WO1992019752A1

WO1992019752A1 - Rna delivery vector

Info

Publication number: WO1992019752A1
Application number: PCT/FR1992/000393
Authority: WO
Inventors: Pierre Meulien; Shivadasan Krishnan; Frédéric Martinon
Original assignee: Transgene S.A.; Pasteur Merieux
Priority date: 1991-05-03
Filing date: 1992-04-30
Publication date: 1992-11-12
Also published as: EP0537326A1; FR2676072A1; CA2086510A1; JPH06502998A; AU1768192A; AU652831B2; FR2676072B1

Abstract

An RNA delivery vector including a liposome and at least one RNA fragment coding for an antigenic determinant which characterizes a tumor or a pathogenic agent, said at least one RNA fragment being encapsulated in said liposome.

Description

RNA delivery vector

The present invention relates to means intended for the transfer within a cell of an RNA fragment coding for an antigenic determinant characteristic of a tumor or of a pathogenic agent as well as to pharmaceutical compositions comprising them. The present invention applies in particular to the field of vaccines.

In general, traditional vaccination consists in sensitizing the immune system of an individual to an infectious agent (bacteria, virus or parasite). To this end, three types of vaccines have been proposed and developed depending on the case, more or less empirically:

- Inactivated (or killed) vaccines obtained from whole bacteria or viruses which have lost their infectivity thanks to chemical treatments;

Live vaccines which are made up of infectious bacteria or viruses whose virulence has been reduced by mutagenesis or by passage through various media which cause mutations; and

Subunit vaccines, based on bacterial toxins or purified infectious agent antigens. On the other hand, it has recently been proposed to apply the principle of vaccines to the prevention of malignant tumors. Indeed, most tumor cells express on their surface antigens which differ either qualitatively or quantitatively from the antigens present on the surface of the corresponding normal cells. These antigens are said to be specific when they are only expressed by tumor cells. When present on both normal and tumor cells, these antigens are said to be associated with the tumor; in this case, they are present either in greater quantity or in a different form in the tumor cells.

Once the vaccine is administered, the cells of the immune system recognize the antigens of the foreign germ, react specifically to this intrusion and keep these antigens in memory; they will protect the body during a subsequent infection.

Generally speaking, there are two main types of immune response: the humoral type response, which is characterized by the production of antibodies by B lymphocytes, and the cell-mediated immune response, which involves effector cells, ie, essentially T8 lymphocytes (cytotoxic lymphocytes). These responses are initially activated by antigen presenting cells and modulated by regulatory cells i.e., T4 lymphocytes (helper T lymphocytes) and suppressor T lymphocytes.

In broad outline, the immune response works like this:

- Antigen presenting cells (monocytes, macrophages and B lymphocytes) capture the antigen, digest it and re-expose fragments on its surface, in association with molecules of the major histocompatibility complex (MHC) of class I or H .

- T4 lymphocytes stimulate the proliferation of antibody-producing B lymphocytes and that of T8 lymphocytes (cytotoxic T lymphocytes or CTL) when they "see" the fragments of antigens associated with the major class II histocompatibility complex. - B lymphocytes produce antibodies which will interact with circulating antigens so as to neutralize them.

- Finally, T8 lymphocytes destroy infected cells when they recognize the fragments of antigens associated with the major class I histocompatibility complex.

It is currently believed that vaccines are likely to induce both a humoral and cell-mediated response to confer strong and lasting immunity.

Among the types of vaccines mentioned above, live vaccines are often the most effective: they simultaneously activate B, T4 and Tg lymphocytes by conferring a good level of immunity. In addition, as they multiply in the body, they act in low doses and generally do not require a booster. Unfortunately, to stay alive, these vaccines usually need to be kept cold. Maintaining the cold chain during transport is a factor which significantly increases the cost of these vaccines. Finally, their attenuated virulence is not zero and they sometimes induce side effects in immunocompromised subjects. Even more serious, they can recover their virulence following reverse mutations, which is extremely rare but strictly intolerable.

Subunit vaccines do not have these defects, but they generally stimulate immune cells less well and require adjuvants (molecules which boost their immunogenic power). The hepatitis B vaccine, consisting of the major surface antigen of the virus, obtained by recombinant DNA technology, is an example of a successfully developed subunit vaccine. However, in other cases, the results have been disappointing.

It is believed that a factor limiting the success of subunit vaccines is that they are not capable of inducing a sufficiently strong cell-type immune response. This probably results from the impossibility for certain antigens to be correctly processed and / or for their fragments, to come into contact with the molecules of the major histocompatibility complex (MHC) of class I.

