AU725723B2

AU725723B2 - Composition containing chitosan

Info

Publication number: AU725723B2
Application number: AU49505/97A
Authority: AU
Inventors: Hanno Kolbe
Original assignee: Transgene SA
Current assignee: Transgene SA
Priority date: 1996-10-23
Filing date: 1997-10-23
Publication date: 2000-10-19
Anticipated expiration: 2017-10-23
Also published as: ATE211489T1; DK0934342T3; JP2001502736A; CA2268804A1; ES2170377T3; FR2754824A1; DE69709891T2; FR2754824B1; EP0934342B1; PT934342E; AU4950597A; EP0934342A1; WO1998017693A1; DE69709891D1

Abstract

The invention concerns a chitosan compound having 5 to 300 glucosamine radicals, a substantially pure preparation of chitosan, a composition containing it and the therapeutic use of the latter for transferring a therapeutically active substance into a host cell. The invention also concerns a method for preparing such a compound or such a preparation.

Description

WO 98/17693 1 PCT/FR97/01897 COMPOSITION CONTAINING CHITOSAN The subject of the present invention is chitosan compounds comprising from 5 to 300 repetitions of glucosamine units and a pharmaceutical composition comprising such compounds. More particularly, the present invention relates to a method for preparing chitosan according to the invention and its use for transferring a nucleic acid into a host cell.

The transfer of genes into a given cell forms the very basis of gene therapy. This new technology, whose field of application is vast, makes it possible to envisage the treatment of serious diseases for which conventional therapeutic alternatives are not very effective or even inexistent and relate to both genetic diseases (hemophilia, cystic fibrosis, myopathy and the like) and acquired diseases (cancer, acquired immunodeficiency syndrome AIDS and the like).

The approach most widely taken consists in using a viral vector to introduce the therapeutic nucleic acid into the cells to be treated and, in particular a retroviral or adenoviral vector. Indeed, viruses have developed sophisticated mechanisms for crossing the cell membranes, for escaping degradation in the lysosomes and for causing their genome to penetrate into the nuclei in order to bring about the expression of the therapeutic gene. However, the viral approach has its limitations, especially a restricted cloning capacity, a potential production of replication-competent viral particles capable of dissemination in the host organism and the environment, a risk of insertional mutagenesis in the case of retroviral vectors and an induction of immune and inflammatory responses in the host which hinder repetitions of treatment in the case of adenoviral vectors. These major disadvantages for human use justify the .search for alternative systems for the transfer of nucleic acids. In this regard, numerous amphiphilic and cationic compounds have already been 2 described in the literature (see for example Behr et al. 1994, Bioconj Chem 5, 382-389).

In this perspective, it may also be advantageous to use polymers (repetition of monomeric units). However, protein polymers should be avoided because of their potentially immunogenic nature.

Neutral polymers such as dextrans, polyvinylpyrrolidone (PVP) and polyethylene glycol (PEG) have the advantage of not being very toxic in vivo but, because of the absence of positive charges, do not complex nucleic acids in a satisfactory manner. Indeed, cationic polymers, such as dendrimers (WO 95/24221), polyethylenimine (WO 96/02655) and polylysine (WO 95/33061) might exhibit hemolytic activity after intravenous administration.

Another cationic polymer consists of chitosan, a natural polysaccharide formed by the linear repetition of glycosamine residues. Commercial preparations of chitosan already exist which are obtained by deacylation of chitin. In general, they contain a long chain of glycosamine units and have a high average molecular mass which may be up to 2000 kDa. As a guide, the preparations having a lower molecular mass which are currently available have about 70 kDa and contain on average 430 glucosamine residues (one glucosamine unit at a molecular mass of 161 Da).

A few prior art documents report use of chitosan for the transfer of medicinal substances and of polypeptides (Berscht et al., 1994, Biomaterials 593-600; Chandry and Sharma, 1990, Biomater. Artif Cells Artif Organs 18, 1-24 and Illum et al., 1994, Pharm. Res. II, 1186-1189) and more recently of nucleic acids (Murata et al., 1996, Carbohydrate Polymers 29, 69-74). The latter document describes the use of chitosan in which the amine residues are quaternized by trimethylation and conjugated with galactose in order to target hepatic cells more particularly. That document does not mention the possibility of attaching DNA without prior quaternization.

3 In addition, the preparations of the state of the art exhibit certain disadvantages which compromise their use for the transfer of genes, especially in humans. In particular, they contain pigments and other contaminants which can be harmful even in a small quantity. Furthermore, their solubility in water or pharmaceutically acceptable conditioning buffers is poor and requires the addition of at least 0.1% acetic acid and the solution obtained remains viscous.

Finally, it is quite likely that the complexing of these long chains of polycations (on average 430 units for the preparations of lower molecular mass) with a long chain of polyanions (nucleic acid) promotes interchain bonds and therefore the formation of largesized complexes which may precipitate in solution. The present invention provides an advantageous solution to these various problems.

Chitosan fragments of low molecular mass: less than 5 kDa, between 5 and 10 kDa and greater than 10 kDa, have now been generated by acid hydrolysis and their particularly advantageous properties for the transfer of genes demonstrated. The chitosan fragments of the present invention have a sufficient size to form, with the therapeutic nucleic acid, a complex of adequate stability to allow its transport in the host cell but also its release in vivo. In addition, the method used makes it possible to reduce the pigments which contaminate the preparations of the prior art and to enhance the solubility in water.

Accordingly, the subject of the present invention is a composition comprising a compound of general formula [X]n in which n represents an integer from 5 to 300 and X has the following formula

,CH

2 OH0 HO 0 and a therapeutically active substance.

4 A first subject of the invention relates to a composition comprising a compound comprising a linear repetition of 5 to 300 glucosamine residues of formula as defined above. This compound according to the invention is advantageously used for its transfecting power for the transfer of a gene or of a vector of interest into a host cell. The case where two glucosamine residues are linked in the following manner is most particularly preferred:

CH

2 OH

NH

0 HO NH 2

O

HO O

SNH

2

CH

2

OH

Advantageously, in the general formula n represents an integer from 5 to 150, preferably from to 75 and most preferably n is between 5 and The compounds particularly preferred within the framework of the present invention are the following: a compound having an average molecular mass of less than 5 kDa, (ii) a compound having an average molecular mass of between 5 and 10 kDa, and (iii) a compound having an average molecular mass greater than 10 kDa and less than 50 kDa.

