that primary amine end groups benefit transduction efficiency when combined with the F108 backbone. On the other hand, the favorable effect observed with compound XV attests to the effect of combining spermine residues with the F127 backbone, while the lower efficiency of compound VIII bearing spermine end groups rules out cationic modifications to the F108-based polymers.

[0258] Overall, only a careful screening of the maximum number of modifications that meet this requirement will allow one skilled in the art to find the most effective adjuvant candidate of this experiment, which should combine several poloxamer backbones with several cationic modifications that meet this requirement.

[0259] Modification matters: Unmodified and cationic poloxamers as adjuvants in lentivirus-mediated transduction experiments The innovative feature of the present invention is based on the cationic modification of the end groups of poloxamers to enhance these adjuvant techniques in transduction experiments. The biological activity of the modified cationic poloxamers with their unmodified counterparts was then compared. Thus, the inventors used compounds 2b and II as modified cationic poloxamers and compared them with Pluronic F108 with or without polybrene adjuvant following a well-described procedure.

[0260] Two cell lines were tested: human embryonic kidney (HEK-293T) and an immortalized mouse microglial cell line (BV2). Cationic poloxamers were combined with an HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene. The results are shown in FIG.

[0261] The results indicated that the three cationic poloxamers efficiently enhanced transduction in both cell lines. F108 in combination with cationic polymer, polybrene, doubled the number of BV2 cells infected by the virus compared to the virus alone. More interestingly, the introduction of cationic end groups into the F108 backbone had a net positive effect on the number of positive GFP cells, which went from 18% when using F108 + polybrene as adjuvant to 26% for compounds 2b and II. On the other hand, the average fluorescence intensity remained unaffected whatever the polymer used. In the case of HEK-293T cells, the positive effect of the use of cationic poloxamers on transduction is also clearly noteworthy. Unmodified and modified cationic poloxamers provide the same enhancement on the number of infected cells. This number goes from about 40% when using LV alone to about 60% when using cationic poloxamer as enhancer. However, the average fluorescence intensity is clearly enhanced by introducing cationic end groups onto the adjuvant. In fact, for both 2b and II, this value is higher compared to F108, gaining almost a third after cationic modification. Altogether, these results clearly demonstrate the effect of cationic modification of the poloxamer backbone on different parameters regarding the global efficiency of the procedure.

[0262] Moreover, it is clear from the previous evaluation that the nature of the cationic functional group introduced has biological importance for the success of the enhanced transduction experiments. As a result, we compared the transduction efficiency of different cationic poloxamers based on the F108 backbone but modified with different polymeric functional groups. Compounds 2b, 4b and IV were evaluated simultaneously on human Jurkat T cells. The cationic poloxamers were combined with an HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene. As before, F108 was used as a control. The results are shown in Figure 15.

[0263] The results confirmed that compound 2b again obtained better results than its unmodified counterpart F108. In fact, both cationic poloxamers induce 20% more cells to be infected with the viral vector, but the cationic modification induces an average increase in intensity of 20% relative to F108. On the other hand, the results with compounds 4b and IV are less efficient. On Jurkat cells, both of them induce only a slight increase in the number of transduced cells compared to the viral vector used alone, and no significant effect was observed in terms of average intensity compared to the virus alone. More importantly, the results observed with compounds 4b and IV are lower than the unmodified cationic poloxamer, highlighting the critical need to design appropriate cationic modifications.

[0264] By establishing that the behavior of cationic poloxamers in transduction experiments depends on the nature of the cationic end group introduced, this experiment supports the fact that careful selection of the modification is key to the success of such procedures. In fact, the introduction of cationic groups is not sufficient to improve transduction over virus alone, even over the same reaction assisted by unmodified poloxamer.

[0265] Transduction optimization: Cell screening and dose response To achieve optimal transduction efficiency, we monitored various parameters such as cationic poloxamer amount, virus titer, cell type, etc. A cationic poloxamer dose-response study was performed using the KG-1a cell line, an immortalized human myeloid acute myeloid leukemia cell that has proven very difficult to infect using classical lentivirus-mediated transduction procedures. Different doses of cationic poloxamer were combined with an HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene. The results are shown in Figure 16.

[0266] The results again highlight the favorable effect of the use of compounds 2b and II in this type of transduction procedure. In fact, lentivirus-mediated transduction alone does not obtain more than 2% of infected cells at an MOI of 2 after 72 hours of incubation, while the use of cationic poloxamer increases this level to values ​​approaching 15% at the same MOI. The results show that in both cases, the dose-response effect is really close between 2b and II, with increasing amounts of adjuvant added.

[0267] This positive effect is confirmed by the analysis of the mean intensity of the fluorescence. With both compounds, this value increases impressively compared to the use of the viral vector alone. The best observed results are obtained when 10 μL of compound 2b is used as additive, with a 9-fold increase compared to the control. The dose-response profile differs slightly between 2b and II, when the first reaches its maximum value at a value of 10 μL, but compound II induces a constant increase in the mean intensity between 2 μL and 20 μL, without reaching a plateau like 2b.

[0268] In conclusion, both cationic poloxamers showed a dose-dependent positive effect on lentivirus-mediated transduction. Despite their structural similarity, compounds 2b and II showed slight behavioral differences depending on the dose added, which again highlights the importance of the choice of cationic functional group versus the global efficiency of the transduction procedure.