Indeed, the association between MHC class I molecules (which participate in the cell type immune response) and peptide fragments takes place in the endoplasmic reticulum of the cell. For this, it is therefore necessary, beforehand, that the antigen reaches this cell compartment. In general, entry into this compartment takes place from the cytosol where the proteins are synthesized by translation of the corresponding messenger RNAs. The passage from the cytosol to the endoplasmic reticulum takes place through two types of mechanisms:

- either a secretion mechanism when the protein is synthesized in the form of a precursor (involving a signal peptide),

- either a peptide "pump" mechanism.

The types of vaccines known up to now absolutely do not make it possible to control this important datum which is the association of MHC class I molecules and peptide fragments. Most often, the infectious elements (bacteria, viruses, parasites or antigens) are indeed absorbed by the phagocytic cells according to the endocytosis (or phagocytosis) mechanism. The endocytosis vesicles fuse with lysosomes whose enzymatic content ensures the degradation of phagocytosed material. These endocytic and lysosomal x vesicles form a separate whole from the endoplasmic reticulum and are not in contact with the latter.

Consequently, even if an antigen is capable of inducing a production of neutralizing antibodies against it, it is absolutely not certain that it will ultimately prove effective for the implementation of a vaccine under- unit.

It has now surprisingly been found that the synthesis of an antigen can be carried out directly in the antigen presenting cells of the individual to be immunized, when the RNA coding for this antigen is administered in the appropriate form eg, in the form of composition liposomal. The fate of the RNA thus injected was indeed highly uncertain given its very short half-life. In addition, it was absolutely not obvious that the RNA was correctly delivered into the cytosol and that it was taken up by the cellular machinery ensuring the translation process.

This new mode of vaccination clearly promotes the association of MHC class I molecules with the peptide fragments resulting from the digestion of the antigen since it has been shown that the individual thus treated is capable of developing an immune response involving cytotoxic T lymphocytes (CTL).

Consequently, the invention proposes:

(i) An RNA delivery vector which comprises a liposome and at least one RNA fragment coding for an antigenic determinant characteristic of a tumor or of a pathogenic agent, said at least one RNA fragment being encapsulated in said liposome;

(ii) A pharmaceutical composition intended for the treatment of a tumor or of a disease induced by a pathogenic agent, for preventive or curative purposes, which comprises, as therapeutic agent, an RNA delivery vector according to the invention ;

(iϋ) The use of an RNA delivery vector according to the invention for the preventive or curative treatment of a tumor or a disease induced by a pathogenic agent;

(iv) The use of an RNA delivery vector according to the invention, for the preparation of a medicament intended for the preventive or curative treatment of a tumor or of a disease induced by a pathogenic agent;

(v) A method of curative or preventive treatment of a tumor or of a disease induced by a pathogenic agent which: comprises the act of administering a sufficient quantity of an RNA delivery vector according to the invention to a patient in need of such treatment. An RNA fragment useful for the purposes of the present invention is by nature a non-infectious RNA fragment, exclusively of the messenger type; that is to say capable of being directly translated into protein by cellular machinery without having to be first replicated or transcribed into DNA. By definition, it does not contain the elements that would allow it to replicate inside a cell. Likewise, it does not contain the genetic information necessary for the reconstitution of a substantially complete pathogenic agent (eg, a bacteria, a virus, or a parasite).

By "antigenic determinant" is meant any peptide comprising at least one epitope characteristic of a tumor antigen or of a pathogenic agent. This antigenic determinant can therefore be the complete native antigen, a precursor or an analog thereof, a fragment of the native antigen or an analog of this fragment. By "analog" is meant a molecule having an amino acid sequence substantially different from that of the native molecule eg, the amino acid sequence of which has a degree of homology of at least about 80%, preferably at least at least 90%, most preferably at least 95% with the sequence of the native molecule.

In general, the antigenic determinant can be characteristic of any type of tumor or any pathogenic agent.

In order to illustrate the above, we cite as an example:

- The nucleocapsid protein (nucleoprotein or NP) of the influenza virus type A affecting humans as defined by the sequence of the mRNA which corresponds to it. This sequence was published in Huddleston & Brownlee, Nucl. Ac. Res. (1982) 10 (3): 1029.

- The fusion protein (F) of the measles virus

- A peptide comprising the epitope of sequence: Thr-Tyr-Glu-Arg-Thr-Arg- Ala-Leu-Val-Arg. This epitope is characteristic of the nucleoprotein of influenza A viruses. - A peptide comprising the sequence: Thr-Tyr-Glu-Arg-Thr-Arg-Ala-Leu-Val-Thr.