Of course a compound which can be used according to the invention may be chemically modified by addition, deletion and/or substitution of chemical radicals or groups so as to enhance its stability, its half-life, the complexing with the nucleic acid and/or its transfecting power. The modification may be carried out on a portion (partial modification) or on all (complete modification) of the glucosamine residues and on one or more radicals of each of the glucosamine residues. According to an advantageous embodiment, a compound which can be used according to the invention is modified by C- and/or O-alkylation, -acylation, -amino-alkylation and/or -polyoxyethylenation (see for 1 i 5 example Dunn et al., 1993, J. Applied Polymer Science 353-365; Boullanger et al., 1995, Carbohydr. Res.

278, 91-101; Muzzarelli et al., 1983, J. Memb. Science 16, 295-308). In this regard, the compounds particularly preferred are poly-N-dodecylpolyglucosamine in which the NH2 radical of formula (I) is replaced by NH-(CH2)11-CH3 and mono-Ca-octadecylpolyglucosamine in which the OH radical of the compound according to the invention at the chain end is replaced by O-(CH2)17-CH3. However, it is also possible to graft an alkyl residue of the hexyl to octadecyl type or a mono nonsaturated alkyl residue of the oleoyl type.

In a particularly advantageous manner, the compound which can be used according to the invention is a chitosanium salt, in particular a sulphate, a phosphate and, more preferably, a chloride.

The compounds which can be used according to the invention may be obtained in various ways. They may for example be chemically synthesized from the glucosamine monomer by polymerization or from a preparation of high molecular mass by controlled enzymatic hydrolysis. However, the use of a method of acid hydrolysis of preparations of chitosan of the state of the art followed by differential diafiltration, as described below, is most particularly preferred.

The present invention also relates to some of the compounds of formula as they are, in particular the compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula

,CH

2

OH

HO

NH

2 characterized in that all or part of the X residues constituting said compound is modified by C- and/or O-alkylation, alcylation, -amino-alkylation and/or 6 -polyoxyethylenation; and the compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula

CH

2

OH

0 HO 0

NH

2 characterized in that it is present in the form of a salt and more particularly of a chloride, sulphate or phosphate; these preferred compounds corresponding to the uses described above.

The present invention also relates to a substantially pure preparation of chitosan. It is in particular obtained by any conventional purification technique and in particular by extraction of the pigments and other residual contaminants. This purification step may be carried out on the commercial preparations of chitosan in order to generate a substantially pure preparation of high molecular mass or otherwise which may be used to complex a nucleic acid.

The present invention relates more particularly to a composition comprising at least one compound or a preparation according to the invention and a therapeutically active substance. It is preferably a negatively charged substance and, in particular, a nucleic acid. Nucleic acid is understood to mean both a sense or antisense deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Moreover, the nucleic acid may be homologous or heterologous to the host cell. It may be natural, hybrid or synthetic sequences derived from genomic DNA, cDNA, mRNA, rRNA, tRNA, viral RNA of any origin (prokaryote, eukaryote, virus, parasite, plant and the like). They may be obtained by conventional molecular biology techniques (cloning, PCR for Polymerase Chain Reaction and the like) or by chemical Ssynthesis.

7 According to a specific embodiment, the nucleic acid used within the framework of the present invention is a vector for the expression of a gene of therapeutic importance. The expression vector is advantageously in the form of a plasmid but it is also possible to use a viral vector (derived from a retrovirus, an adenovirus, a poxvirus, a herpesvirus, an adeno-associated virus and the like). As regards the preferred embodiment, the choice of the plasmids which can be used in the present invention is vast. It is possible to use a plasmid/vector of the state of the art or to construct it by genetic engineering techniques (Maniatis et al., 1989, Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY). Advantageously, it possesses the genetic elements allowing it to replicate and to be maintained in a host cell and/or microorganism, such as for example at least one replication origin and a selectable gene. In the latter case, it may be a gene encoding an auxotrophy or conferring a resistance to an antibiotic (ampicillin, tetracyclin, puromycin, G418, chloramphenicol and the like). Of course the plasmid used in the present invention may also comprise additional elements which enhance its maintenance, its stability in the host cell or alternatively its integration into the chromosomes of the host. In general, such elements are known to persons skilled in the art.

In accordance with the aims pursued by the present invention, the gene of therapeutic interest used in the present invention may encode a ribozyme, an antisense RNA or alternatively an mRNA which will then be translated into a polypeptide. It may be a mature polypeptide, a precursor and in particular a precursor intended to be secreted and comprising a signal peptide, a native, truncated, chimeric polypeptide obtained from the fusion of sequences of various origins or of a mutated polypeptide having enhanced and/or modified biological properties. Among the 8 polypeptides whose use may be envisaged, there may be mentioned more particularly: cytokines or lymphokines P- and yinterferons, interleukins and in particular IL-2, IL-6, IL-10 or IL-12, tumor necrosis factors (TNF), colony-stimulating factors (GM-CSF, C-CSF, M-CSF and the like); cell or nuclear receptors (receptors recognized by pathogenic organisms (viruses, bacteria or parasites) and preferably by the HIV virus (Human Immunodeficiency Virus) or their ligands; proteins capable of complementing a deficient cellular activity especially in the context of a genetic disease (factor VII, factor VIII, factor IX, dystrophin or minidystrophin, insulin, CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) protein, growth hormones (hGH); enzymes (ureas, renin, thrombin and the like); enzyme inhibitors (al-antitrypsin, antithrombin III, viral protease inhibitors and the like); polypeptides with antitumor effect which are capable of at least partially inhibiting the initiation or the progression of tumors or cancers (antisense RNA, antibodies, inhibitors acting on cell division or transduction signals, products of expression of tumor suppression genes, for example p53 or Rb, polypeptides or peptides stimulating the immune system and the like); proteins of the major histocompatibility complex Sclass I or II or regulatory proteins acting on the expression of the corresponding genes; 9 polypeptides capable of inhibiting a viral, bacterial or parasitic infection and/or its development (antigenic polypeptides having immunogenic properties, antigenic epitopes, antibodies, trans-dominant variants capable of inhibiting the action of a native protein by competition and the like); toxins (herpes simplex virus 1 thymidine kinase (HSV-1-TK), ricin, cholera toxin, diphtheria toxin and the like) or immunotoxins; and markers (p-galactosidase, luciferase and the like).

It should be mentioned that this list is not limiting and that other genes may also be used.

Advantageously, the therapeutic gene is placed under the control of elements necessary for its expression in the host cell. "Elements necessary" is understood to mean all the elements allowing its transcription into RNA and the translation of an mRNA into a polypeptide. Among these, the promoter is of particular importance.