[0269] The next experiments had to be carried out to enhance the efficiency of the lentivirus-mediated transduction procedure using a wide variety of cell types, with the aim of validating the performance of the cationic poloxamer.

[0270] Compound 2b was tested against six different cell lines: human cervical cancer (HeLa), adult mouse hypothalamic cell line (CLU-500), immortalized mouse microglial cell line (BV2), rat glioma (C6) cell line, human immortalized Jurkat T cells, and CD34+ KG-1a human myeloid acute myeloid leukemia cell line. An HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene was used as the viral vector, and an optimized MOI for each cell line was selected to highlight the cationic poloxamer effect. The results are shown in Figure 17.

[0271] This test on various cells clearly demonstrates the consistency of cationic poloxamers used as adjuvants in lentivirus-mediated transduction experiments. In fact, whatever the cell line tested, the introduction of compound 2b always results in a clear increase in the number of infected cells. This ranges from an increase in the number of infected cells of about 10% compared to the virus alone (Jurkat) to a 650% effect of KG1-a, again depending on the cell line used. The uniformity of these excellent results can also be observed when looking at the average fluorescence intensity. Also in this reading, the use of cationic poloxamers as adjuvants significantly enhances the results obtained with lentivirus alone in the cell lines tested, and this enhancement is highly cell-dependent. When considering the BV-2 cell line, the effect of the intensity is only a factor 1.5, whereas the transduction of KG1-a with compound 2b is more than 2.5-fold compared to the virus alone. In conclusion of this series of experiments, the effect of cationic modification of the poloxamer backbone as an enhancer for lentivirus-mediated transduction experiments has been clearly demonstrated. Cationic modifications of poloxamers affect their adjuvant properties, resulting in a panel of adjuvants that are well suited to the requirements in gene therapy applications.

[0272] Finally, the efficiency of cationic poloxamers in enhancing lentivirus-induced transduction was confirmed in primary human CD34+ stem cells released from umbilical cord blood. A dose-response study assessed the ability of cationic poloxamers to infect primary human CD34+ cells with an HIV-1 vector encoding a green fluorescent protein (GFP) reporter gene. The results are shown in Figure 18 and show the favorable effect of using compounds 2b and II, which increase the transduction efficiency to 35% at an MOI of 5 after 72 hours of incubation, compared to lentivirus alone, which gives about 7% of infected cells at the same MOI of 5 after 72 hours of incubation.

[0273] As previously observed, cationic poloxamers were also able to dramatically improve the mean intensity of fluorescence when compared to the use of viral vectors alone, with the best observed results being a 5-fold increase compared to the control when 20 μL of compound 2b was used as an additive.

[0274] In conclusion, these results demonstrate the ability of cationic poloxamers to enhance lentivirus-mediated transduction of primary CD34+ stem cells in a dose-dependent manner.

[0275] Use of cationic modified poloxamers as adjuvants in adenovirus-mediated transduction experiments. To extend the scope of the present invention, the effect of cationic poloxamers as efficient adjuvants in adenovirus-mediated transduction experiments was also investigated. As described in the introduction, unmodified poloxamers were used as adjuvants in adenovirus-mediated transduction experiments, but very high concentrations were required. In these conditions, the polymer acts as a "pseudogel" agent that reduces the diffusion of viral particles in the biological medium. The main result is that the virus diffuses less and is more likely to be in contact longer with the cells to be infected. This prolonged contact mechanically increases the uptake of the virus and therefore the efficiency of the entire procedure. It was possible to formulate cationic poloxamer 2b at a concentration of 25% (wt / vol), close to its gelation point, and the cationic modification did not inhibit the "gel" behavior observed with the corresponding unmodified poloxamer. The main objective of this experiment was to check the pseudogel efficiency of modified cationic poloxamers and also to monitor whether cationic modification leads to a complement of the efficiency for the adenovirus-mediated transduction procedure when lentiviruses are used.

[0276] To do so, C6 and HEK-293 cell lines were transduced with adenovirus encoding GFP (AdGFP) in the presence or absence of commercial F108 and its cationic counterpart 2b at a final concentration of 7.5% (weight / volume) with an MOI of 5. The results of this experiment are shown in FIG.

[0277] This experiment clearly demonstrates the effect of using modified cationic poloxamers as transduction adjuvants in combination with adenovirus. In fact, adenovirus used alone on either HEK-293 or C6 cells was unable to infect 5% of the cells. On the other hand, cationic modification results in a significant enhancement of adenovirus transduction efficiency. In fact, when compound 2b is used against its unmodified counterpart, the number of infected cells increases from 46% to 55% for C6 cells and from 32% to 46% for HEK293 cells. When considering the average fluorescence intensity, which almost doubles with compound 2b for both cell lines, the favorable effect of cationic modification is even more outstanding. This experiment demonstrates that cationic modification of poloxamers opens a wide range of applications for virus-mediated transduction experiments and that the observed improvement is not limited to lentiviruses.

[0278] To confirm these favorable results, this experiment was further extended with several other poloxamers based on both the F108 or F127 backbone. The main results are shown in the following table:

[0279] [Table 27] Table 27A and B: Cationic poloxamer difference in adenovirus infection and transduction enhancement. C6 and HEK293 cell lines were infected with adenovirus encoding GFP protein at an MOI of 4 in the presence or absence of cationic poloxamer (final concentration 7.5%). 72 hours after infection, the % of GFP positive cells (top table) and the mean intensity (bottom table) were analyzed by flow cytometry.

