EP1583560A1

EP1583560A1 - Composition for intracellular transport of biological particles or macromolecules

Info

Publication number: EP1583560A1
Application number: EP03815710A
Authority: EP
Inventors: Alain Prochiantz; Edmond Dupont; Alain Joliot; Alain Trembleau; Michel Volovitch
Original assignee: Centre National de la Recherche Scientifique CNRS; Ecole Normale Superieure
Current assignee: Centre National de la Recherche Scientifique CNRS; Ecole Normale Superieure
Priority date: 2003-01-07
Filing date: 2003-12-31
Publication date: 2005-10-12
Also published as: FR2849603B1; FR2849603A1; US20070054401A1; AU2003303902A1; WO2004069279A1; AU2003303902A8

Abstract

The invention relates to a composition comprising a macromolecule or particle having one or several hydrophobic domains on the surface thereof whereby at least one transducer peptide is adsorbed thereon. Said composition can be used to introduce the macromolecule or particle into living cells.

Description

COMPOSITION FOR INTRACELLULAR TRANSPORT OF BIOLOGICAL MACROMOLECULES OR PARTICLES.

The present invention relates to new means of intracellular transfer of macromolecules or particles of interest.

The importation of macromolecules, and in particular of polynucleotides or proteins, into living animal cells constitutes a basic approach, both for basic research and in the context of various applications, for example in gene therapy.

One of the major difficulties of this approach results from the need to transport these macromolecules across the cell membrane.

This problem has been the subject of a great deal of research, which has led to the development of different methods of intracellular transfer and of different types of vectors.

Thus, the introduction of polynucleotides into cells is currently essentially based on transfection techniques (calcium phosphate, electroporation), lipofection (liposomes, charged lipids) or viral infection (lentivirus, adenovirus, l virus). herpes, etc.) or on the use of nanoparticles.

More recently, it has been proposed to use transducing peptides. This term denotes peptides comprising, or constituted by, a sequence called

"transduction domain" conferring on them the capacity to penetrate inside a living cell, independently of the presence of specific transporters or receptors. Review articles concerning transducer peptides have been published recently by LIDGREN et al. ,

TiPS, 21, 99-102, (2000); SCHWARZE and DOWDY TiPS, 21, 45-48,

(2000); SCHWARZE et al. Trends Cell. Bάol., 10, 290-295,

(2000); PROCHIANTZ Current Opinion in Cell Biology, 12, 400-406, (2000); Cell-Penetrating Peptides. Processes and applications. Ed. Ulo Langel. CRC Press (2002). By way of examples of transducing peptides, there may be mentioned in particular:

- penetratins, which are peptides derived from the third helix of a homeodomain; peptides of the penetratins family are described for example in the publications of JOLIOT et al. Proc. Natl. Acad. Sci. USA, 88, 1864-1868, (1991); DEROSSI et al. J. Biol. Chem. , 269, 14, 10444-10450, (1994); BRUGIDOU et al. Biophys. Biochem. Res. Co. , 214, 685-693, (1995), as well as in US Patent 5,888,762, US Patent 6,080,724, or PCT Application WO 00/01417; peptides derived from the Tat protein of HIV1, and in particular from the 48-60 fragment of said protein; such peptides are described for example by FAWELL et al. Proc. Natl. Acad. Sci. USA., 91, 664-668, (1994) or by VIVES et al. J. Biol. Chem., 272, 16010-16017,

(1997) .

- peptides derived from the HS22 protein VP22; such peptides are described for example by ELLIOTT and O'HARE Cell, 88, 223-233, (1997); peptides derived from a signal sequence conjugated to a nuclear localization sequence; such peptides are described for example by LIN et al. J. Biol. Chem., 270, 14255-14258, (1995); J. Biol. Chem., 271, 5305-5308, (1996), LIU et al. Proc. Natl. Acad. Sci. USA, 93, 11819-11824, (1996), MORRIS et al. Nucleic Acids Res., 25, 2730-2736, (1997), CHALOIN et al. Biochemistry, 36, 11179-11187, (1997); Biochem. Biophys. Res. Commun., 243, 601-608, (1998), ZHANG et al. Proc. Natl. Acad. Sci. USA, 95, 9184-9189, (1998);

