IE911914A1 - Novel neurotropic growth factors comprising a homeobox¹peptide - Google Patents

Abstract

New class of cellular growth factors which are active in particular on nerve cells, the said factors comprising at least one homeobox peptide, optionally linked to another peptide sequence.

Description

The present invention relates to use of homeobox peptides, or peptides derived from these, for obtaining medicaments.

Under the name of homeobox peptides, there is designated a family of related peptide sequences, which are found in different animal species in the products of genes involved in embryogenesis.

There are in fact known genes which express different stages of embryonic development, and the expression of which controls the phenoma of migration and cellular differentiation implied in the morphogenesis of the organism.

These genes are called homeotic genes and their translation products are called homeoproteins.

One of these genes which has been particularly studied is the gene Antennapedia of the Drosophile; analysis of this gene has permitted putting in evidence a sequence of DNA of about 180 pb, called a homeobox sequence. - 2 This homeobox sequence has the property of being highly conserved in numerous homeotic genes, this being not only true for the Drosophile but also in the course of evolution, in different animal species. Homeobox sequences homologous to that of the Drosophile have therefore been found in all of the vertebrates and are comprised in mammals [ACAMPORA et al., NUCLEIC ACID RES., 17, 10385, (1989)].

The homeobox sequence code for a polypeptide sequence of 60 aminoacids, which corresponds to a region structurally and functionally conserved is present in all the homeoproteins, the homeodomain. The sequence of the homeo domain code or the homeobox sequence of the gene Antennapedia is indicated hereinafter by way of example.

(I) NH2Arg Lys Arg Gly Arg Gin Thr Tyr Thr Arg Tyr Gin Thr Leu Glu Leu Glu Lys Glu Phe His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg lie Gly lie Ala His Ala Leu Cys Leu Thr Glu Arg Gin He Lys He Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys Glu AsnCOOH.

The role and the mechanism of activity of the homeo domain sequence has been the object of much research. It is thus in fact known that this sequence enables the fixation of homeoproteins to DNA, at the level of consensus sequence including the motif ATTA, present in the promoters or the amplification sequences for different genes and comprised of homeobox genes themselves.

MULLER et al [EMBO J., 1, 4299,, (1988)] have cloned the homeobox sequence Antennapedia and purified the corresponding polypeptide or homeobox polypeptide (pAntp). In the presence of a reducing agent, they have obtained the polypeptide in the form of a monomer, of a sedimentation coefficient of about 1 S and of a molecular weight apparently 9040 Da (theoretical molecular weight, according to the peptide sequence = 8545 Da). - 3 »Ε9ΐ1θ14 In the absence of a reducing agent, the preparation of a polypeptide contained significant proportion of dimers, corresponding to the homeo domains lying between them by the disulphate bridges.

These same authors have also shown that the polypeptide purified in monomeric form links to DNA, at the level of a sequence ANNNNCATTA, containing thus the consensus sequence ATTA.

Given the very high degree of conservation of homeobox sequences from one species to another, it is considered that the properties of the peptide pAntp can be generalised in other homeobox peptides, enabling differentiation in their sequence by some aminoacids, but having functional properties substantially identical; in the following text, the terms homeobox peptide designates without distinction both the peptide pAntp as well as every other member of that family, or a related family, for example the engrailed family.

Other works [OTTING et al, EMBO J., 1, 4305, (1988)] have established that the homeo domain had a particular structure (helix/tower B/helix) which was involved in the bond to the DNA.

The homeobox peptide to DNA link was the result of : a bond of high affinity (KD « 10θ-10"^θΜ), involving the consensus sequence ATTA, and a link of low affinity (Κθ » 10~θ-10-^^), involving the large spiral of the double helix of DNA. A publication of Kissinger et al. [Cell, 63, 579-590, (1990)1 describes a study carried out by crisialography on the homeo domain engrailed DNA complex. This study shows that the terminal C part of the homeo domain, comprising in particular the structure code helix 3 (aminoacids 42-58 of the engrailed homeo domain) is fixed at the level of the large spiral of the DNA. The bond is essentially assured by hydrophilic interaction; this bond was indpendent of the presence of the consensus sequence.