- The H23-ETA tumor antigen associated with breast cancer in humans, as described in Wreschner et al, J. Biochem. (1990) 189: 463.

- A peptide comprising the major epitope of the H23-ETA antigen, having the sequence: Pro-Gly-Ser-Thr-Ala-Pro-X-Ala-His-Gly-Val-Thr-Ser-Ala-Pro- Asp-Y-Arg-Pro-X, where X is Pro or Ala and Y is Thr or Asn.

An RNA delivery vector according to the invention can contain different RNA fragments; each of these fragments coding for a particular antigenic determinant. For example, an RNA delivery vector according to the invention intended for the preventive treatment of influenza can contain both:

A fragment of RNA coding for the nucleoprotein of influenza virus type A and a fragment of RNA coding for the nucleoprotein of influenza virus type B or else,

- A RNA fragment coding for the nucleoprotein of influenza A viruses and one or more RNA fragment (s), each of these coding for the hemaglutinin of a particular strain of the influenza virus.

For the purposes of the present invention, an RNA fragment can be obtained in a conventional manner e.g., by chemical synthesis or by in vitro transcription of the corresponding DNA fragment.

By "liposome" is meant a vesicle essentially formed by a membrane made up of lipids in a double layer. Such a vesicle may have one or more membrane strata. In the first case, we speak of a unilamellar vesicle while in the second case it is an oligo- or plurilamellar vesicle. Typically, the liposome membrane is made up of amphiphylic lipids such as phospholipids, in combination or not with other lipid constituents. Phospholipids suitable are, for example, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cardiolipin, phosphatidylinositol, phosphatidic acid and phosphatidylglycerol. In addition, provision may also be made to use synthetic phospholipids, whether or not derived from the phospholipids mentioned above, containing for example various groups such as hydroxyl, imidazole, amine, amides, sulphydiyl groups, etc.

Preferably, other lipids such as steroids, cholesterol, aliphatic amines, can be mixed with the phospholipids. Such lipids are in particular useful as stabilizing or antioxidant agents.

For the purposes of the present invention, a first advantageous lipid mixture consists of phosphatidylcholine (PC) and cholesterol (CH) in variable proportion. However, it is indicated that satisfactory proportions PC: CH are of the order (in terms of share) of 5: 1 to 5: 5. Such a mixture in equal parts is particularly suitable. A second advantageous mixture consists of phosphatidylcholine, cholesterol and phosphatidylserine (PS) in variable proportion, for example CH / PC / PS in 5: 4: 1 proportion.

The preparation of the liposomes as well as the packaging of the RNA can be carried out according to known techniques. In particular, the DNA packaging techniques which are commonly used are applicable in the present case.

In addition, the membrane can also contain various proteins inserted into the membrane, attached by covalent bond to a lipid of the membrane, directly or indirectly via a binding agent, or simply in interaction with a phospholipid. When a protein is linked by covalent bond to a lipid, the latter is preferably a lipid having an amino function such as, for example, phosphatidylethanolamine. Advantageously, these proteins can be used as a targeting agent for liposomes on a particular class of cells. These are, for example, monoclonal antibodies or lymphokines. For the purposes of the present invention, the RNA fragment (s) encoding an antigenic determinant of a tumor or of a pathogenic agent is (are) advantageously accompanied by an RNA fragment coding for an interleukin-1, an RNA fragment coding for an interleukin-4 or a RNA fragment coding for an interleukin-2; the latter being particularly preferred.

In accordance with the aims pursued by the present invention, it should be understood that an RNA fragment coding for an antigenic determinant or an interleukin comprises, in addition to the coding sequence, all the elements necessary for the translation of the latter.

When different RNA fragments make up a vector or a pharmaceutical composition according to the invention, these can be partially or totally linked to each other, so as to form at least one polycistronic RNA fragment.

Finally, a pharmaceutical composition according to the invention can be manufactured in a conventional manner. In particular, the therapeutic agent or agents according to the invention are combined with a diluent or a support which is acceptable from a pharmaceutical point of view. A composition according to the invention can be administered by any conventional route in use in the field of vaccines, in particular by the subcutaneous route, by the intramuscular route or by the intravenous route, for example in the form of a suspension. injectable. It is indicated that the subcutaneous route is particularly well suited for administration of a composition according to the invention. The administration can take place in single dose or repeated one or more times after a certain interval of interval. The appropriate dosage will vary depending on various parameters, for example, the individual being treated or the mode of administration.