- the transportanes which are derived from a fusion between a portion of a neuropeptide, galanine, and a peptide of the wasp venom POOGA et al. , FASEB J., 12, 67-77,

(1998); Ann. New York Acad. Sci., 863, 450-453, (1998). The transducing peptides can import into living cells, in particular animal cells, molecules or molecular complexes of varied nature (acids nucleic acids, proteins, peptides / nucleic acids, nucleotide analogs, liposomes).

These molecules or molecular complexes are usually designated by the general term "cargo ships". It has been reported that certain transducing peptides could import large cargo ships.

Thus, LEWIN et al. (Nat. Biotech, 18, 410-414, 2000) conjugated a derivative of the TAT 48-60 transducing peptide to nanoparticles consisting of an iron oxide nucleus coated with a dextran envelope, and observed that the nanoparticles thus modified (45 nm in diameter) were imported into living cells.

EGUCHI et al. (J. Biol. Chem., 276, 28, 26204- 26210, 2001) constructed recombinant λ phages expressing on their surface a chimeric protein comprising the TAT transducer peptide fused to the N-terminal end of the phage protein D , and containing a marker gene. They observed, after incubation of these phages with COS-1 cells in culture, an intracellular expression of the marker gene in a proportion of these cells of up to 30%.

It is however generally considered that one of the major limitations of the transducer peptides mentioned above, such as the penetratins or the TAT peptides, lies in the need to couple by covalent bond (s) the transducer peptide and the cargo .

In order to overcome this limitation, peptides designed to bind by ionic or hydrophobic interactions either with nucleic acids or with proteins, have been constructed. One of these peptides, called MPG, is intended for the intracellular transport of nucleic acids (MORRIS et al., Nucl. Acids Res., 2730-2736, 1997; Nucl. Acids Res., 3510-3517, 1999); it comprises two distinct regions, separated by a binding peptide: an N-terminal hydrophobic region derived from the glycine-rich signal sequence of the gp41 protein of HIV1, allowing the fusion with the cell membrane, and a hydrophilic region derived from the nuclear localization sequence of the SV40 T antigen, allowing the interaction of the peptide with the nucleic acid, and its nuclear addressing. The other, called Pep-1 (MORRIS et al., Nature

Biotech, 19, 1173-1176, 2001) is intended for the transport of proteins. It differs from MPG in the nature of the N-terminal hydrophobic region, which consists of a sequence rich in tryptophan, intended to allow addressing to the cell membrane and the formation of hydrophobic interactions with proteins.

These two types of peptides are also described in PCT application WO 02/10201, which generally proposes to use, for importing proteins into living cells, peptides of 16 to 30 amino acids comprising two distinct successive domains: a hydrophobic domain, containing 3 to 5 tryptophan residues including at least one Trp-Trp pair, alternating with glutamic acid and threonine residues; a hydrophilic domain containing 4 or 5 consecutive basic residues (lysine or arginine), these two domains being optionally separated by a spacer domain containing a proline residue or a glutamine residue. Effective importation was only observed, however, in the case of peptides additionally carrying a cysteamine group. Furthermore, in the context of work on the properties of the transducing peptides of the penetratins family, the inventors have evaluated the capacity of these peptides to import cargoes of large size. For this purpose, they tested one of these peptides, using a λ phage as cargo. They then found not only that this peptide was capable of importing the phage into an animal cell, but also that, contrary to what was previously assumed, the importation could be carried out without it being necessary to couple the peptide and the phage by covalent bond. In addition, the inventors found that the efficiency of this import was much higher than that observed by EGUCHI et al. with the transducing peptide TAT coupled by peptide bond to the N-terminus of protein D of phage λ.