[0280] In the case of F108 derivatives, we monitored the number of transduced cells relative to the virus alone. On C6 cells, all tested poloxamers clearly show an effect on infected cells ranging from a 10-fold increase for compound XXX to a 50-fold increase for compound 2b. This one also shows a better adjuvant effect than the unmodified poloxamer F108, which is less than 10% efficient.

[0281] The results observed in HEK-293 are similar, with all compounds showing clear effects when compared to the virus alone. Here again, poloxamer 2b demonstrates once again the best potentiation potential, being more efficient than its unmodified counterpart. These results are another proof of the favorable impact brought about by the cationic modification presented in this invention.

[0282] F127 derivatives were evaluated by monitoring by fluorescence intensity, with adenovirus as a control. Between the two selected compounds III and 5b, the latter shows an almost two-fold increase in fluorescence intensity against adenovirus alone, but against the two selected cell lines. Furthermore, unmodified F127, which was tested simultaneously in the experiment, did not show a favorable effect when combined with adenovirus, again validating our cationic modification strategy. Finally, it is interesting to note that compound 5b, which is not optimally efficient when combined with lentivirus, proved to be a powerful adjuvant when combined with adenovirus.

[0283] This round of experiments was completed by testing the effect of using serum in combination with poloxamer in the adenovirus-mediated transduction procedure. NIH-3T3 cells were transduced with adenovirus and poloxamer II at different concentrations with or without serum. The results are shown in Figure 20.

[0284] The results highlight the possibility that the addition of serum may have a favorable effect of cationic poloxamer on the efficiency of adenovirus-mediated transduction. In fact, when using poloxamer at a concentration of 12.5%, the presence of serum allows a three-fold increase in the efficiency of transduction, but the effect is less visible. At a concentration of 6.5% of compound II, the effect of serum is still visible, with a two-fold increase to a barely three-fold increase when serum is used. These results provide evidence of the full compatibility between the use of serum and compounds of the present invention, making them compatible with procedures routinely used in cell biology processes.

[0285] Use of cationic poloxamers as adjuvants in AAV-mediated transduction experiments. To further expand the scope of the present invention, the effect of cationic poloxamer was further tested as an effective adjuvant in AAV-mediated transduction experiments. Compound II at a final concentration of 15% (weight / volume) was tested in combination with AAV (GFP) on HEK-293 cells. The results of this experiment are summarized in Figure 21.

[0286] Again in this procedure, the use of modified cationic poloxamers proved to be a real source of enhancement of the efficiency of the transduction procedure. The addition of 15% (weight / volume) compound II allows AAV to infect 57% of the cells, whereas AAV used alone only infects 12%. When considering the average potency, the improvement is less significant, nevertheless a net increase of 5% compared to the virus alone. In the end, this experiment demonstrated that cationic poloxamers are both useful and effective as adjuvants in AAV-mediated transduction experiments.

[0287] Poloxamers 5b and 2b were also tested in these experimental conditions. They were combined with AAV to infect HEK-293 at different concentrations. The results are shown in FIG. 22.

[0288] Compound 5b exhibits similar adjuvant behavior compared to compound II, with a net increase in the number of transduced cells of almost 60% when poloxamer is used at a concentration of 15%. It is interesting to note that compound II exhibits its maximum enhancement factor at a concentration of 7.5%, highlighting the clear difference in adjuvant behavior. The effect produced by compound 2b, which is based on F108 like compound II, is less excellent, but remains very favorable when compared to the virus alone, with more than 40% of the cells transduced at the end of the experiment.

[0289] When considering the average fluorescence intensity, compounds 2b and 5b were less efficient than compound II. Overall, this set of experiments clearly confirmed the ability of cationic poloxamers to effectively enhance AAV-based transduction experiments.

[0290] Furthermore, the value of many of the methods described herein is clearly demonstrated by the use of several types of viruses in combination with a wide range of different cationic poloxamers. The cationic modification of poloxamers is undoubtedly a step towards further increased efficiency in transduction procedures. Some compounds have proven to be particularly efficient when combined with one type of viral vector, while some polymers appear to be more all-around enhancers.

[0291] Cationic poloxamer and magnetic nanoparticle formulations: DLS characterization of cationic poloxamer coated MNPs As mentioned above, magnetic nanoparticles (MNPs) have been demonstrated to be powerful for boosting, accelerating and synchronizing viral infection. As a result, it can be foreseen that the combination of magnetic nanoparticles and unmodified poloxamers will lead to a strong synergistic effect. Unfortunately, the non-ionic properties of unmodified poloxamers lead to nanoparticle destabilization or precipitation, which hinders their use as nanoparticle coating agents or formulations.

[0292] To investigate the effect of poloxamer cationic modification on magnetic nanoparticle formulation and stabilization, we first focused on DLS (Dynamic Light Scattering) to measure the size and charge of the resulting nanoparticles. Size and net charge (zeta potential) measurements are currently key parameters to check when focusing on MNP synthesis and characterization. In fact, a stable colloidal suspension is primarily characterized by its propensity to maintain magnetic objects in a non-aggregated state. That is, it is crucial that the nanoparticles present in the solution retain their nanometric size. This is even more important if this colloidal suspension is intended to have biological applications, since aggregated objects have proven in the past to have disastrous consequences for cell viability. Zeta potential is a direct electrical consequence of the presence of charged chemical functional groups at the surface of nanometric objects. Thus, due to electrostatic repulsion that limits the propensity of the magnetic core to stack into micrometric objects, a suspension consisting of charged nanoparticles (either positive or negative) is less likely to evidence aggregation of its components. Furthermore, the net positive charge allows these beads to form stable complexes with negatively charged viruses (such as many lentiviruses or adenoviruses), which appears to be a favorable step in magnetically induced transduction experiments.