It may be derived from any gene (eukaryotic, viral, natural promoter of the gene of interest in question and the like) or may be hybrid. Moreover, it may be constitutive or regulatable. Alternatively, it may be modified so as to enhance the promoter activity, suppress a region inhibiting transcription, modify its mode of regulation, introduce a restriction site and the like. There may be mentioned, by way of examples, the viral promoters CMV (Cytomegalovirus), of the TK gene of the HSV-1 virus, SV40 virus (Simian Virus early promoter, early adenoviral promoter (ElA) or late adenoviral promoter (MLP for Major Late Promoter) or alternatively the eukaryotic promoters of the murine or Shuman PGK (Phospho Glycerate Kinase) gene, -1- 10 antitrypsin genes (liver-specific), immunoglobulin genes (lymphocyte-specific).

Of course the nucleic acid may, in addition, comprise additional elements such as intron sequence, signal sequence, nuclear localization sequence, sequence for termination of transcription, site for initiation of translation of the IRES type and the like. It is also indicated that the vector may comprise several genes of interest placed under the control of independent or common regulatory elements.

According to an optimum embodiment, the respective proportions of the compound according to the invention and of the nucleic acid are preferably determined such that the charge ratio of said nucleic acid to said compound (R DNA/compound) is between 1/10 and 10/1 and, preferably, between 1/1 and A composition according to the invention may be used as it is or in combination with other compounds and, in particular, an adjuvant capable of enhancing its transfecting power. The adjuvants preferably used in the composition according to the invention are the cationic amphiphilic compounds and/or the neutral or zwitterionic lipids. In the first case, a mono-, di- or tri-alkylated or -acylated compound carrying from 1 to 4 positive charges such as a cationic lipid is preferred. By way of examples, there may be mentioned dioctadecylamide (DOGS), (N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC- Chol), (2,3-droleylocyl-N-[2-(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate) (DOSPA), spermine cholesterol and spermidine cholesterol. As regards a neutral or zwitterionic lipid, there may be mentioned phosphatidylethanolamine, phosphatidylcholine, phosphocholine, sphyngomyelin, ceramide and cerebroside or their derivatives.

Preferably, it is dioleylphosphatidylethanolamine

(DOPE).

A composition according to the invention is S more particularly characterized in that the lipid to 11 DNA charge ratio is between 25/1 and 1/1 and preferably 5/1 and 2/1.

It is also possible to combine the composition according to the invention with other substances in order to further enhance the transfection efficiency or the stability of the complexes.

The present invention also relates to the use of a composition according to the invention for preparing a medicament for curative, preventive or vaccinal purposes intended for transferring nucleic acid into a host cell in vivo, ex vivo or in vitro. A composition according to the invention may be used in various types of host cells, for example a microbial, yeast, insect or plant cell, but it is preferably a mammalian cell and, in particular, a human cell. Said cell may be a primary or tumor cell of a hematopoietic origin (totipotent stem cell, leukocyte, lymphocyte, monocyte, macrophage and the like), hepatic origin, renal origin, of the central nervous system, fibroblast origin, epithelial origin and in particular a pulmonary epithelial cell or a muscular epithelial cell (myocyte, myoblast, cardiomyocyte, satellite cell and the like).

A composition according to the invention may be administered by the systemic route or by aerosol. It is possible to envisage more particularly the gastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumor, intrapulmonary, intranasal or intratracheal route. The administration may be made as a single dose or as a dose repeated once or several times after a certain time interval. The appropriate route of administration and the appropriate dosage vary according to various parameters, for example the individual or the disease to be treated or alternatively the gene(s) to be transferred. The formulation may also include a pharmaceutically acceptable excipient.

A composition according to the invention is in particular intended for the preventive or curative treatment of genetic diseases (hemophilia, cystic 12 treatment of genetic diseases (hemophilia, cystic fibrosis, Duchenne's and Becker's myopathy and the like), cancers and viral diseases (AIDS, papilloma infections, herpesvirus infections and the like) Finally, the present invention also relates to a method for preparing a compound which can be used according to the invention from a preparation of chitosan of high molecular mass by acid hydrolysis, precipitation with ethanol and extraction of the pigments in the presence of an organic solvent.

The high-molecular mass chitosan preparations are commercially available (for example Fluka, references 22741, 22742 and 22743). Within the framework of the present invention, the use of a preparation having an average molecular mass of about kDa which corresponds to the lowest molecular mass commercially available (Fluka reference 22741) is preferred. The acid hydrolysis is carried out in the presence of hydrochloric acid having a molarity of about 4 M at a temperature of about 100 0 C and for about 4 hours. Of course persons skilled in the art are capable of adjusting the experimental conditions for hydrolysis according to the starting preparation. After gross filtration in order to remove the insoluble matter and cooling, the hydrolysate is precipitated with ethanol at a final concentration of about 50% at cold temperature. The precipitated material is harvested by centrifugation or any conventional means.

It is preferable to carry out several washes in ethanol before taking it up in water or an appropriate buffer.

The dissolved material is subjected to an extraction in the presence of solvent. Any polar and water-immiscible solvent may be used and, in particular, C 4 to C 8 alcohols and esters of the latter.

A preferred solvent within the framework of the invention is 2-butanol. This step which has the advantage of considerably reducing the pigments contained in the starting preparation preferably 13 comprises a first extraction with a third of the volume of 2-butanol and then a second extraction carried out in an aqueous phase with a third of the volume of a 2butanol/hexane (50/50) mixture.

According to a particularly advantageous embodiment, the method according to the invention comprises an additional step intended to separate the compounds of the invention according to their size.

Although various fractionation methods can be envisaged (gel permeation, membrane filtration and the like), the use of differential diafiltration using membranes of increasing cut-off is preferred. The compound of the invention having an average molecular mass of less than kDa is obtained in the filtrate resulting from a first diafiltration through a membrane having a cut-off of 5 kDa. The concentrate is then recovered and subjected to a second diafiltration through a membrane having a cut-off of 10 kDa so as to obtain, in the filtrate, the compound having an average molecular mass of 5 to 10 kDa and, in the concentrate, the compound of the invention having an average molecular mass greater than 10 kDa and less than the starting chitosan. Of course persons skilled in the art are capable of adapting the technique according to the desired average molecular mass. It is also possible to carry out one or more additional diafiltrations on the last concentrate generated. For example, passing the latter through a membrane having a cut-off of 15 kDa will make it possible to harvest compounds according to the invention having an average molecular mass of 10 to kDa.

The present invention is more particularly described with reference to the following figures: Figure 1 illustrates a graph of cytotoxicity on L132 cells obtained with the polymers pcTG12 N1, pcTG12 N2, pcTG12 N3, dextran and polylysine.

Figure 2 illustrates a graph of cytotoxicity on CCRF-CEM cells obtained with the polymers pcTG12 N1, pcTG12 N2, pcTG12 N3, dextran and polylysine.