To explain these results, surprising in view of the difference in structure between the penetratins and the peptides of PCT application WO 02/10201, the inventors propose the following hypothesis: the penetratins have a transduction domain capable of adopting a secondary structure (in helix α or in β sheet) amphiphilic, having a face having hydrophobic residues, and a charged face comprising a tryptophan residue framed by 2 basic residues ensuring the interaction with the membranes and the formation of a reverse micelle allowing 1 ' internalization of the peptide in the cell. For example, in the case of pANTP-type penetatin, the Ile, Trp, Phe residues at positions 3, 14, and 7 of the peptide sequence, form in the α-helix a hydrophobic triplet; this hydrophobic triplet is distant from the charged zone constituted by the Lys residues (position 13 of the peptide sequence) and Arg

(position 10 of the peptide sequence), which in the α helix surround the Trp residue in position 6 of the peptide sequence (DEROSSI et al. J. Biol. Chem, 271, p 18188-18193, 1996).

It is assumed that the hydrophobic face of the transduction domain allows the formation of interactions of sufficient strength to ensure stable attachment of the transducing peptide to the cargo. Interaction with the membrane would take place via the face charged with the transduction domain; Trp framed by two charged amino acids can be inserted into the membrane (this insertion has been observed by fluorescence studies of tryptophan), destabilizing it and allowing the passage of the vector and its cargo.

The subject of the present invention is a process for preparing a composition making it possible to introduce into a living cell, in particular a eukaryotic cell, and in particular an animal cell, a cargo ship constituted by a macromolecule or a molecular assembly (for example a particle), of size less than or equal to about 1 μ in its largest dimension, said cargo having on its surface one or more hydrophobic domains, which method is characterized in that it comprises the adsorption on said hydrophobic domain (s), of at least one transducing peptide, with the exception of peptides described in PCT Application WO 02/10201.

According to a preferred embodiment of the present invention, said cargo is a protein or a particle having a surface of protein nature. It is also possible to use liposomes, nanoparticles, glycolipids, or any natural or artificial macromolecular combination as cargo.

According to another preferred embodiment of the present invention, said cargo is generally of size less than or equal to 500 nm in its largest dimension.

This includes, for example, viral or pseudoviral particles, in particular phage particles.

According to yet another preferred embodiment of the present invention, said transducing peptide is a peptide from the family of penetratins.

We define here as "peptide of the penetratins family" any peptide comprising a transduction domain capable of adopting an amphiphilic secondary structure (in α helix or in β sheet) having a face comprising hydrophobic residues allowing interaction with the cargo ship , and a face allowing interaction with the membranes, comprising a tryptophan residue surrounded by basic residues.

This includes in particular the penetratins described in PCT application WO 00/01417, and more particularly those comprising a transduction domain defined by one of the formulas below:

Xl-X2-X3-X4 ~ X5-X6 ~ X7-X8-X9 ~ Xl0 ~ Xll-Xl2-Xl3-X-.4 ~ Xl5 ^_ Xl6 (I) X; L6-Xl5 ~ Xl4 "^'" Xl3 ~ l2 -Xll-Xl0-X9 ~ X8-X7-X6 ~ 5-X4- 3 ~ 2- -. (there) in which X ₆ represents a tryptophan residue, Xi, X ₂ , X, X ₉ , X _i5 , Xi6, are amino acids non-hydrophobic and X ₃ , X, and Xχ, are hydrophobic amino acids.

Fields of transduction which are particularly preferred for the implementation of the present invention are those in which Xχo and Xχ ₃ are basic amino acids.

It is also possible to use penetratin derivatives, for example some of the truncated or substituted penetratins described in PCT Application WO 00/01417, or PCT Application WO 00/29427. It is also possible to use a transducer peptide comprising, in addition to the transduction domain, one or more other functional domains; by way of example, mention will be made of the peptides comprising a transduction domain and a nuclear export sequence described in PCT application WO 02/39947.

The adsorption of the transducing peptide is carried out in a simple manner, by incubation for at least 15 minutes, preferably for 30 to 60 minutes, of said transducing peptide with the cargo. Incubation can be carried out ex vivo or in vivo, in a very wide range of temperatures, generally between 15 and 40 ° C. It will preferably be carried out at ambient temperature, that is to say around 20 to 25 ° C, or at physiological temperatures (around 37 ° C), in a medium with neutral pH; it can be for example a culture medium for cells, or a NaCl solution (9 g / 1).