[0293] The lack of charged chemical functional groups in the native unmodified poloxamer backbone prohibits repulsive interactions when combined with magnetic beads, resulting in destabilization and / or aggregation of these nanoparticles. One objective of the present invention was to demonstrate that cationic modified polymers are capable of stabilizing magnetic MNPs by creating electrostatic repulsive interactions and providing the synthesized nanometric objects with sufficient positive charges on their surface that can maintain colloidal suspension and allow the achievement of enhanced viral transduction. As a result, the resulting MNPs will stack several features useful for efficient magnetic NP-induced transduction experiments: - Ability to bind viruses on the NP surface according to Magnetofection™ requirements - The presence of cationic poloxamer in the complex which acts as an adjuvant during transduction - Stabilization of magnetic NPs to avoid the fibrosis of micrometric objects detrimental to cell viability.

[0294] Following these guidelines, several magnetic nanoparticles were synthesized using a well-described co-precipitation strategy. The resulting iron core consists mainly of magnetic crystals that give the object its strong magnetism. The strategy involves the use of a pre-coating molecule before the introduction of the cationic poloxamer. This pre-coating molecule must be negatively or positively charged to stabilize the magnetic core and may contain hydrophobic regions that would be useful for binding the NP surface with the cationic poloxamer by hydrophobic interactions. Examples of pre-coatings include a wide range of well-known molecules such as dextran, starch, Zonyl FSA, linoleic acid, oleic acid, silicon oxide polymers, polyethyleneimine, chitosan, Tween, Span, etc. In the following studies, we chose to use an oleic acid-based pre-coating.

[0295] The MNPs were resuspended using different unmodified or cationically modified poloxamer solutions, while the amount of iron was kept constant.

[0296] Coating of magnetic cores with dilute and concentrated aqueous solutions of cationic poloxamer.

[0297] The MNPs were first resuspended with a 0.25% (w / v) cationic polymer solution, and then, after further decantation, the MNPs were formulated in the corresponding 1% (w / v) cationic poloxamer solution. This intermediate dilution decantation step appeared to be a reliable solution to avoid direct aggregation of the resulting MNPs. Only the NPs suspended in the 1% (w / v) solution were evaluated by DLS.

[0298] A wide range of modified polymers were tested to find out whether other interactions such as those provided by cationic end groups (hydrophobic, π / π...) could be advantageous over short (5 days) and long (1 month) incubations. The size measurements are shown below in Table 28:

[0299] [Table 28] Table 28: Size measurements of magnetic NPs resuspended in various 1% (wt / vol) cationic poloxamer solutions. Sizes are expressed in nm and were recorded at two different time points ("average time" of 5 days (d), 1 month) after resuspension in cationic poloxamer solution. The "Agg" tag indicates possible aggregation of beads, with no nanometric objects remaining that could have been measured.

[0300] This experiment highlights the inability to stably formulate or coat magnetic NPs with dilute solutions of native (non-ionic) or unmodified poloxamers: in fact, this leads to rapid, systematic and irreversible aggregation of the beads, whatever the polymer chosen, either F108 or F127.

[0301] In the case of MNPs coated or formulated with cationic F108 derivatives (2b, II, 4b, XXX and XIV), all cationic poloxamers obtain colloidally stable nanoparticles after 5 days after resuspension. Amine (2b), triazole derivative (4b) and histidine (XIV) seem to be the most promising candidates as stabilizers for MNPs resulting in MNPs with a size in the full range for transduction applications, i.e., between 200 and 300 nm. This result was confirmed after 1 month of fabrication. When compared to non-coated NPs, the introduction of cationic polymers at the NP surface induces a small increase in the overall size of the constructs with a distribution peak that remains thin after the redispersion / sonication procedure, highlighting the binding of the cationic polymer and the bead surface.

[0302] However, despite these cationic modifications, some cationic poloxamers, such as II, showed characteristic behavior of aggregation that was not complete after 5 days (size of 677 nm) but was complete after 1 month. MNPs formulated with various cationic poloxamers based on the F127 backbone also showed to be powerful stabilizers for magnetic nanoparticles (see Table 29); one cationic poloxamer (VII) led to a stable MNP formulation even after 1 month.

[0303] In summary, it was not possible to formulate stable MNPs coated with commercially available unmodified and non-ionic poloxamers, but the cationic functional groups introduced on the poloxamer end groups proved to be a necessary and essential condition for the corresponding coated MNPs. Furthermore, depending on the properties of the cationic end groups, the stability of the MNPs seems to be variable. Secondary interactions such as hydrogen bonding (compound 2b), π / π stacking (compound XXX) or both (compounds VII and 4b) may be involved as well. Concurrently with these measurements, the zeta potential was evaluated with the same compounds at the same time, and the results are shown in Table 29 below:

[0304] [Table 29] Table 29: Zeta potential (Zp) measurements of magnetic NPs resuspended in various 1% (wt / vol) non-ionic and cationic poloxamer solutions. The potential is expressed in mV and was recorded at two different time points (5 days and 1 month "average time") after resuspension in non-ionic or cationic poloxamer solutions. The "Agg" tag indicates that no nanometric objects remain that could have been measured, indicating possible aggregation of beads.