Although one now has a wealth of data obtained in vitro and in acellular systems, on the homeobox peptide to DNA link, up to the - 4 present, the consequences on the cellular functions have been ignored. It has even been ignored whether the homeobox peptides were capable of having their own activity, or if their role reduced simply to facilitating the homeoprotein to DNA bond.

However, in studying the action of homeobox synthetic peptides on cellular cultures, the inventors have discovered surprising properties of these peptides, properties which have never been suspected up to now.

They have in fact confirmed that homeobox synthetic peptides, when they are added to nerve cells in a culture, penetrate into all the cells of the culture and that the entry of the peptides into the neurones is followed by an accumulation in the nucleus.

This accumulation is not only blocked by preincubation with an oligonucleotide containing the consensus sequence ATTA, but also by preincubation with fragments of a non-specific double branched DNA (that is to say not containing the consensus sequence).

The inventors, in analysing the penetration process, have shown the importance in this of the region corresponding to the 3+4 helix (27 final aminoacids of the homeobox peptide).

They have also observed penetration of polypeptides comprising this homeobox peptide.

They have also observed this phenomenon although to a lesser degree in cell cultures other than nerve cells.

The inventors have further shown that the accumulation of homeobox peptides in the nucleus was accompanied by growth and an intense cellular differentiation.

These properties of homeobox peptides, demonstrated by the inventors, enable their utilisation for obtaining medicaments, as well as their utilisation in vitro as an active agent on cellular cultures.

In fact, it follows from the work of the inventors that the homeobox peptides or fragments of them both furnish new growth factors, in particular neurotropes, and/or new vectors for transmembrane and intercellular transport of active molecules on cellular functions, in particular of peptides and oligonucleotides.

However, both of these applications relate to actual needs. They have been made the object of different studies, a brief summary of which is given below.

The relevance of vectors to intercellular transport of peptides or of oligonucleotides is apparent following demonstration that certain peptides and oligonucleotides, while fixing themselves specifically to certain regions of DNA, are capable of acting on the cellular functions (for example, proteins activating or retarding the expression of a gene, anti-sense oligonucleotides, etc.).

However, effective use, in particular for therapeutic purposes, of the properties of these molecules, involves making them appear at site while making them cross numerous barriers separating the extracellular region of the DNA, and in particular the cytoplasmic membrane. However, very often, these molecules, on account of their charge and their high molecular mass, cannot of themselves cross this barrier. Different solutions to this problem have been proposed; certain of them which concern oligonucleotide sequences are cited for example in the introduction to the publication of LEMAITRE et al. [Proc. Natl. Acad. Sci. USA 84, 648-652(1987)]. These authors themselves propose an approach consisting of linking in a covalent manner an oligonucleotide sequence complementary to an RNA sequence of a vesicular stomatitic virus (VSV) to a polymer of (L-lysine).

The conjugate obtained penetrates into the cells and specifically inhibits synthesis of the proteins of VSV in the infected cells.

These results show the involvement of the association between an active macromolecule and a transport vector for said c --7macromolecule. It is however, particularly desirable to study new vectors.

In regard to active growth factors on the survival and differentiation of neurons, only a small number of them are actually known; the first to have been put in evidence is the nerve growth factor (NGF). The action of the nerve growth factor is exercised essentially on the sensorial neurons and the neurons of the sympathetic nervous system; an action on certain cells of the central nervous system and of the immune system has also been discovered. The neurotropic activity of the NGF is carried by a sub-unit (sub-unit β) of 118 amino acids.

Other substances for neurotropic action have also been described; there are, for example, the Ciliary Neurotropic Factor (CNTF) [LIN et al, SCIENCE, 246. 1023-1026, (1989); STOCKLI et al. NATURE, 342. 920-923, (1989)], the Brain Derived Neurotropic Factor (BONF) [LEIBROCK et al., NATURE, 341. 149-152, (1989)], the Glial Derived Nexin (GDN), comprising the extracellular matrix [GLOOR et al·, CELL, 47, 687-693, (1986)].