The transducer peptide / cargo molar ratio in the incubation medium depends in particular on the size of the cargo vessel, for example, in the case of a bacteriophage, a molar ratio corresponding to 1,000 to 500,000 peptide molecules per bacteriophage can be used.

The present invention also relates to a composition comprising a freighter on the surface of which a transducer peptide is adsorbed, capable of being obtained by the process according to the invention. The compositions according to the invention can be used immediately after their preparation; where appropriate, they can also be stored for at least 3 days in the incubation medium, at temperatures between approximately 4 ° C. and 37 ° C.

The present invention also relates to the use of compositions in accordance with the invention for introducing a cargo ship, as defined above, into a living cell. In particular, the present invention thus relates to a method for introducing a cargo ship into a living cell, characterized in that it comprises bringing said cell into contact with a composition according to the invention comprising said cargo ship. The process according to the invention can be carried out on cells in culture, by adding to the culture a composition according to the invention, and incubation for 1 to 14 hours, preferably for 2 to 6 hours. Preferably, the composition in accordance with the invention is used at a rate of 10,000 to 20,000 cargo / peptide transducer complexes per cell.

The process according to the invention can also be implemented in vivo, for example by injecting a composition according to the invention into an animal.

A subject of the present invention is also the use of a composition in accordance with the invention for obtaining a medicament, and in particular as a carrier of an active principle consisting of the cargo or contained in it .

The present invention has the advantage of making it possible to introduce into living cells any hydrophobic cargo ship or the surface of which has at least one hydrophobic domain, without it being necessary to carry out prior coupling by covalent bond between the cargo ship and the transducer peptide. The present invention is of particular interest for introducing into cells living viral or pseudoviral particles, in particular bacteriophages, containing polynucleotides of interest which it is desired to express in said cells.

These particles can thus be used for example as gene therapy vectors, in vivo or ex vivo. It is also possible to prepare compositions in accordance with the invention from phage libraries containing polynucleotides coding for various polypeptides capable of modifying the behavior of certain cells (migration, proliferation, differentiation, etc.), and to use these compositions for bring these phage banks into tissues, in culture or in vivo, and identify regulatory sequences for these behaviors.

The present invention will be better understood with the aid of the additional description which follows, which refers to nonlimiting examples, illustrating the implementation of the present invention, for introducing phages into living cells. EXAMPLE 1: ABSORPTION OF A TRANSDUCER PEPTIDE ON IAMDA BACTERIOPHAGES

Phage preparation:

The autofluorescent EGFP gene

(CLONTECH) or that of the chicken homeoprotein En2 (Engrailed2) (LOGAN et al., 1992, Dev Genetics 13: 345-358) were placed under the control of the CMV promoter and upstream of the polyadenylation sequence of SV40 , in a plasmid derived from pBK-CMV (STRATAGENE) having a unique EcoRI site upstream of the CMV promoter, and a unique SalI site downstream of the polyadenylation signal. The functionality of these constructs was verified by electroporation and transient expression in COS cells, and detection of autofluorescence of GFP or im unocytochemical detection of the Engrailed 2 protein using a polyclonal antibody directed against this protein (don by Dr S. SAULE, UMR 146, Institut Curie, OrsayJ). The fragment framed by the two unique sites

(EcoRI and Sali) was then transferred into the phage genome La bda-ZAP (STRATAGENE), between the EcoRI and Xhol sites, and the recombinant DNA was packaged in vi tro using the GIGAPACK PLUS reagents (STRATAGENE ). The resulting phages

(respectively called Lambda-ZAP-GFP and Lambda-ZAP-En2) allowed the infection of competent bacteria (strain