[0305] The results shown in this table correlate very well with the previous size measurements. The commercial natural and non-ionic poloxamers F108 and F127, when used as coating agents, shield part of the negative charge of the original MNPs, reducing their stabilization by electrostatic interactions and promoting their aggregation. On the other hand, all the cationic poloxamers provide the obtained nanoparticles with a positive surface charge that clearly aids in colloidal stabilization over time, highlighting the large behavior of compounds 2b, 4b, XIV, VII even one month after the resuspension procedure. These experiments verify the fact that the introduction into the structure of cationic end groups of poloxamers made it possible to stabilize NPs coated with these modified polymers, in contrast to their natural counterparts.

[0306] Using this strategy, we further extend the coating and formulation of MNPs with higher concentrations of cationic poloxamer (5% and 10% (w / v)). For this study, we selected compounds 2b, 4b and XXX derived from the F108 backbone, and compound VII derived from the F127 backbone. To do this, we used a resuspension procedure that includes an intermediate resuspension / decantation step with a less concentrated cationic poloxamer solution.

[0307] [Table 30] Table 30: Size measurements of magnetic NPs resuspended in various 5% (wt / vol) non-ionic and cationic poloxamer solutions. Sizes are expressed in nm and were recorded at two different time points ("average time" of 5 days (d), 1 month) after resuspension in non-ionic and cationic poloxamer solutions. The "Agg" tag indicates that no nanometric objects remain that could have been measured, indicating possible aggregation of beads.

[0308] As previously mentioned, unmodified and neutral poloxamers (F108 and F127) resulted in instantaneous aggregation of MNPs, whereas cationic poloxamers were able to stably coat MNPs at this higher concentration. On the other hand, the four selected compounds had a range of about 200 nm, appropriate for transduction applications, resulting in successful stable beads after 5 days minimizing cytotoxicity. The nature of the selected compounds does not appear to have an effect on the recorded sizes, except for the beads from compound XXX, which appeared slightly larger than their relatives. The 1-month stability test showed no obvious differences; most of the NPs retained the same size compared to the size recorded 5 days after resuspension. Here again, the beads from compound XXX showed differences, with sizes in the range of more than 500 nm. This net increase in size may be a sign of prior stacking leading to aggregation, even if there is no sign of such a phenomenon evident at this time point. In conclusion, here again, the electrostatic repulsive interactions incorporated by the cationic modification of these poloxamers allowed the generation of stable suspensions of magnetic NPs even when the concentration of the cationic poloxamer solution used for resuspension was increased. The corresponding zeta potential values ​​recorded at the same time points are shown in Table 31 below.

[0309] [Table 31] Table 31 shows the zeta potential (Zp) measurements of magnetic NPs resuspended in various 5% (wt / vol) unmodified cationic poloxamer solutions. The potential is expressed in mV and was recorded at two different time points (5 days (d) and 1 month) after resuspension in neutral or cationic poloxamer solutions. The "Agg" tag indicates that no nanometric objects remain that could have been measured, indicating possible aggregation of beads.

[0310] The zeta potential measurements correlate perfectly with the observed results for size. All nanoparticles show a high positive charge after formulation with cationic poloxamer, without significant changes after 5 days or 1 month. Only beads obtained from polymer XXX showed a slight decrease in zeta potential, probably due to the increase in size. These data confirmed that highly concentrated solutions of cationic poloxamer may be suitable for resuspension of decanted magnetic NPs. The cationic modification at this concentration still allowed electrostatic repulsive interactions that could stabilize the colloidal nanostructure.

[0311] The final part of this detailed study involved the resuspension of the magnetic cores in a 10% (wt / vol) cationic poloxamer solution. This concentration is the preferred concentration for best results in lentivirus-mediated transduction experiments using cationic poloxamer as an adjuvant. Stable suspension of beads at such concentrations may accentuate the adjuvant effect of cationic poloxamer. We selected the same compounds (2b, 4b, VII, XXX) than previously resuspended the beads. Here again, a sequential decantation / resuspension procedure was performed to avoid direct aggregation of the beads. This time, the only time point selected was 14 days. Size results are summarized in Table 32 below.

[0312] [Table 32] Table 32: Size measurements of magnetic NPs resuspended in various 10% (wt / vol) unmodified or cationic poloxamer solutions. Sizes are expressed in nm and were recorded at one time point (14 days (d)) after resuspension in unmodified or cationic poloxamer solutions. The "Agg" tag indicates that no nanometric objects remain that could have been observed, and indeed possible aggregation of beads.

[0313] At this concentration, once again a 10% (w / v) solution of commercially available unmodified poloxamer was unable to efficiently resuspend the magnetic nanoparticles, in contrast to the cationic modification that successfully stabilized the MNPs. Compounds 2b and VII have only a slight increase in the size of the beads coated with 2b and VII, yet impressively stabilize the colloids, without any sign of aggregation at this time. These 2b and VII cationic poloxamers are clearly the best candidates for resuspension at this concentration, as highlighted by the analysis of the zeta potential recorded at the same conditions. Nevertheless, some cationic poloxamers, such as compound XXX, did not allow to obtain a stable nanometric NP solution, inducing stacking of beads at this concentration, probably due to stronger π / π interactions. Results with compound 4b showed that a size of 453 nm clearly indicated the onset of aggregate behavior, which was not completed after 14 days.