Very common growth factors such as the Fibroblast Growth Factor (FGF) [PARK et HOLLENBERG, DEV. BIOL., 134. 201-205, (1989)] or 1‘Epidermial Growth Factor (EGF) [MORRISON et al., SCIENCE, 238. 72-74, (1987) also have a neurotropic action.

The action mechanism of these factors is actually poorly understood. It has been shown that the NGF penetrates into the neurons by means of a specific receptor, which is a phosphorylsed glycoprotein (CHAO et al, Science, 232. 518 (1986)]. In the interior of the nerve cell, the NGF stimulates synthesis of RNA, by means of the second messenger. Numerous experiments show the potential therapeutic relevance of neurotropic growth factors.

Utilisation of NGF has for example, been suggested for Alzheimer's disease. It has in fact been shown that the NGF permits enhancing the acetyltransferase choline activity of cholinergic neurons, and of preventing their degeneration (MOBLEY et al, Science, --8229. 284 (1984)], [KROMER, Science, 235. 214, (1987)]. However, it is understood that Alzheimer's disease is associated with a degeneration of cholinergic neurons and reduction of choline acetyltransferase activity.

Experiments carried out on adult rats in which the cholinergic paths connecting hippocamp and septum had been destroyed in advance, that which involves a degeneration of the septal neurons, has shown that intra-ventricular injection of NGF permits survival of the septal neurons, as well as restoration of a normal choline acetyltransferase activity [WILL & HEFTI, Behav, Brain.. Res., 17,17 (1985)]. Utilisation of neurotropic growth factors has also been envisaged for Parkinson's disease, linked with a degeneration of dopaminergic neurons.

Another approach to treatment of illnesses associated with neuron degeneration has recently been proposed and seems to promise, in the near future, a signficant development: it concerns intra-cerebral grafts of cells enabling supplementation of deficient neuronal functions; use of foetal neurons [LINDVALL et al., SCIENCE, 247. 574-577, (1990] or of transformed cell lines [HORELLOU et al., EUR. J. NEUROSCI., 2, 116-119, (1990)] has also been suggested.

Recently, transformed cells producing a recombinant NGF have been implanted into the brain of rats, at the same time as cholinergic neurons of foetal orgin. It has been confirmed that in these conditions, the survival of the grafted neurons as well as the neogenesis of nerve fibres has been considerably enhanced [ERNFORS et al, Proc. Natl. Acad. Sci.. USA, 86, 4756, (1989)].

These studies show therefore that the neurotropic growth factors can find particularly relevant application in the treatment of problems resulting from neuronal lesions or degeneration.

A restriction on use of neurotropic growth factors is however constituted by the small number of growth factors actually known, as well as by their specificity to a relatively narrow action, limited to certain types of neurons. In addition, the majority of β911914 - 9 these growth factors cannot actually be obtained in sufficient quantity for therapeutic usage.

It would thus be particularly desirable to make use of 5 neurotropic growth factors which do not have the disadvantages which have been mentioned.

The present invention by showing the unexpected properties of homeobox peptides proposes a new answer to both of the two problems which have been set forth above.

The subject of the present invention is a polypeptide chosen from the group consisting of homeobox peptides and fragments of these, for use as medicaments.

In the sense of the present invention, there is understood by homeobox peptides, any peptide relating to the families defined above, and in particular any sequence of aminoacids having, with the peptide pAntp of Drosophile, a homology which permits fixation of this peptide to a DNA double helix.

According to a preferred mode of realisation for the present invention, said polypeptide comprises the sequence corresponding to the 3 helix of a homeobox peptide.

According present invention, to a preferred manner of realisation the homeobox peptide is the pAntp peptide. of the According to a preferred manner of realisation of the present invention, the homeobox peptide or its fragment, such as defined above, are connected to another peptide sequence. According to another preferred embodiment of the present invention, the homeobox peptide or its fragment are connected to a 35 nucleotide sequence.