14 Figure 3 illustrates the hemolytic activity tested on rat red blood cells of the polymers pcTGl2 N1, pcTG12 N2, pcTGl2 N3, dextran and polylysine after 1 hour of contact.

Figure 4 illustrates the hemolytic activity tested on rat red blood cells of the polymers pcTGl2 N1, pcTG12 N2, pcTGl2 N3, dextran and polylysine after 5 hours of contact.

Figure 5 illustrates the expression of luciferase in A549 cells transfected with the plasmid pTG11033 complexed with DC Chol/DOPE (control) or DC Chol/DOPE/chitosan at a concentration of 10, 30 and Figure 6 illustrates the expression of luciferase in primary myoblasts transfected with the plasmid pTG11033 complexed with DC Chol/DOPE (control) or DC Chol/DOPE/chitosan at a concentration of 10, and

EXAMPLES

EXAMPLE 1 Preparation of the chitosan fragments.

In general, the commercial preparations of chitosan of varied molecular mass may be used and the ones most appropriate for carrying out the present invention are those of low molecular mass (for example the Fluka preparation under the reference 22741 whose average molecular mass is about 70 kDa). The acid hydrolysis is carried out in a Schott DURAN bottle having a capacity of 1 liter, provided with a magnetic stirrer. After weighing, 25 g of chitosan (batch 340315/1 295) are placed in the presence of 625 ml of 4 M hydrochloric acid (HC1) (analytical grade HC1 such as R.P. Normatur, Prolabo 22 252.290, min After having exchanged the gaseous phase with nitrogen for min (in order to avoid any oxidation) and closed the bottle, the reaction mixture is placed under regular magnetic stirring (500 rpm) on an oil bath kept at 100°C. After equilibration of the temperature (15 min 15 under the abovementioned experimental conditions), the reaction is allowed to continue for 4 hours at 100 0

C.

It is observed that this treatment is accompanied by a change in the color of the solution which appears light brown. Of course the hydrolysis conditions are adjusted to the starting chitosan preparation. If the latter has a high molecular mass (Fluka, 750 or 2000 kDa), the hydrolysis will be more stringent (increase in the HCI concentration or in the reaction time) The reaction mixture is immediately filtered (filter having pores of about 0.6 mm) in order to remove the insoluble aggregates and impurities. The filtrate is transferred into a 2-liter Schott DURAN bottle and cooled for about 1 hour on ice before being precipitated by addition of 625 ml of absolute ethanol (analytical grade, for example sds, reference 013A0516) and stored overnight at 2-4 0 C. The next day, the precipitated material is harvested by centrifugation (10,000 g, 2-4 0 C, 10 min) and subjected to two washes in an ethanol/water (50/50) mixture. The pellets are taken up in 20 to 60 ml of demineralized water on a Milli Q cartridge (Milli Q water), frozen at -80 0

C

while waiting to be lyophilized. 12 to 16 g of chitosan fragments are usually obtained in the form of hydrochloride salts.

The extraction of the pigments from the preparation of chitosan x nHC1- is then carried out in the following manner. 14.9 g of the preceding lyophilized preparation are dissolved in 300 ml of Milli Q water and then treated with 100 ml of 2-butanol (analytical grade). The mixture is then vigorously stirred and left at room temperature in a separating funnel until the phases are separated. The top phase which is brownish in color is removed and the aqueous phase is subjected to another extraction in the presence of 100 ml of a 2-butanol/hexane (50/50) mixture. As above, the top phase is recovered from which the aqueous medium is evaporated by rotation under vacuum.

16 The dried material is finally dissolved in to 100 ml of Milli Q water and subjected to two consecutive diafiltration steps using membranes of increasing cut-off in order to purify the chitosan fragments of corresponding molecular mass. The solution is first of all filtered through a membrane with a cutoff of 5 kDa (for example YM-5, Amicon) The latter operation is repeated three times, adjusting with Milli Q water to the initial volume and the filtrates thus obtained are grouped together and frozen while waiting for lyophilization. A dry preparation, which is white in color, of chitosan chloride having a molecular mass of less than 5 kDa (designated pcTG12 N1) is obtained. The concentrate is, for its part, subjected to a second diafiltration on a membrane with a cut-off of 10 kDa (for example YM-10, Amicon). After 3 washes with the initial volume, the filtrates are grouped together, frozen and lyophilized so as to give a dry preparation, which is white in color, of chitosan chloride having a molecular mass of between 5 and kDa (designated pcTG12 N2) The concentrate, which is also frozen and lyophilized, is recovered, from which a slightly yellowish dry preparation of chitosan chloride having a molecular mass greater than 10 kDa (designated pcTG12 N3) is obtained.

The preparations thus generated may be characterized by conventional analytical methods, for example by assaying the free amine groups (Curotto and Aros, 1993, Analytical Biochemistry 211, 240-241). The length of the polymers was also determined by laser desorption mass spectrometry and a distribution in the form of a Gaussian curve is observed. The polymers of the pcTG12 N1 preparation 5 kDa) consist of a repetition of 5 to 35 glucosamine residues, the maximum being about 10. The polymers of the pcTG12 N2 preparation (5-10 kDa) exhibit a repetition of 10 to 50 monomers, the maximum being between 30 and 35. As regards the pcTG12 N3 preparation, a number of glucosamine residues greater than 15 is observed. °|greater than 15 is observed.

17 EXAMPLE 2: Complexing with DNA and results on a gel The capacity of the preceding polymeric preparations to complex the reporter plasmid pCH11ON is evaluated. It contains the promoter/enhancer block of the SV40 virus directing the expression of the LacZ gene encoding the P-galactosidase of Escherichia coli provided, at the N-terminus, with a nuclear localization signal derived from 500 ng of plasmid DNA are placed in the presence of increasing quantities of each of the pcTG12 N preparations in a DNA/chitosan mass ratio varying from 1/0.5 to 1/4 (the precise DNA/chitosan ratios are: 1/0.5, 1/1, 1/1.5, 1/2, 1/3 and 1/4) in a total volume of 10 pl optionally adjusted with TE (10 mM Tris, 1 mM EDTA), pH 7.5. The negative control consists of plasmid DNA simply taken up in TE. The mixtures are analyzed on a 0.9% agarose gel (migration buffer TAExl pH 7.8 and 2 hours migration at 50V) and visualized with ethidium bromide. The free plasmid DNA migrates in the form of several bands corresponding to the different topoisomers (supercoiled, circular DNA) whereas once complexed with chitosan, it no longer penetrates into the gel and is visualized in the loading slots.