[0314] [Table 33] Table 33: Zeta potential measurements of magnetic NPs resuspended in various 10% (wt / vol) neutral or cationic poloxamer solutions. The potential is expressed in mV and was recorded at one time point (14 days) after resuspension in neutral or cationic poloxamer solutions. The "Agg" tag indicates that no nanometric objects remain that could have been measured, indicating possible aggregation of beads.

[0315] This last set of data generally follows the previous one. Compounds (2b, VII), which are able to maintain a positive charge on the particle surface, are not likely to induce aggregation during the resuspension step. In contrast, beads coated with 4b become almost neutral over time. Compound XXX appears to be an exception to this embodiment, since its positive charge would not counterbalance the aggregation possibly caused by π-stacking of the triphenylphosphonium moieties. This highlights the fact that secondary interactions, if properly balanced and not contributing to stacking or aggregation, can have a favorable effect on particle stabilization.

[0316] In conclusion, we have clearly demonstrated the favorable impact of cationic modification of poloxamers on the stabilization of resuspended magnetic nanoparticles. Contrary to commercially available neutral poloxamers, some modified cationic poloxamers result in a positive charge on the bead surface to ensure colloidal stabilization by electrostatic repulsion interactions. With careful selection of chemical functional groups and an optimized decantation / resuspension strategy, MNPs with appropriate iron / cationic poloxamer content were stably obtained. These were tested in lentivirus-mediated magnetically induced transduction experiments.

[0317] Biological evaluation of modified magnetic nanoparticles coated with cationic poloxamer The present invention clearly highlights the reliable effect of using cationic poloxamers to ensure colloidal stabilization of magnetic nanoparticles.The aim is then that such MNPs can combine the favorable adjuvant effect described earlier in this specification with the properties of cationic nanoparticles to ensure the efficiency of lentivirus-mediated transduction experiments.Therefore, the inventors compared the transduction efficiency of the combined lentivirus vectors on the KG1a cell line with either: ViroMag nanoparticles from OZ Biosciences (Marseille, France), a long-standing reference in enhancing lentivirus-mediated transduction; The cationic poloxamer 2b, which was shown earlier in this document to be efficient as an adjuvant in transduction experiments, ViroMag nanoparticles coated with cationic poloxamer 2b according to the strategy developed in this paper.

[0318] KG1a cells were transduced with lentivirus encoding GFP with an MOI of 5 in combination with ViroMag magnetic nanoparticles (VM), ViroMag NPs coated with a 5% (weight / volume) solution of cationic poloxamer 2b or cationic poloxamer 2b (VM+2b). After 72 hours of incubation, the % of GFP-positive cells (A) and the mean intensity of genetically modified cells (B) were assessed by flow cytometry. The results are shown in FIG. 23.

[0319] The results showed the effect of cationic poloxamers. ViroMag MNPs succeeded in enhancing the expected and published transduction, since it allowed to double the number of cells infected with lentivirus. Compound 2b mentioned above, when used as an adjuvant, also allowed to boost the transduction efficiency, since the number of infected cells reached almost 40% on average, with a 1.5-fold higher intensity than LV alone. Combining cationic poloxamer 2b with ViroMag resulted in the highest efficiency. The synergistic effect brought about by cationic poloxamer and MNPs was demonstrated. In fact, VM+2b allowed the effect of adjuvant 2b to have the effect of magnetically induced transduction experiments resulting in a higher number of transduced cells and a higher average intensity. This experiment not only confirmed the effect of the introduction of cationic functional groups on the terminal groups of the poloxamers, which enhances the behavior of these adjuvants, but also demonstrated the intrinsic enhancing properties of modified cationic poloxamers, which allows this cationic modification to be combined with the effect of magnetically induced lentivirus transduction in only one formulation. This is of critical importance in facing today's new challenges of viral-mediated gene therapy or the use of viruses to generate cell therapy products for clinical trials.

Claims

1. A method for transducing a viral vector into a target cell using a cationic poloxamer.

2. 2. The method of claim 1, wherein the cationic poloxamer is used alone or formulated with nanoparticles.

3. 3. The method according to claims 1 and 2, characterized in that the cationic poloxamer is used either alone or in a composition comprising the cationic poloxamer.

4. The method of any one of claims 1 to 3, comprising contacting the target cell with a viral vector and a cationic poloxamer.

5. 5. The method of claim 4, wherein the target cells are contacted with the cationic poloxamer prior to, simultaneously with, or after contact with a virus.

6. 5. The method of claim 4, wherein the cationic poloxamer and the viral particles are added as a mixture onto the cells simultaneously or sequentially.

7. 7. The method according to any one of claims 1 to 6, characterized in that the contacting of the virus, the cationic poloxamer and the cells is in the range of 5 seconds to 3 months, preferably in the range of 10 minutes to 2 weeks, more preferably in the range of 20 minutes to 1 week, even more preferably in the range of 0.5 hours to 120 hours.

8. 8. The method of any one of claims 1 to 7, wherein the cationic poloxamer is provided at a stock concentration of 0.01 to 500 mg / ml, preferably 0.05 to 300 mg / mL, more preferably 1 to 200 mg / mL, even more preferably 5 to 150 mg / mL.