The homeobox peptides or fragments of these, as well as their fusion products with another polypeptide sequence can be easily - w obtained by processes known in themselves, for example by peptide synthesis, or even by genetic engineering. The fusion products of a homeobox peptide and an oligonucleotide sequence can be obtained for example by the technique described by LEMAITRE et al. [Proc. Natl.

Acad. Sci. USA, 84, 648-652 (1987)].

The intracellular penetration properties of homeobox peptides and of their fragments permits their utilisation to introduce into cells active molecules on the cellular functions, in particular other peptides, or nucleotide sequences having pharmacological properties.

There may be utilised a homeobox peptide or a fragment of a homeobox peptide containing the 3 helix, which recognises independently of the sequence, the structure of the molecule of DNA (large spiral); the bond to a specific sequence of DNA or RNA is then assured by the peptide or the oligonucleotide linked to the homeobox peptide or to the fragment of the homeobox peptide.

According to the invention, polypeptides comprising a homeobox peptide or a fragment of this are also usable as growth factors, in particular neurotropes, both in vivo and in vitro.

Contrary to the neurotropic growth factors known in the prior art, the homeobox peptides are active on a large number of types of neurons. The inventors have in particular observed the activity of pAntp on nerve cells prepared from various regions of the central nervous system of the embryo, in particular the spinal cord, the rhombencephalon, the ventral mesencephalon, the tectum and the cortex. The activity on the cortical cells is particularly surprising, since at present no expression of homeoproteins has been revealed in this region of the brain.

The large sphere of action of homeobox peptides gives them great interest as pharmacological agents, particularly in the treatment of neuronal lesions or degeneration. They can also be used in the field of intracerebral neuron grafts, which are called on to develop and for which it is essential to assure the survival and the ,E 911914 io - ΤΓ most rapid and the most extended development in volume of the cellular graft.

This can be effected, for example, by pre-incubating the 5 neurons for grafting with a growth factor according to the invention, or even by effecting a graft together with embryonic cells with transformed cells capable of synthesising and secreting the homeobox peptides, or fusion peptides containing a homeobox sequence.

The subject of the present invention is also an invitro treatment process for neurons intended for grafting, this procedure being characterised in that said neurons are incubated in the presence of at least one homeobox peptide, possibly linked to another peptide or oligonucleotide sequence.

The present invention also has as its subject a cellular composition for use in neuron graft techniques, in which said composition is characterised in that it contains a group of neurons which is it desired to graft and transformed cells suitable for synthesising and secreting a homeobox peptide.

The inventors have already studied the action of homeobox peptides not only on cell cultures but also on multicell organisms. They have therefore confirmed that thanks to the speed of penetration of homeobox peptides, these enter in localised fashion into the cells adjacent to the point of injection.

This property offers the advantage that in the case of use on a living organism, it is possible to apply, as desired, a very precise localised treatment to a group of cells.

The present invention will be best understood by reference to the description which follows, which refers to examples demonstrating the activity of homeobox peptides.

It will however be appreciated that the examples are given only by way of illustration of the subject of the invention, and that they do not constitute any kind of limitation. \( - r 1) OBTAINING GROWTH FACTORS ACCORDING TO THE INVENTION EXAMPLE 1; Obtaining a pAntp peptide.