The results show that the plasmid DNA is completely complexed with the polymers pcTG12 N1 and N2 from a DNA/chitosan ratio of 1/2 and 1/3 respectively.

The formation of the complex is more rapid with the polymers having a molecular mass greater than 10 kDa for which the absence of free DNA is observed from equimolarity (R As a whole, these results show the capacity of the 3 preparations of the invention to bind the plasmid DNA.

EXAMPLE 3: Cytotoxic tests A. In vitro The cytotoxicity of the polymers pcTG12 N1, N2 and N3 was evaluated in vitro in relation to the human cell lines L132 (ATCC CCL5) derived from human embryonic lungs and CCRF-CEM (ATCC CCL119) derived from 18 a human lymphoblastoma. These lines were selected on the basis of their different behavior in cell culture, the first growing in an adherent manner (L132) and the second in suspension (CCRF-CEM).

The CCRF-CEM cells are cultured in a 96-well microtiter plate having a "v"-shaped bottom (Costar) in an amount of 1x104 cells per well and cultured at 37 0

C

in a 5% CO 2 atmosphere in RPMI 1640 medium (Gibco BRL) containing 10% fetal calf serum (FCS) (Gibco BRL).

After 24 hours, the cells are centrifuged at 1000 g for min and the cellular pellet is taken up in the same medium in the presence of the polymers to be tested (dissolution of a sufficient quantity of each of the polymers in RPMI 1640-10% FCS medium so as to obtain a final concentration of 0.1 pg/ml to 1 mg/ml and then sterilizing filtration on 0.2 pm filters (Acrodisk)).

Medium containing dextran having a molecular mass of 72 kDa (Sigma) is used at concentrations identical to those used for the pcTG12 molecules as a negative control and polylysine having a molecular mass of kDa (Sigma) as positive control. The control cells are cultured under the same conditions but in the absence of polymers.

The L132 cells are placed in culture in a 96-well microtiter plate (Costar) in an amount of 1x10 4 cells per well and cultured at 37 0 C in a 5% CO 2 atmosphere in E199 medium (Gibco BRL, reference 22340- 012) containing 5% FCS. After 24 hours, the culture supernatant is replaced with fresh E199-5% FCS medium in which appropriate concentrations of each of the polymers of the invention or of the controls have been dissolved as indicated above.

The viability of the L132 and CCRF-CEM cells is determined after 67 hours of exposure to the different polymers by the MTT test according to the technique described in Sgouras and Ducan (1990, J. of Materials Science: Materials in Medicine 1, 61-68). After a period of incubation of 5 hours, the cellular supernatant is removed (optionally after low-speed 19 centrifugation in the case of the lines in suspension) and replaced with 100 1l of optical grade DMSO (Sigma) and the absorbance at 550 nm is determined on each culture well (Titerteck plate reader). A low absorbance is a sign of a high mortality. The viability is expressed as percentage relative to the absorbance read for the cells cultured in the absence of polymers.

Figures 1 and 2 present the results obtained for the L132 and CCRF-CEM cells respectively and show that the 3 polymers according to the invention (chitosan fragments having a molecular mass 5, 5-10 and 10 kDa) do not affect the cellular viability to any great extent and this at doses as high as mg/ml, just like dextran (nontoxicity recognized).

At 1 mg/ml, a reduction in viability by a factor of about 20% is observed. The IC50, that is to say 50% of viable cells, corresponds to a concentration of about 2 mg/ml. On the other hand, polylysine exerts a cytotoxic effect at doses which are 100 times lower (IC50 reached at a concentration of 10 pg/ml) As a whole, these data confirm the absence of cytotoxicity for the chitosan preparations of the invention which is a prerequisite for their use for the transfer of genetic material in humans.

Recently published data (Carrino-Gomez and Ducan, 1996, Proc. 1st International Symposium on Polymer Therapeutics: From the Laboratory to Clinical Practise. January 10-12 1996, London) appear to indicate a degree of cytotoxicity of the chitosan preparations of high molecular mass. This cytotoxic effect could be due to the molecular mass or to a charge effect. It is also indicated that the chitosangalactose conjugate described by Murata et al. (1996, supra) exhibits a cytotoxic activity greater than the compounds of the present invention (50% of cytotoxicity for about 200 pg/ml if reference is made to Figure 1).

B. In vivo 20 200 p.g of each of the chitosan preparations of the invention were administered to rabbits by the intravenous route and no toxic effect is observed.

EXAMPLE 4: Hematocompatibility tests A. Hemolysis of red blood cells The polymers pcTG12 N1, N2 and N3 were diluted to concentrations ranging from 1 to 107 ng/ml in PBS buffer, placed in the presence of a fresh suspension of rat red blood cells weight/volume) and incubated at 37 0 C for 1 and 5 hours. The mixture is centrifuged at 1500 g for 10 min at room temperature and the quantity of hemoglobin present in the supernatant is evaluated by spectrophotometric assay at 550 nm. The procedure is carried out in the same manner with the dextran and polylysine controls. The results are expressed as percentage lysis relative to a control treated with Triton X-100 (Sigma) which corresponds to 100% lysis.

The data obtained after 1 and 5 hours of contact are illustrated in Figures 3 and 4. The three polymers of the invention show no membrane activity up to 100 ig/ml (105 ng/ml) and above a lytic activity of less than 10% after 5 hours of contact. Under the same conditions, polylysine at a concentration greater than ug/ml affects membrane integrity.

B. Electron microscopy studies Rat red blood cells are incubated for 1 or hours in the presence of 10 jg/ml of pcTGl2 N1, N2, N3, dextran or polylysine and then suspended overnight in a solution of glutaraldehyde (0.25% vol/vol) reconstituted in PBS (Oxoide). The cells are centrifuged at low speed (1200 g 1 min at room temperature; MSE Microcentour centrifuge) and, after removing the supernatant, are taken up in 1% osmium tetroxide (weight/vol) in PBS. After 1 hour at room temperature, centrifugation is carried out under the same conditions as above and the cells are treated with 50% ethanol diluted in PBS for 30 min. The latter operation is repeated with 60%, 70%, 80%, 90% and finally absolute ethanol. The cellular pellet is suspended in hexa- 21 methyldisilazane (HMDS, Sigma) and, after evaporation of the HMDS, the samples are coated with gold (50 LA EMTECH Gold Coater). Examination by electron microscopy (Philips XL) shows that the polymers of the invention do not disrupt the morphology of the red blood cells (a morphology comparable to the cells treated with PBS alone or with dextran) whereas a high percentage of the cells incubated in the presence of polylysine exhibit an impaired morphology (having the shape of a star).