9. The method according to any one of claims 2 to 8, wherein when the cationic poloxamer is used in a formulation with nanoparticles, the nanoparticles are magnetic nanoparticles.

10. 10. The method according to any one of claims 2 or 9, characterized in that a further step of a magnetic field is applied when the virus, the cells and the adjuvant have been in contact for between 10 seconds and 96 hours, preferably between 1 minute and 48 hours, more preferably between 5 minutes and 4 hours.

11. The method of any one of claims 1 to 10, comprising a further step of spinoculation or centrifugation before or after contacting the target cells with the virus and the cationic poloxamer.

12. Formula I or Formula II: 【Chemistry 1】 【Chemistry 2】 The novel linear or branched cationic poloxamer according to the formula P is of formula IIIa or b: 【Chemistry 3】 or P is according to formula IV or b: 【Chemistry 4】 In accordance with the above formula, "a" represents the number of hydrophilic units repeated in said polymer backbone P and is an integer ranging from 2 to 10,000, preferably from 5 to 1,000, more preferably from 20 to 200; "a-1" is the "a" minus 1 unit and is an integer ranging from 2 to 10000, preferably from 5 to 1000, more preferably from 20 to 200; "b" is the number of hydrophobic units repeated in said polymer backbone P and is an integer ranging from 2 to 1000, preferably from 5 to 500, more preferably from 15 to 80; "b-1" is the "b" minus 1 unit and is an integer ranging from 2 to 1000, preferably from 5 to 500, more preferably from 15 to 80; -X １ , X ２ , X ３ and X ４ are the same or different and are preferably selected from nitrogen, phosphorus, silicon, sulfur and oxygen, more preferably nitrogen, phosphorus, silicon or sulfur, even more preferably nitrogen, phosphorus or sulfur, and each of R １ , R ２ , R ３ and R ４ represents a heteroatom covalently bonded to; n is an integer comprised between 1 and 20, more preferably between 2 and 6; - R １ , R ２ , R ３ and R ４ may, simultaneously or independently, 1 to 6 hydrogen atoms, preferably 2 to 4 hydrogen atoms, 1 to 8 heteroatoms, preferably 2 to 4 heteroatoms, selected from, for example, nitrogen, phosphorus, silicon, sulfur and oxygen, preferably nitrogen, phosphorus, silicon or sulfur, more preferably nitrogen, phosphorus or sulfur, - 1 to 24, preferably 1 to 12, more preferably 1 to 6 linear, branched and / or cyclic saturated or unsaturated hydrocarbon groups containing 1 to 24 carbon atoms, with or without one or more heteroatoms such as oxygen, nitrogen, sulfur, phosphorus, - 1 to 6, more preferably 1 to 3, naturally occurring or non-naturally occurring amino acid residues, Any combination of these definitions is a non-polymeric chemical moiety selected from: - A １ , A ２ , A ３ and A ４ For example, - halogen anions, such as, for example, iodide, bromide, chloride or fluoride; organic groups having a negative charge, whether or not concentrated on a carbon atom, such as, for example, methanesulfonate, trifluoromethanesulfonate, trifluoroacetate, acetate, formate, paratoluenesulfonate, carbonate, hydrogen carbonate; Inorganic anions, such as, for example, sulfate, phosphate, nitrate, hydrogen sulfate, hydrogen phosphate; inorganic non-coordinating inorganic anions such as tetrafluoroborate, hexafluorophosphate or perchlorate, carba-closo-dodecaborate; Boron-centered organic anions based on the tetrakis[3,5-bis(trifluoromethyl)phenyl]borate skeleton may represent the same or different counterions, which may be selected from one or several organic groups such as In the formula, except for the F108 skeleton, a is an integer in the range of 130 to 135, b is an integer in the range of 48 to 52, a-1 is an integer in the range of 129 to 134, and X １ R １ =X ２ R ２ =NH ２ , or X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -S-S-C ５ H ４ N or X １ R １ =X ２ R ２ =-OC(O)-NH-NH ２ F127 backbone, a is an integer in the range of 98 to 103, b is an integer in the range of 52 to 58, a-1 is an integer in the range of 97 to 102, and X １ R １ =X ２ R ２ =NH ２ , or X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -NH ２ , or X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -NH-C １９ -H １８ -N ７ -O ５ , or X １ R １ =X ２ R ２ =-OC(O)-(CH ２ ) ２ -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH ２ , or X １ R １ =X ２ R ２ =-OC(O)-CH(NH ２ )-CH-(CH ３ ) ２ , or X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -NH-(CH ２ ) ２ -NH-(CH ２ ) ２ -NH ２ , or X １ R １ =X ２ R ２ =-OC(O)-CH(NH ２ )-CH ２ -SH; F68 backbone, a is an integer in the range of 72 to 78, b is an integer in the range of 25 to 32, a-1 is an integer in the range of 71 to 77, and X １ R １ =X ２ R ２ =NH ２ , and X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -S-S-C ５ H ４ N or X １ R １ =X ２ R ２ =-OC(O)-NH-NH ２ P123 skeleton, a is an integer in the range of 17 to 23, b is an integer in the range of 67 to 73, a-1 is an integer in the range of 16 to 22, X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -NH ２ L121 backbone, and a is an integer in the range of 8 to 12, b is an integer in the range of 64 to 72, a-1 is an integer in the range of 7 to 11, and X １ R １ =X ２ R ２ =-CH=N-(CH ２ ) ２ -NH ２ Or X １ R １ =X ２ R ２ =-CH=N-(CH ２ ) ４ -NH ２ Or X １ R １ =X ２ R ２ =-CH=N-(CH ２ ) ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH ２ P85 backbone, a is an integer in the range of 24 to 28, b is an integer in the range of 38 to 42, a-1 is an integer in the range of 23 to 27, and X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ３ -NH ２ P105 skeleton, a is an integer in the range of 34 to 39, b is an integer in the range of 52 to 60, a-1 is an integer in the range of 33 to 38, and X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -S-S-C ５ H ４ N or X １ R １ =X ２ R ２ =-OC(O)-NH-NH ２ F88 backbone, a is an integer in the range of 94 to 100, and b is an integer in the range of 34 to 42, a-1 is an integer in the range of 93 to 99, X １ R １ =X ２ R ２ =-OC(O)-NH-(CH ２ ) ２ -S-S-C ５ H ４ N or X １ R １ =X ２ R ２ =-OC(O)-NH-NH ２ P124 backbone, a is an integer in the range of 8 to 14, b is an integer in the range of 16 to 24, a-1 is an integer in the range of 7 to 13, and X １ R １ =X ２ R ２ =-OC(O)-CH ２ -C １２ H ２０ N ３ O ８ , or X １ R １ =X ２ R ２ =N((CH ２ ) ３ -NH ２ )-(CH ２ ) ４ )-NH ２ P104 skeleton, a is an integer in the range of 24 to 30, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 23 to 29, X １ R １ =X ２ R ２ =-OC(O)-(CH ２ ) ２ -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH ２ P103 skeleton, a is an integer in the range of 14 to 20, b is an integer in the range of 56 to 64, a-1 is an integer in the range of 13 to 19, X １ R １ =X ２ R ２ =-OC(O)-(CH ２ ) ２ -NH-(CH ２ ) ３ -NH-(CH ２ ) ４ -NH-(CH ２ ) ３ -NH ２ L64 backbone, a is an integer in the range of 10 to 16, b is an integer in the range of 26 to 34, a-1 is an integer in the range of 9 to 15, and X １ R １ = N ３ and X ２ R ２ = -C １７ H １５ N ２ O ２ T908 skeleton, a is an integer in the range of 116 to 122, b is an integer in the range of 15 to 20, a-1 is an integer in the range of 115 to 121, X １ R １ =X ２ R ２ =X ３ R ３ =X ４ R ４ =NH ２ That is, Cationic poloxamer.