The coding sequence for the homeodomain of the gene Antennapedia of the drosophile was synthesised using the PCR techique, starting from the plasmid p9O3G which contains, between the locations BamHl and PVUII, a fragment of 600bp of DNAc of Antp (GARBER et al., 1983). The two beginnings for which the sequence is indicated below are used. The first beginning (5,GGGGGAATTCCATATGCGCAAACGCGCAAG 31) contains a restriction location Ndel upstream of the starting codon, and the second beginning (5'GGGGAAGCTTGGATCCTCAGTTCTCCTTCTTCCACTTCAT 3') contains a terminating codon followed by a BamHl location. A plasmid named pAHl was formed by ligation of an Ndel-BamHl fragment of 200 bp obtained by PCR on the plasmid pET3a (ROSENBERG et al., 1987). The polypeptide was then expressed in E. COLI BL 21 (Lys S). The transformed cells were cultivated at 37°C in the the LB medium in the presence of ampicillin and chloramphenicol (100 ug in each case), up to an optical density DO^qq = 1.2. After an induction of five hours in the presence of 1 mM of IPTG these cells were collected by centrifugation (16000 g, 15 minutes) washed three times in a phosphate buffer 50nW, pH7,5, Nacl 400 mM, EDTA 2 mM, PMSF 1 mM, DTT 1 mM (plug A), then sonically.

After centrifuging (16000 g, 15 minutes), the supernatant was precipitated with streptomycin sulphate (20 mg/ml) under mild agitation for 15 minutes at ambient temperature, then centrifuged (16000 g, 15 minutes). The supernatant was directly charged on an S-SEPHAROSE Fast Flow column (PHARMACIA), which was previously equalised with the buffer A. The column was then washed with a large quantity of phosphate buffer 50 mM pH 7, 5, NaCl 0,5 M and the pAntp peptide was eluted by application of a Nacl gradient (0,5 to 1M).

The eluted peptide (3 mg/1 of culture) was then dialysed for 24 hours against the phosphate buffer 50 mM pH 7,5, NaCl 150 mM.

The sequences of the amplified DNA segment and of the tv - 13 peptide were determined (APPLIED BIOSYSTEM 477) and it was verified that they correspond to those of the homeobox of Antennapedia. Analysis of the peptide by electrophoresis on a gel in the presence of SDS showed the presence of a single band of molecular weight corresponding to that of the homeopeptide of Antennapedia.

The peptide sequence obtained is the following: (I) NH2Arg Lys Arg Gly Arg Gin Thr Tyr Thr Arg Tyr Gin Thr Leu Glu Leu Glu Lys Glu Phe His Phe Asn Arg Tyr Leu Thr Arg Arg Arg Arg lie Glu lie Ala His Ala Leu Cys Leu Thr Glu Arg Gin He Lys He Trp Phe Gin Asn Arg Arg Met Lys Trp Lys Lys Glu AsnCOOH It has also been verified by retardation on preincubated peptide gel with an oligonucleotide duplex corresponding at the bonding location to a homeobox protein of the Antennapedia type and obtained from the promoter Hoxl.3 [Hoxl.3p, (ODENWALD et al., GENES AND DEV., 3, 158, 1989)]: 3' CAGAGCACGTGATTACCCCCTCAACC 5' ' GTCTCGTGCACTAATGGGGGAGTTGG 3' that the peptide recognises in vitro the consensus fixation sequence of the proteins of the Antennapedia type.

II) Demonstration of the activity of growth factors according to the invention Example 2 : Demonstration of the biological effect of the peptide pAntp on neuron cultures Neuron sampled from different regions of the brain (mesencephalon, spinal cord, cortex) of embryos (E14 to E16) of the rat were put in culture in a defined medium (CHAMAK and PROCHIANTZ, - 44 DEVELOPMENT, 106, 483, 1989) enabling the survival of single neuronal cel Is (CDM medium).

The pAntp peptide was renatured by incubation for ten minutes at 60°C in the presence of dithiothreitol (0.1 mM) and magnesium (lOmM) in an isotonic phosphate buffer pH 7.2 containing 33 mM of D-glucose. The peptide was added to nerve cells at a concentration of 9 ug/ml (such as 1.3 uM).

The effects observed after addition of the peptide are illustrated in Figure 1 (A and B). Figure 1A shows the appearance of sample cells cultivated for 24 hours in the absence of the peptide. Figure IB shows the appearance of cells cultivated for 24 hours in the presence of the peptide. It may be noted that 24 hours after the addition there is a considerable augmentation of the neuritic growth in the cells cultivated in the presence of the peptide pAntp.