EXAMPLE 5: Transfection of the chitosan/nucleic acid complexes The plasmid pTG11033 (1 mg/ml) is used to evaluate the transfection efficiency of the compounds of the invention. It comprises the luc reporter gene which encodes luciferase placed under the control of the CMV promoter and the gene for resistance to kanamycin. However, any other plasmid may also be used.

DC-Chol is obtained by chemical synthesis on a scale according to the method described by Gao et al.

(1991, BBRC 179, 280-285). The colipid DOPE is commercially available (Sigma, reference P5078). The stock solutions for the preparations of chitosan pcTG12N are stored at 10 mg/ml in chloroform. The DC-Chol/DOPE mixture is obtained by mixing the two compounds in a 1/1 weight ratio and a stock solution is made in chloroform in an amount of 10 mg/ml.

A. Transfection of pulmonary cells The transfections are carried out in 96-well plates in which 2x104 A 549 cells (ATCC CCL185) derived from human pulmonary carcinoma, in Dulbecco's modified Eagle medium (DMEM) containing 10% FCS, are cultured.

After 24 hours of culture at 37 0 C in a CO2 atmosphere the cells are placed in the presence of DNA/DC Chol/DOPE complexes in the presence or in the absence of pcTG12 N1 polymers. The preparation of the complexes is carried out in polypropylene Nunc tubes and under a laminar flow cabinet so as to preserve the sterility of the preparations. Briefly, the lipids are first mixed with the polymer by injection of DC-Chol/DOPE either in 22 water (control 0% of chitosan) or in water supplemented with chitosan (10, 30 or 60%) with the aid of a Hamilton syringe and while the whole is maintained gently stirred. It is indicated that the percentages of chitosan are calculated relative to the charge provided by the cationic lipid DC-Chol. The DNA is then added to the lipid/polymer mixture.

More precisely, 2 mg of DC-Chol/DOPE (200 pl of stock solution) are dried and the dry lipids are taken up in 150 1l of ethanol until complete dissolution is obtained (a sonication of 10 seconds can improve the dissolution) and the following mixtures are generated: DC Chol/DOPE/DNA: 40.5 l1 (540.5 pg) of the preceding lipid solution are added to 500 01 of water of which 455 pl (that is 455 pg) are placed in the presence of 30 pg of pTG11033 dissolved in a volume of 145 l1 of water, DC Chol/DOPE/10% Chitosan/DNA: 40.5 pl (540.5 pg) of the preceding lipid solution are added to 498 p1 of water and 2 l1 (20 pg) of chitosan of which 455 pl are placed in the presence of 30 pg of pTG11033 dissolved in a volume of 145 pl of water, DC Chol/DOPE/30% Chitosan/DNA: 40.5 pl (540.5 pg) of the preceding lipid solution are added to 494 pl of water and 6 pl (60 pg) of chitosan of which 455 pl are placed in the presence of 30 pg of pTG11033 dissolved in a volume of 145 pl of water, DC Chol/DOPE/60% Chitosan/DNA: 40.5 pl (540.5 pg) of the preceding lipid solution are added to 488 pl of water and 12 pl (120 pg) of chitosan of which 455 pl are placed in the presence of 30 pg of pTG11033 dissolved in a volume of 145 pl of water, After 24 hours at 4 0 C, 19 l1 of 5 M NaC1 are added to the preceding mixtures so as to adjust the concentration to 0.9% (154 mM). 86 pl of each of the mixtures thus generated are collected and placed in line A of a 96-well microtiter plate, adjusted to 100 pl with the aid of the DMEM medium and diluted 1/2 serially in DMEM medium. The volume in each well is 23 adjusted to 235 1l with the culture medium and 225 p1 removed and added to the A549 cells which have been in culture for 24 hours. 25 pl of FCS are added 4 hours after the beginning of the transfection. The culture is continued for 48 hours, the medium eliminated and after having been washed with 100 p1 of PBS buffer, the cells are lysed with 50 pl of lysis buffer (Promega) and frozen at -80 0 C until the luciferase activity is measured. The luciferase activity is visualized on 20 p1 (that is 2/5 of the sample) by measuring the emission of photons with the aid of a luminometer (LB96P, Berthold). The results are expressed in RLU/min/sample. As a guide, the protein concentration of the sample determined is 24 to 32 pg.

As shown in Figure 5, the presence of chitosan in the DC Chol/DOPE/DNA complexes is found to be advantageous, the transfection level reflected by the luciferase activity being greater with the complexes containing chitosan than with the complexes lacking it.

Indeed, the luciferase activity is significantly higher by a factor of about 1.5 in the presence of chitosan and by a factor of 2 in the presence of larger quantities (30 and B. Transfection of primary myoblasts The myoblasts are obtained from biopsies performed on quadricep muscles of male dogs. The samples are fragmented into segments about 1 mm 3 and placed in culture in Petri dishes (Falcon) coated with 1% human plasma gelatin. The culture is carried out in Ham F14 medium (Gibco BRL) supplemented with growth factors [2 mM Glutamine (BioMerieux), FGF 10 ng/ml (Peprotech-TEBU), EGF 10 ng/ml (Sigma), insulin pg/ml (Sigma)], gentamicin 40 pg/ml (Scherring Plough) and calf serum at a concentration of 10%. Under these conditions, an extensive growth of the mononucleated cells, mainly myoblasts, is observed (confirmed by cytochemical studies). After removal of the tissue fragments, the layer of myoblasts is dissociated in the presence of trypsin this 24 being before fusion into muscle fibers or myotubes. The culture is continued during at most 15 passages and the cells frozen in liquid nitrogen in DMEM medium containing 10% FCS and 8% DMSO.

For the transfections, after thawing, the cells are distributed in 96-well plates in an amount of 103 cells per well and cultured under standard conditions for 24 hours before being transfected in a serum-free medium with variable quantities of plasmid DNA 1, 0.5, 0.25, 0.125, 0.063, 0.032 or 0.016 [ig) complexed with DC Chol/DOPE alone (by way of comparison) or with the DC Chol/DOPE/Chitosan mixture at a chitosan concentration of 10, 30 or 60%. As above, the preparation pcTG12Nl is used and the percentages of chitosan are expressed relative to the charge provided by DC-Chol. The various complexes are prepared in Ham F14 medium supplemented with gentamicin and glutamine according to conventional methods or as indicated in the preceding example. Once the culture medium has been aspirated, the cells are transfected with 225 [il of each test sample. Four hours after the transfection, the cells are re-placed in the presence of serum by addition of 25 il of FCS to each well. The luciferase activity is determined 48 hours posttransfection after washing the cells in phosphate buffer and lysis (Promega buffer). The measurement is carried out with the aid of a Microlumat LB96P luminometer (Berthold).