The invention is illustrated below with reference to the following figures:

Figure 1 shows the plasmid pGEM-3Z. FIG. 2 diagrams the inserts of the plasmids pTG3003 (b.ase pGEM- 3Z), pPM2 (base pUC18), pPM3 (base pUC18), pPM4 (base pUC18), pPM5 (base pGEM-3Z) and pPM6 (base pGEM-3Z ). NP = nucleoprotein; IL-2 = interleukin-2; S. signal = signal sequence of the precursor of IL-2; 3'NTRβG = 3'untranslated end of the rabbit β-globin gene.

FIG. 3 diagrams the inserts of the plasmids pPM9, pPM10, pPM11 and pPM12 (each based on pGEM-4Z). 5'NTRβG = 5'untranslated end of the rabbit β-globin gene.

EXAMPLE 1 Preparation of liposomes loaded with RNA coding for the nucleoprotein of the influenza virus type A.

1A. RNA preparation.

An EcoI-Sali fragment comprising the cDNA coding for the influenza virus type A nucleoprotein, strain A / NT / 60/68 (Huddleston & Brownlee, Nucl. Ac. Res. (1982) 1Q (3): 1029) is obtained by digestion of the plasmid pNP28 (Jones & Brownlee, Gene (1985) 25: 333).

This EcoRf-Sali fragment is inserted into the plasmid pGEM-3Z (Promega Corp; FIG. 1) digested with EcoRI and Sali so as to place the cDNA coding for the nucleoprotein under the control of a promoter recognized by the RNA polymerase of phage T7 . The plasmid pTG3003 is thus obtained.

The plasmid pTG3003 is then linearized by digestion with SalI; then transcribed using an in vitro transcription kit (Stratagene; catalog ref.

200341) according to the supplier's instructions. The RNA thus obtained is capped by adding to the transcription buffer 10 μl of a solution of m7G (5 ') ρpp (5') G 5 mM (Pharmacia; product number 27-4635).

1B. Preparation of liposomes.

A cholesterol solution is prepared by dissolving the powdered product (Sigma) in chloroform at the rate of 100 mg / ml. Then in a 30 ml Corex tube _. 58 μl of the cholesterol solution and 110 μl of a phosphatidylcholine solution at 100 mg / ml (Sigma) are mixed. The final mixture contains approximately

30 μmoles of lipids.

This mixture is vigorously stirred with a vortex. Then the solvent is allowed to evaporate at 40 ° C. The drying operation is completed by lyophilization for approximately 1 hr.

The lipid layer deposited at the bottom of the tube is taken up in 1 ml of water. The mixture is homogenized with a vortex and sonicated at the minimum frequency using a Braun Labsonic L sonicator. The operation is carried out for 10 min. by intermittent passages of 15 seconds, spaced by 30 seconds of cold rest.

The suspension thus obtained is centrifuged at 16,000 rpm, at 4 ° C for 15 min. to sediment titanium particles and larger vesicles

(Biofuge / Heraeus in eppendorff tube). Then the supernatant containing a majority of unilamellar vesicles having a diameter of approximately 50 microns, is recovered in 15 ml Corex tubes.

To the vesicular suspension obtained in B), 135 μl of the RNA preparation obtained in A) are added; or approximately 40 μg of RNA. The mixture is gently immersed in liquid nitrogen, rolling the tube kept vertical between his fingers. When the mixture is completely frozen, it is lyophilized overnight.

The lyophilisate is gradually rehydrated in 100 μl of distilled water. Then added dropwise 0.9 ml of Hepes buffer pH 7.05 of composition: 140 mM NaCl, KC15 mM, Na2_HPθ4 2H2O 0.75 mM, Hepes 25 mM. The vesicular suspension is then calibrated by successive extrusions through a first and second filter whose pores have a diameter of 400nm and 200nm respectively. The suspension thus obtained contains a majority of oligolamellar vesicles which have encapsulated the RNA.

In order to substantially eliminate the non-encapsulated RNA, the suspension is deposited on a Sepharose 4B column (10 ml; column of section 1 cm) pre-balanced in Hepes buffer. Elution is continued in Hepes buffer. The eluted fractions of white appearance which contain the vesicles are collected together; this corresponds to a volume of approximately 2 ml. EXAMPLE 2 Induction of a CTL response initiated by liposomes loaded with RNA coding for the nucleoprotein of the influenza A virus.