XLlBlue-MRF ', STRATAGENE), then were titrated and stored after a first amplification round. To control the quality of the recombinants obtained, the phagemids internal to the genomes of the recombinant Lambda phages were automatically excised by co-infection of XL1Blue-MRF 'bacteria with an auxiliary phage (ExAssist, STRATAGENE). After culture in a liquid medium, the bacteria still alive and the Lambda virions are destroyed by heating, and the recombinant filamentous phages are recovered. The plasmid forms of these recombinant phagemids are recovered after infection of non-permissive bacteria for the replication of the filamentous phage (strain SOLR, STRATAGENE), and the functional integrity of the excised plasmids is verified by electroporation in COS cells. After this verification, the recombinant Lambda phages are amplified to reach a titer of at least 10 ¹¹ particles per ml, then concentrated with PEG, dialyzed against PBS supplemented with Ca ⁺⁺ and Mg ⁺⁺ , and stored at 4 ° C. .

Adsorption of the transducing peptide:

The transducing peptide used is a penetatin of sequence:

RQIKIWFQNRRMKWKK (SEQ ID NO: 1) corresponding to helix 3 of the peptide pAntp

(homeodomain of the Antennapedia protein of Drosophila).

The biotinylated penetatin is mixed with the recombinant phages, at a rate of 10 μg of peptide for 10 ⁹ phage particles, in 50 to 100 μl of appropriate medium (DMEM / F12 medium (1: 1) or PBS-Dulbecco). The mixture is incubated at room temperature for 30 min. EXAMPLE 2: INTRODUCTION OF PHAGES IN CULTIVATED CELLS

The cells used are dog kidney epithelial cells (MDCK). The phages used are marked with fluorochrome

Cy3 (AMERSHAM) by covalent linkage of fluorochrome to capsid proteins, according to the manufacturer's instructions. They are then incubated in the presence of penetatin, as described in Example 1 above. As a negative control, phages labeled with fluorochrome Cy3 are used, incubated under the same conditions in the absence of penetatin.

The phage / penetatin preparation, or the control preparation is added to the cell culture or suspension medium (depending on whether the treated cells have already been seeded or whether they have just been dissociated) at the rate of 10,000 phages / cell, and left in contact with cells for 4 hours.

The cells are then washed and re-cultured in fresh medium, then fixed in 4% parafor aldehyde in PBS for 10 min at room temperature, rinsed in PBS and mounted in mounting medium for DAKO fluorescent specimens containing lμg / ml of DAPI

(4 '-6-diamidino-2-phenylindole). They are then observed under a confocal Leica TCS epifluorescence microscope. Images are analyzed and processed using software

Adobe PHOTOSHOP.

The results are illustrated in Figure 1: Figure 1 A: control preparation with phage without penetatin Figure 1 B: phage / penetatin preparation

A significant intracellular fluorescence is observed in the cells which have received the phage / penetatin preparation. On the other hand, in the case of cells which have received only the phage preparation, no fluorescence is observed. EXAMPLE 3 INTRODUCTION OF LAMBDA IN VIVO PHAGES IN MOUSE BRAIN CELLS

Different phage / penetatin preparations

(recombinant phages expressing GFP; recombinant phages expressing En2; phages labeled with fluorochrome Cy3) are administered to adult mice by infusion into the lateral ventricle of the brain.

A day before infusion, the phages (6.5.10 ⁸ pfu / μl solution) are dialyzed against 0.9% NaCl containing 10 M MgCl2 (for phage stability) at 4 ° C overnight. On the day of the infusion, the phage / penetatin mixture is produced: 70 μl of the dialysis phage solution against 0.9% NaCl (i.e. 6.5.10 ¹⁰ pfu) + 3 μl of 9% Nacl + 27 μl of the stock solution of penetrine (i.e. 162 μg), or approximately 5 x 10 ⁵ molecules of penetrine per phage particle. 100 μl of mixture are loaded into an osmotic micro-pump (ALZET 1003D) connected by a catheter to a cannula which will be implanted in the lateral ventricle. The entire micro-pump is immersed in 0.9% NaCl at 37 ° C for 4 hours in order to initiate the flow.