The results presented in Figure 6 show that the best transfection rates are obtained with the complexes incorporating chitosan, the luciferase activity being 4 to 9 times higher than when the transfection uses DNA complexed with DC Chol/DOPE alone. In conclusion, the beneficial transfection effect of chitosan was observed in a lung adenocarcinoma cell line and, at an even higher level, in a primary line of myoblasts.

25 EXAMPLE 6: Biodistribution of the polymers pcTG12 N1, N2 and N3 The preparations of chitosan pcTG12 N1, N2 and N3 are labeled with iodine with the aid of the Bolton/ Hunter reagent (Amersham Life Science; IM5861) (Bolton and Hunter, 1973, Biochem J. 133, 529-539). The preparations are adjusted to 10 mg/ml in borate buffer (for 1 1: 3.094 g of boric acid, 3.728 g of potassium chloride, pH 6.5) and 500 1l placed in the presence of triethylamine (193 il for pcTG12 N1 and 163 1l for pcTG12 N2 and N3) allowing deprotonation of the -NH 3 groups to -NH 2 Each polymer thus activated is added to mCi of Bolton/Hunter reagent (100 1l) previously dried and the reaction is allowed to continue for 15 min on ice.

The labeling efficiency is determined on an aliquot of 5 pl by electrophoresis on chromatographic paper (Whatman, ref. 3001 845) and in barbital buffer pH 8.6 (Sigma, ref. B6632). Na1 25 I (Amersham, ref. IMS30) is used as control. The migration of the labeled preparations relative to the control makes it possible to quantify the preparation of free and bound 125I (see thesis by Ruth Duncan 1979, Keele University).

The percentage of free 1251 is estimated less than 1%.

The remainder of the preparation is subjected to dialysis against a solution of 1% HC1 in water (dialysis tubes Sigma D7884) until radioactivity can no longer be detected in the dialysate.

200 p1 of the labeled preparation (5 x 104 cpm) is administered intravenously to Wistar rats (3 rats per preparation). The animals are sacrificed 5 min or min after the injection and the radioactivity is determined on the urine, feces and a selection of organs after digestion in 1 N sodium hydroxide NaOH containing 0.1% Triton X-100 The counting is carried out with the aid of a gamma counter (LKB Wallac) for 10 min per sample. The quantity of radioactivity in each organ relative to the total radioactivity is determined.

26 The results presented in the tables below are expressed as percentage of the dose recovered per organ standard derivation) and show a recovery of 32% min) and 16% (60 min) of the doses injected for the preparation N1 and 36% (5 min) and 47% (60 min) for the preparation N3.

Biodistribution of pcTG12 N1 Organs 5 min 60 min Heart 1.3 0.6 1.0 0.6 Liver 13.7 1.3 26.5 9.6 Lungs 27.8 9.0 7.2 0.8 Kidneys 10.4 1.9 11.7 3.3 Spleen 0.8 0.3 0.9 0.2 Blood 45 6.0 32.2 10.5 Urine 0 19.1 25.9 Feces 0 0.2 0.2 Thyroid 0.8 0.1 1.5 Biodistribution of pcTG12 N3 Organs 5 min 60 min Heart 0.3 0.2 0.1 0.1 Liver 54.7 12.5 82.7 1.9 Lungs 23.5 8.4 2.1 Kidneys 5.6 1.0 10.5 0.2 Spleen 4.4 1.9 2.1 0.1 Blood 11.4 3.6 2.6 Urine 0 0 Feces 0 0 Thyroid 0.2 0.2 0.1 0.1 EXAMPLE 7: Study of the degradation of the chitosan/DNA complexes 1 mg of calf thymus DNA is complexed with increasing quantities of the preparations pcTG12 N1, N2 and N3 in a charge ratio (polymers/DNA) of 1:1 to 1:0.01. The complexes thus formed are placed in the presence of deoxyribonuclease II (DNAase II) according CD/00370 2 4 7 .2 27 to the standard protocol and the state of the DNA is estimated on an agarose gel. The degradation is notably reduced (relative to a noncomplexed DNA control) when the DNA is complexed with chitosan in a high charge ratio.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

Claims

1. Composition comprising at least one compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula ,CH 2 OH HO O O 1 NH 2 and a therapeutically active substance, characterized in that all or part of the X residues constituting said compound is modified by C- and/or 0-alkylation, alcylation, -amino-alkylation and/or -polyoxy- ethylenation.

2. Composition according to Claim 1, characterized in that n represents an integer from 5 to 150.

3. Composition according to Claim 2, characterized in that n represents an integer from 5 to 75, and in particular 5 to

4. Composition according to one of Claims 1 to 3, characterized in that said compound has an average molecular mass of between 5 and 10 kDa.

5. Composition according to one of Claims 1 to 3, characterized in that said compound has an average molecular mass greater than 10 kDa and less than kDa.

6. Composition according to one of Claims 1 to characterized in that said compound is in the form of a poly-N-dodecyl-polyglucosamine or a mono-Ca-octadecyl- polyglucosamine.

7. Composition according to one of Claims 1 to 6, characterized in that said compound is in the form of a salt and more particularly a chloride, sulfate or phosphate.

8. Composition according to one of Claims 1 to 7, characterized in that said compound is substantially free of pigments and of residual impurities. AMENDED SHEET 29

9. Composition according to Claim 1 to 8, characterized in that said therapeutically active substance is a nucleic acid. Composition according to Claim 9, characterized in that said nucleic acid is a vector for the expression of a gene of therapeutic importance placed under the control of elements necessary for its expression in a host cell.

11. Composition according to Claim 10, charac- terized in that the therapeutic gene encodes a ribozyme, an antisense RNA or alternatively a poly- peptide selected from a cytokine (interleukin-2, interleukin-12, alpha-, beta- or gamma-interferon, tumor necrosis factor, colony-stimulating factor, antigen of the major histocompatibility complex and the like), a cell surface or nuclear receptor, a clotting factor (FVII, FVIII, FIX and the like), CTFR (for Cystic Fibrosis Transmembrane Conductance Regulator) protein, dystrophin, insulin, a growth hormone, an enzyme (renin, thrombin, urease and the like), an inhibitor, a viral antigen, an antigenic epitope, a tumor suppressor polypeptide, an antibody, a toxin (thymidine kinase of the herpes simplex-1 virus, ricin, diphtheria toxin and the like), a polypeptide having an antitumor or antiviral activity and a marker poly- peptide.

12. Composition according to one of Claims 9 to 11, characterized in that the charge ratio of said nucleic acid to said compound is between .10/1 and 1/10, and in particular between 1/1 and

13. Composition according to one of Claims 8 to 12, characterized in that it comprises, in addition, at least one adjuvant capable of enhancing the transfect- ing power of said composition.