Three series of BALB / c mice corresponding respectively (i) to the test, (ii) positive control and (iii) to the negative control are prepared as follows:

(i) Test: the mice receive 200 μl of the liposome preparation obtained in Example 1 intravenously.

(ii) Positive control: the mice receive 130 hemaglutinating units of the influenza virus type A, strain Caen (A-Caen) intraperitoneally. The different type A strains sometimes differ from each other for certain proteins such as hemaglutinin or neuraminidase but never for nucleoprotein. However, there are variations regarding the nucleoprotein between type A and B viruses.

(iii) Negative control: the mice receive intravenously 200 μl of a preparation of liposomes prepared as in Example 1, the addition of RNA having been omitted.

15 days later, the mice of (i) and (iii) receive an injection identical to the first and under the same conditions.

15 days after the second injection, the spleen of the mice is removed and dilacerated. The red cells are eliminated by osmotic shock. Then the spleen cells are cultured in plates with cups at a rate of 5.10 "cells / cups in a volume of 2 ml in DMEM culture medium (Gibco) supplemented with 10% fetal calf serum, 2 mM L- glutamine, 50 μM β-mercaptoethanol, 10 mM Hepes, 1 mM sodium pyruvate, 10 units / ml penicillin, 10 μg / ml streptomycin, 2.5 μg / ml amphotericin and non-amino acids -essential (Flow labs).

This medium also contains 5 μM of peptide NP 147-158R ^" of formula: Thr-Tyr-Glu-Arg-Thr-Arg-Ala-Leu-Val-Thr-Gly. This peptide corresponds to an epitope systematically recognized by CTL BALB / c mice when they are immunized with influenza A virus (Bodmer et al, Cell (1988) 52: 253).

The culture is continued at 37 ° C. under an atmosphere containing 7% CO 2. After 7 days, the initial culture medium is replaced by fresh medium. In each well, 5 10 ⁶ spleen cells of BALB / c mice not subjected to immunization are added, these cells having been irradiated with 4000 Rad of gamma radiation.

On the ^12th day of culture, the activity of the CTLs is detected by the standard ^{5 **} Cr release test, especially described by Martinon et al, J. Immunol. (1989) 142: 3489. Details are given as follows:

1 to 10 × 10 ⁶ mouse mastocytoma cells DBA / 2 (H-2d) histocompatible with BALB / c mice (P815 cells), in DMEM medium, are incubated in the presence of 100 μCi of Na2 H 1-O4 (CEA, France ) for 1 hour at 37 ° C. The cells are washed twice, then taken up in DMEM medium.

The operation is repeated using P815 cells infected with the influenza virus type A, Bangkok strain (P815 / A-Ban) or by the influenza virus type B Yamagata strain (P815 / B-Yam) L infection of the P815 cells is first carried out as follows: 10 "cells in 0.5 ml of DMEM medium are infected with 100 haemaglutinating units of virus. After 90 min at 37 ° C., the cells are washed in 20 ml of DMEM medium supplemented with 10% VF to stop infection Expression of virus proteins is continued 3 hr at 37 ° C before the cells are used in the cytolysis test.

The P815 cells (target cells) are then distributed in the wells of a microtiter plate at the rate of 3000 cells per well. In the three cases (i), (ii) and (UT), the spleen cells (effector cells), in DMEM medium, are added to each well so as to constitute series forming a range of effector / target cell ratio included between 100/1 and 0.3 / 1 approximately. The peptide NP 147-158R ^" is finally added to the final concentration of 2.5 μM. In each well, the final volume is 200 μl. The plate is then incubated 4 hr at 37 ° C and then centrifuged. From each well, take 100 μl of supernatant. The radioactivity of each sample is measured and the results are expressed in the table below, as a percentage of the release of chromium due to the activity of CTL:

(observed release - spontaneous release) lOOx

(total amount of chromium incorporated - spontaneous release)

KXHMPLE 3: Second preparation of liposomes loaded with RNA coding for the nucleoprotein of the influenza A virus.

3A. The RNA is obtained as previously described in 1A.

3B. Preparation of liposomes.

A cholesterol solution is prepared by dissolving the powdered product in chloroform at the rate of 100 mg / ml. Then, in a 30 ml Corex tube, 58 μl of the cholesterol solution are mixed, 176 μl of a dipalmitoyl phosphatidylcholine solution at 50 mg / ml and 219 μl of a phosphatidylserine solution at 10 mg / ml. The final mixture contains approximately 30 μmoles of lipids.

This mixture is vigorously stirred with a vortex. Then the solvent is allowed to evaporate at 40 ° C. The drying operation is completed by lyophilization for 1 hr.