13. "-X １ R １ ", -X ２ R ２ ", -X ３ R ３ " and "-X ４ R ４ "teeth, Primary, secondary or tertiary amine cationic moieties. Amine derivatives such as guanidine, hydrazine, guanidinium, hydrazinium etc. may be preferred entities according to the invention; ○ organic quaternary phosphonium moieties such as, for example, substituted tri-n-butylphosphonium, substituted triphenylphosphonium, substituted triethylphosphonium; o Quaternary ammonium salts based on a nitrogen atom covalently bonded to a four carbon moiety, such as, for example, substituted quaternary trimethylammonium, substituted quaternary tri-n-butylammonium, substituted quaternary tri-n-octylammonium; organic tertiary sulfonium salts based on a sulfur atom covalently bonded to a three carbon moiety, such as, for example, substituted dimethylsulfonium, substituted di-n-butylsulfonium, substituted di-n-octylsulfonium; organic heterocycles incorporating at least 1 to 6 similar or different heteroatoms, such as oxygen, nitrogen, sulfur, phosphorus, and containing at least one unsaturation that provides them with aromaticity, and having a delocalized or non-delocalized net positive charge on the ring; Basic amino acid residues as the source of cationic charge, for example lysine, arginine, histidine, ornithine, tryptophan, natural or non-natural; Natural non-polymeric polyamines, such as, for example, non-polymeric spermine, spermidine or thermospermine derivatives 13. The novel linear or branched cationic poloxamer according to claim 12, characterized in that it is a non-polymeric entity selected from among

14. The cationic poloxamer of claim 13, characterized in that the organic heterocycle having a net positive charge is selected from pyridine and its cationic counterpart pyridinium; imidazole and its cationic counterpart imidazolium; triazole and its cationic counterpart triazolium; piperidine and its cationic counterpart piperidinium; morpholine and its cationic counterpart morpholinium.

15. formula: 【Chemistry 5】 【Chemistry 6】 【Chemistry 7】 【Chemistry 8】 【Chemistry 9】 15. The cationic poloxamer according to claim 13 or 14, selected from the compounds according to

16. A composition, preferably a therapeutic or cosmetic or life science composition, comprising a compound according to formula I or II as defined in any one of claims 12 to 15.

17. A composition comprising a cationic poloxamer and a virus for transducing a target cell.

18. A kit comprising a cationic poloxamer formulated as defined in any one of claims 1 to 15, alone or further comprising a virus, and optionally further comprising instructions for use.

19. A population of transduced cells obtainable by the method according to claims 1 to 11.

20. 20. A method of treating a subject in need of treatment with cellular gene therapy, said method comprising administering to said subject an effective amount of the transduced cell population of claim 19.

21. Use of a cationic poloxamer according to any one of claims 12 to 15 for the use of a viral vector in the transduction of cells.

22. The method according to any one of claims 1 to 11, wherein the cationic poloxamer is as defined in any one of claims 12 to 15.

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