The pumps are placed in a subcutaneous pocket at the scapular region of the animal, and the cannula implanted in the lateral ventricle of the brain according to the following stereotaxic coordinates: lateral 0.8 mm, anteroposterior 0mm, dorso- ventral 2mm with respect to the Bregma of the skull taken as the origin of the coordinates.

The infusion is carried out for three days at a flow rate of Iμl / hour. The infused animals are then euthanized by anesthesia followed by an intracardiac perfusion of paraformaldehyde 4% in PBS; brains are removed and post-fixed overnight at 4 ° C in this fixative. The next day, they are cut with vibrato and into 50μm thick frontal sections.

The sections are either observed immediately after mounting in mounting medium (DAKO + DAPI) in the case of direct fluorescence (GFP or CY3), or used for the immunodetection of the expressed heterologous protein by phage (in the case of phage expressing GFP or En2). Penetatin is detected by streptavidin coupled to fluorochrome Cy3 (IMMUNOTECH)

For immunodetection and / or detection of penetatin, the sections are preincubated for approximately one hour in PBS buffer 5% SVF 0.25% Triton X-100 (PBST) at room temperature. The antibodies are diluted in the same buffer, at 1/5000 for the anti-En2 polyclonal antibody, and at

1/500 for the anti-GFP polyclonal antibody (SANTA-CRUZ), and incubated with the sections overnight at 4 ° C. The sections are then rinsed 3x15min in PBS buffer; a secondary fluorescent anti-i rabbit munoglobulin antibody coupled to FITC (JACKSON) is then added after dilution to 1 / 500th in PBST. For the detection of penetatin, the fluorescent streptavidin is diluted to 1 / 500th in the

PBST.

After incubation for one hour at room temperature and three rinses of 15 min in PBS, the sections are mounted in DAKO + DAPI medium, and observed by confocal epifluroescence microscopy.

The results are illustrated in Figures 2 and 3:

FIGS. 2 A and 2 B represent markings on front sections 50 μm thick.

Figure 2A: Detection of phage cy3 in the cerebral parenchyma of an adult mouse after infusion of the penetatin / phage mixture into the lateral ventricle.

Figure 2B: Detection of GFP fluorescence in the cerebral parenchyma of an adult mouse after infusion of the penetatin / GFP phage mixture in the lateral ventricle

Figure 3: Co-localization of the engrailed 2 protein and penetatin in the brain parenchyma of an adult mouse after infusion of the penetatin / phage mixture coding for En 2. Figure 3A: Immunodetection of the Engrailed 2 protein encoded by the phage

Figure 3 B: detection on the same section of penetatin using fluorescent streptavidin Figures 3 D and 3 E are enlargements of Figures 3 A and 3 B respectively

Claims

1) Process for preparing a composition making it possible to introduce into a living cell, a cargo ship constituted by a macromolecule or a molecular assembly of size less than or equal to approximately 1 μm in its largest dimension and having on its surface one or more hydrophobic domains , which method is characterized in that it comprises the adsorption on the said hydrophobic domain (s), of at least one transducing peptide, with the exception of the transducing peptides of 16 to 30 amino acids comprising a hydrophobic domain containing 3 to 5 tryptophan residues including at least one Trp-Trp pair, alternating with glutamic acid and threonine residues, and a hydrophilic domain containing 4 or 5 consecutive basic residues.

2) Method according to claim 1, characterized in that the cargo is a protein or a particle having a surface of protein nature.

3) Method according to claim 2, characterized in that the cargo is a viral or pseudoviral particle.

4) Method according to claim 3, characterized in that the cargo is a bacteriophage.

5) Method according to any one of claims 1 to 4, characterized in that the transducing peptide is a peptide of the family of penetratins.

6) Method according to any one of claims 1 to 5, characterized in that the adsorption of the transducing peptide is carried out by incubation for at least 15 minutes of said transducing peptide with the cargo. 7) Composition comprising a freighter on the surface of which a transducing peptide is adsorbed, capable of being obtained by a process according to any one of claims 1 to 6.

8) Use of a composition according to claim 7 for introducing said cargo into a living cell in culture. 9) Use of a composition according to claim 7 for obtaining a medicament.