The lipid layer deposited at the bottom of the tube (approximately 30 μmoles) is taken up in 300 μmoles of octyl-β-glucoside. The solution should be clear.

To this preparation, approximately 50 μg of RNA prepared in 3A is added. In order to remove the detergent, this mixture is dialyzed against 10 mM Hepes buffer pH 7.05 containing 150 mM NaCl and 2.7 g of Biobeads-SM2 adsorbent (9 mg of adsorbent / μmole of detergent). The dialysis is continued with stirring for 16 hr at room temperature.

The vesicular suspension is then calibrated by three successive extrusions at 40 ° C through membranes whose pores have a diameter of 200 nm; then finally eluted in Hepes buffer on a Sepharose CL4B column. EXAMPLE 4 Preparation of liposomes loaded with RNAs coding for (i) the nucleoprotein of the influenza virus type A and (ii) human rinterleukin-2.

4A. RNA preparation.

The plasmid pTG3003 described in 1A is partially digested with SphI and treated with Klenow polymerase. We religion by inserting a MluI linker and selecting a plasmid having lost the SphI site located 3 ′ of the sequence coding for NP, with replacement by MluI; or the plasmid pPM1.

On the other hand, the sequence coding for IL-2 is recovered, in the form of the EcoRI-JCbal fragment of a phage M13 marketed by British Biotechnology under the reference BBG3. This fragment is inserted into pUC18 (Viera & Messing, Gene (1982) 19: 259) to give the plasmid pPM2.

A .Xbal-HindIII fragment comprising the 3 'untranslated end of the rabbit β-globin gene (Kafatos et al, PNAS (1977) 72: 5618) is synthesized by PCR using the primers OPM1 and OPM2.

OPM1:

Xbal ₅ ^/ TTTTTCTAGAGCCCTGGCTCACAAATACCACTGAC 3 '

OPM2:

"HindIII MluI 5 'AAAAAAGCTTACGCGTTTTTTTTTTTTTTTTGCAATGAAAATAAATTTCC 3'

This fragment is inserted into pPM2, previously digested with Xbal and HindIII. The plasmid pPM3 is thus obtained.

A synthetic DNA fragment comprising the sequence coding for the signal peptide of human IL-2 is inserted into pPM3 previously digested with EcoRI and -Xhol to give the plasmid pPM4. This synthetic DNA fragment is formed by hybridization of the following oligonucleotides:

OPM3:

EcoRI Sali

5 'AATTCGCGCGTCGACATAGGCCTATCCACC ATG TAC AGG ATG CAA

CTC GTG TCA TGC ATT GCA CTA AGT CTT GCA CTT GTC ACA AAC

AGT GCT CCT ACT AGC 3 'X ol

OPM4:

Xhol 5 'TCGAGCT AGT AGG AGC ACT GTT TGT GAC AAG TGC AAG ACT TAG TGC AAT GCA TGA CAC GAG TTG CAT CCT GTA CAT GGTGGATAGGCCTATGTCGACGCGCG 3'

Sali EcoRI

Finally, the assembly coding for the precursor of IL-2 and the 3 'untranslated region of the rabbit β-globin gene is recovered from pPM4 in the form of a SalI - MluI fragment to be inserted into pPM1 previously digested with Sali and MluI in order to give the plasmid pPM5.

The plasmid pPM5 is then linearized by MluI digestion; then transcribed using an in vitro transcription kit (Megascript from Ambion). The RNA thus obtained is capped by adding to the transcription buffer 10 μl of a solution of m7G (5 ') ppρ (5') G 5 mM (Pharmacia; product number 27-4635).

4B. The preparation of the liposomes is carried out as previously described in 3B.

About 66 μg of RNA obtained in 4A / 30 μmol of lipids is used. EXAMPLE 5 Third Preparation of Liposomes Loaded with RNA Encoding the Influenza Virus Type A Nucleoprotein

5A. RNA preparation.

The plasmid pPM5 is digested with StuI and Hpal so as to eliminate almost all of the region coding for the precursor of IL-2; then religated to give the plasmid pPM6.

pPM6 is then linearized by MluI digestion; then transcribed using an in vitro transcription kit (Megascript from Ambion). The RNA thus obtained is capped by adding to the transcription buffer 10 μl of a solution of m7G (5 ') ppp (5') G 5 mM (Pharmacia; product n ° 27-4635).

5B. The preparation of the liposomes is carried out as previously described in 3B

About 66 μg of RNA obtained in 5A / 30 μmol of lipids is used.