14. Composition according to Claim 13, charac- terized in that said adjuvant is a cationic amphiphilic compound. AMENDED SHEET 30 Composition according to Claim 14, charac- terized in that said cationic amphiphilic compound is a mono-, di- or tri-alkylated or -acylated compound carrying from 1 to 4 positive charges.

16. Composition according to Claim 15, charac- terized in that said compound is a lipid selected from dioctadecylamide (DOGS), (DC-Chol), (2,3-droleylocyl-N-[2-(sperminecarboxamido)- ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate) (DOSPA), spermine cholesterol and spermidine cholesterol.

17. Composition according to one of Claims 13 to 16, characterized in that said adjuvant is a neutral or zwitterionic lipid.

18. Composition according to Claim 17, charac- terized in that said neutral or zwitterionic lipid is or derives from a phosphatidylethanolamine, phosphatidylcholine, phosphocholine, sphingomyelin, ceramide or cerebroside.

19. Composition according to Claim 18, charac- terized in that said neutral or zwitterionic lipid is dioleylphosphatidylethanolamine (DOPE). Composition according to one of Claims 14 to 19, characterized in that the cationic amphiphilic compound to DNA charge ratio is between 25/1 and 1/1 and preferably 5/1 and 2/1.

21. Use of a composition comprising at least one compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula ,CH 2 OH HO -0 S NH 2 and a nucleic acid as therapeutically active substance for preparing a medicament for curative, preventive or SAMENDED SHEET 31 vaccinal purposes intended for transferring said nucleic acid into a host cell in vivo, ex vivo or in vitro.

22. Use of a composition according to Claim 21, characterized in that n represents an integer from 5 to 150, preferably from 5 to 75 and still more preferably from 5 to

23. Use of a composition according to Claim 21 or 22, characterized in that said compound has an average molecular mass of less than 5 kDa.

24. Use of a composition according to Claim 21 or 22, characterized in-that said compound has an average molecular mass of between 5 and 10 kDa. Use of a composition according to Claim 21 or 22, characterized in that said compound has an average molecular mass of between 10 and 50 kDa.

26. Use of a composition according to one of Claims 9 to 20, for preparing a medicament for curative, preventive or vaccinal purposes intended for transferring said nucleic acid into a host cell in vivo, ex vivo or in vitro.

27. Use according to Claim 26, characterized in that said host cell is a microbial, yeast, insect, plant, human, animal and, in particular, mammalian cell.

28. Use according to Claim 27, characterized in that said human host cell is a muscle cell or a pulmonary epithelial cell.

29. Composition comprising at least one compound compound of formula [X]n in which n represents an integer from 5 to 300 and X has- the following formula ,CH 2 OH H NH 2 0 AMENDED SHEET 32 and a therapeutically active substance, characterized in that said compound has an average molecular mass of less than 5 kDa. Composition according to Claim 29, charac- terized in that n represents an integer from 5 to 150.

31. Composition according to Claim 30, charac- terized in that n represents an integer from 5 to and in particular 5 to

32. Compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula ,CH 2 OH HO SNH 2 characterized in that all or part of the X residues constituting said compound is modified by C- and/or O-alkylation, acylation, -amino-alkylation and/or -polyoxyethylenation.

33. Compound according to Claim 32, in the form of a poly-N-dodecyl-polyglucosamine or a mono-Ca- octadecyl-polyglucosamine.

34. Compound of formula [X]n in which n represents an integer from 5 to 300 and X has the following formula ,CH 2 OH HO ^O H NH 2 characterized in that it is present in the form of a salt and more particularly a chloride, sulfate or phosphate. Compound according to Claims 32 to 34, characterized in that n represents an integer from 5 to

150. AMENDED SHEET 33 36. Compound according to Claim 35, characterized in that n represents an integer from 5 to 75, and in particular 5 to 37. Compound according to one of Claims 32 to 36, characterized in that said compound has an average molecular mass of less than 5 kDa. 38. Compound according to one of Claims 32 to 36, characterized in that said compound has an average molecular mass of between 5 and 10 kDa. 39. Compound according to one of Claims 32 to 36, characterized in that said compound has an average molecular mass greater than 10 kDa and less than kDa. Method for preparing a compound of formula [X]n in which n represents an integer from S to 300 and X has the following formula H 2 OH 0 HO NH 2 0 NH2 characterized in that a preparation of chitosan of high molecular mass is subjected to acid hydrolysis, precipitation with ethanol and a step of extraction of the pigments in the presence of a solvent. 41. Method according to Claim 40, characterized in that said solvent is 2-butanol. 42. Method according to Claim 40 or 41, charac- terized in that it comprises, after the step of extraction of the pigments, a step of differential diafiltration. 43. Method according to Claim 42, characterized in that said step of differential diafiltration comprises a first diafiltration through a membrane having a cut- off of 5 kDa and a second diafiltration through a membrane having a cut-off of 10 kDa carried out on the concentrate of said first diafiltration. AMENDED SHEET CD/00370247.2 34 44. .A method for transferring a nucleic acid as a therapeutically active substance into a host cell in vivo, ex vivo or in vitro for curative, preventative or vaccinal purposes using a composition comprising at least one compound of. the formula [X]n in which n represents an integer from to 300 and X has the following formula ,CH 2 OH 0 HON O NH2 45. A method according to claim 44 in which n represents an integer from 5 to 150. 46. A method according to claim 44 in which n represents an integer from 5 to 47. A method according to claim 44 in which n represents an 15 integer from 5 to a 48. A method according to any one of claims 44 to 47 in which the compound has an average molecular mass of less than *o .g 49. A method according to any one of claims 44 to 47 in which the compound has an average molecular mass of between S" and 50. A method according to any one of c lims 44 to 47 in which the compound has an average molecular mass of between and 51. A composition according to claim 1 substantially as herein described with reference to the examples. CDI00370247 .2 I -o 52. Use of a composition according to claim 21 substantially as herein described with reference to the examples. 53. A method according to any one of claims 44 to 50 in which the host cell is a microbial, yeast, insect, plant, human, animal or mammalian cell. 54. A method according to claim 53 in which the host cell is a mammalian cell. A claim according to claim 52 in which the host cell is a human muscle cell or a human pulmonary epithelial cell. 56. A composition according to claim 29 substantially as herein described with reference to the examples. 57. A method according to claim 40 substantially as herein described with reference to the examples. 9 *S S. Transgene S.A. By its Registered Patent Attorneys Freehills Carter Smith Beadle 16 August 2000