EXAMPLE 6 Preparation of liposomes loaded with RNA coding for the fusion protein of the measles virus.

6A. RNA preparation.

The plasmid pTG2148 described in patent application EPA 305 209 comprises the DNA fragment coding for the fusion protein (Buckland et al, J. Gen. Virol. (1987) £ 8: 1695). pTG2148 is digested with HindIII and Hpal from mother to eliminate the 5 'untranslated region of the gene coding for the fusion protein. This is replaced by the 5 'untranslated region of the rabbit β-globin gene reconstructed by hybridization of the oligonucleotides OPM5 and 6. The plasmid pPM7 is thus obtained. OPM5 î HindIII 5 'AGCTTACACTTGCTTTTGACACAACTGTGTTTACTTGCAATCCCCCAAAACAGC CACCATGGGTCTCAAGGTGAACGTCTCTGCCATATTCATGGCAGTACTGTT 3' Hpal

OPM6: Hpal 5 'AACAGTACTGCCATGAATATGGCAGAGACGTTCACCTTGAGACCCATGGTGG CTGTTTTGGGGGATTGCAAGTAAACACAGTTGTGTCAAAAGCAAGTGTA 3' HindIII

On the other hand, the plasmid pGEM-4Z (Stratagene) is digested with EcoRI, then treated with Klenow polymerase. We religion by inserting a MluI linker to obtain the plasmid pPM8.

Finally, the 5 'untranslated region of the β-globin gene and the sequence coding for the fusion protein are recovered from pPM7 in the form of a HindIII-Sacl fragment. This is inserted into pPM8 previously digested with HindIII and Sac1. The plasmid pPM9 is thus obtained in which the sequence coding for the fusion protein is placed under the control of the T7 promoter.

pPM9 is linearized by MluI digestion; then transcribed using an in vitro transcription kit (Megascript from Ambion). The RNA thus obtained is capped ^* by adding to the transcription buffer, 10 μl of a solution of m7G (5 ') ρpp (5') G 5 mM (Pharmacia; product number 27-4635).

6B. The preparation of liposomes is carried out as previously described in 3B.

About 60 μg of RNA obtained in 6A / 30 μmol of lipids is used. EXAMPLE 7 Second preparation of liposomes loaded with RNA coding for the fusion protein of the measles virus.

7A. RNA preparation.

A synthetic DNA fragment, of sequence (double strand) is prepared:

EcoRI Xbal MluI

5 'GAATTCCACTCTAGACACA 3' 3 'TCGACTTAAGGTGAGATCTGTGTGCGC 5' cohesive end Sali

This DNA fragment is inserted into pPM9, probably digested with SacI and MluI, to give the plasmid pPM10 which no longer has a SacI site.

The .Xbal - MluI fragment of pPM5 which comprises the 3 'untranslated end of the β-globin gene is recovered, then inserted into pPM10 previously digested put Xbal and MluI to give the plasmid pPM11.

pPMll is then linearized by MluI digestion; then transcribed using an in vitro transcription kit (Megascript from Ambion). The RNA thus obtained is capped by adding to the transcription buffer, 10 μl of a solution of m7G (5 ') pρp (5') G 5 mM (Pharmacia; product number 27-4635).

7B. The preparation of the liposomes is carried out as previously described in 3B1

About 60 μg of RNA obtained in 7A / 30 μmol of lipids is used. EXAMPLE 8 Preparation of liposomes loaded with RNAs coding for (i) the fusion protein of the measles virus and (ii) human interleukin-2.

8A. RNA preparation.

The assembly coding for the precursor of IL-2 and the 3 'untranslated region of the β-globin gene of the plasmid pPM4 is recovered in the form of an EcoIU-Mul fragment. This fragment is inserted into pPM11 previously digested with EcoRI and MluI to give the plasmid pPM12.

pPM12 is then linearized by MluI digestion; then transcribed using an in vitro transcription kit (Megascript from Ambion). The RNA thus obtained is capped by adding to the transcription buffer, 10 μl of a solution of m7G (5 ') pρp (5') G 5 mM (Pharmacia; product number 27-4635).

8B. The preparation of liposomes is carried out as previously described in 3B.

About 87 μg of RNA obtained in 8A / 30 μmol of iipids is used.

EXAMPLE 9 Induction of a CTL response initiated by the liposomes prepared in Examples 3, 4 and 5.

The CTL response is implemented and analyzed as described in Example 2, except for 2 modifications: it is specified that 500 μl of each of the liposomal preparations obtained in Examples 3, 4 and 5 are used to inject a mouse ( first injection as well as booster) and that the injections are made subcutaneously.

Some tests are performed 15 days after the first injection.

On the other hand, parallel experiments make it possible to test (i) the RNA mixed with liposomes but not encapsulated and (ii) the pure RNA. In case (i), 50 μg of RNA prepared in 1A mixed with empty liposomes prepared according to the method described in 3B with the exception of the addition of RNA, corresponding to approximately 30 μmol of lipids. In case (ii), 50 μg of RNA prepared in 1A is injected in a volume of approximately 0.5 - 1 ml.

The results are presented in the table below.

Injection Effectors P815 / - P815 P815 P815

/ target / NP / A-Ban / B-Yam

These results show in particular that:

- RNA alone is ineffective in inducing an immune response;

- RNA mixed with liposomes but not encapsulated is ineffective in inducing an immune response;

- the RNA coding for NP alone, encapsulated in liposomes, is capable of inducing an immune response only after booster (comparison tests # 1 and 3); and

- the RNA coding for NP and IL-2, encapsulated in liposomes is capable of inducing an immune response after a single injection (comparison tests

# 2 and 3).

EXAMPLE 10 Immunization of Mice Against Measles Virus Using the Liposomes Prepared in Examples 6, 7 and 8.

The animal model which is implemented is substantially as described in Drillien et al, PNAS (1988) 8: 1252.

5 batches of 8 BALB / c mice are constituted.

A batch is reserved for the positive control. These mice receive 4 x 10 'plaque forming units of the vaccinia virus WTGFM1173 intraperitoneally in a single injection. WTGFM1173 contains the measles virus fusion protein gene. It has been described as an active immunogenic agent

(Drillien et al, supra).

A batch is reserved for the negative control. These mice receive, subcutaneously, approximately 30 moles of lipids in the form of empty liposomes prepared according to the method described in 3B (with the exception of the addition of RNA) as a first injection and as a booster 15 days later.

The other 3 lots are reserved for tests. The mice receive a first injection subcutaneously and a booster 15 days later. The first injection and the booster each correspond to approximately 0.4 - 0.6 ml of the liposomal preparations of Examples 6, 7 and 8. About 15 days after the booster, the mice are tested by intracerebral injection of 50 l of a 10% suspension of brain cells of newborn mice previously infected with the SSPE isolate of the measles virus (Wild et al, J. Med. Virol. (1979) 4: 103).

This test indicates that the liposomes of Examples 6, 7 and 8 have an immunogenic action similar to that of VVTGFM1173.

Claims

CLAIM

i. An RNA delivery vector which comprises a liposome and at least one fragment of messenger RNA coding for an antigenic determinant characteristic of a tumor or of a pathogenic agent, said at least one fragment of RNA being encapsulated in said liposome .

2. An RNA delivery vector according to claim 1, which comprises a liposome, at least one fragment of RNA coding for an antigenic determinant characteristic of a tumor or of a pathogenic agent and a fragment of RNA coding for an interleukin-2; said at least one RNA fragment and said RNA fragment coding for an interleukin-2 being encapsulated in said liposome.

3. An RNA delivery vector according to claim 2, wherein said at least one RNA fragment and said RNA fragment coding for interleukin-2 being linked to each other so as to form a fragment of polycistroπ RNA.

4. An RNA delivery vector according to one of claims 1 to 3, wherein said at least one DNA fragment codes for an antigenic determinant characteristic of an influenza virus which is selected from the protein of the nucleocapsid of said virus and hemaglutinin of said virus.

5. An RNA delivery vector according to claim 4, wherein a first RNA fragment codes for the nucleocapsid protein of the influenza virus and a second RNA fragment codes for the hemaglutinin of the influenza virus flu; said first and second RNA fragments being linked to each other to form a polydstronic RNA fragment which is encapsulated in said liposome.

6. A pharmaceutical composition intended for the treatment or prevention of a tumor or of a disease induced by a pathogenic agent, which comprises, as therapeutic agent, a vector of RNA delivery according to one of claims 1 to 5, with a pharmaceutically acceptable diluent or carrier.

7. The use of an RNA delivery vector according to one of claims 1 to 6, for the preparation of a medicament intended for the preventive or curative treatment of a tumor or of a disease induced by an agent pathogenic;

8. A method of curative or preventive treatment of a tumor or of a disease induced by a pathogenic agent which comprises the act of administering a sufficient quantity of an RNA delivery vector according to one of claims 1 at 6, to a patient in need of such treatment.