The same results on the cell growth have also been observed from experiments in which the cells are preincubated with peptide pAntp, then put in culture for 24 hours in a CDM environment devoid of peptide. 30 minutes incubation in the presence of pAntp already permits one to observe the effects described above; these effects are maximised after an incubation of 2 hours and are not augmented by prolongation of the preincubation time.

The preincumbation of the homeobox peptide with the consensus fixation sequence obtained starting from the promotor Hoxl.3, or even with a mixture of fragments (of aleatory sequence) of double helix DNA, blocks its biological effect.

It has also been shown in utilising a peptide marked with fluoresceine, that the homeobox peptide is rapidly (less than 2 hours) captured by all the neurons, then transported into the nucleus (Figure 2A), and that this transport is blocked after preincubation with the conssensus oligonucleotide obtained starting from the promoter Hoxl.3 or even with a mixture of fragments (of aleatoiry sequence) of double helix DNA (Figure 2B).

Comparison between the entry and the intrace1lular repartition of the pAntp peptide and of the polyornithine has also been effected.

The cells have been incubated for 1 hour in the presence of pAntp or polyornithine, marked with fluoresceine.

The results are shown in Figure 3 which shows that the pAntp marked (3A) is found principally in the nucleaoplasm, contrary to the polyornithine present in the soma and the nucleoles (3B).

Example 3: Penetration of pAntp into culture fibroblasts.

The fibroblasts are obtained starting from the skin of rat embryos, mechanically associated and trypsinised; the cells are cultivated on glass boxes covered with poly-DL-ornithine (SIGMA, PM 40.000, 15 Ug/ml), in the culture medium DMEM-F12 (GIPCO-PRL), and in the presence of 10% of foetal cow serum. After 24 hours of culture, the cells are incubated for one hour in the presence of the peptide pAntp marked with fluoresceine. The marked peptide pAntp penetrates into the fibroblasts, but is present in the nucleus in a lesser quantity than in the nerve cells.

Example 4: Penetration of the pAntp peptide into the embryonic cells in-vivo. 0.2 ul of solution (1 Ug/Ul) of pAntp peptide marked with fluoresceine are injected into the mesencephalon of a quail embryo for two hours. After four hours of incubation in-ovo, the embryo is extracted from the egg and sections of the mesencephalon are effected. Observation of these cuts under the microscope shows that the fluorescent marking is localised at the nucleus of the cells situated in immediate proximity to the point of injection.

As well as relating to that which precedes, the present invention is not limited only to those of its manners of implementation, embodiment and application which have been described explicitly; it also embraces all variants which come within the field η - κτ of knowledge of the man skilled in the art, without limiting the scope or field of the present invention.

Claims (19)

1. Polypeptide, selected from the group consisting of homeobox peptides and fragments of these, for use as a medicament.

2. Polypeptide according to claim 1, characterised in that it comprises the 3 helix sequence of a homeobox peptide.

3. Polypeptide according to either of claims 1 or 2, 10 characterised in that the homeobox peptide is the peptide pAntp.

4. Polypeptide according to any of claims 1 to 3, characterised in that it is linked to another polypeptide sequence. 15

5. Polypeptide according to any of claims 1 to 3, characterised in such that it is linked to an oligonucleotide sequence.

6. Polypeptide according to any of claims 1 to 3, for use as a cellular growth factor, in particular a neurotrope.

7. Polypeptide according to any of claims 1 to 3, for use as a vector for intercellular transport of active molecules on the cellular functions. 25

8. Use of a polypeptide such as defined in claims 1 to 5 as an active agent on cell cultures.

9. Use according to claim 8, characterised in that said polypeptide is used as a growth factor.

10. Use according to claim 8, characterised in that said polypeptide is used as intercellular transport vector for active molecules on the cellular functions. 35

11. Use according to any of claims 8 to 10, characterised in that the cell cultures are neurons. i - 18

12. Process for invitro treatment of neurons intended for grafting, the process being characterised in that said neurons are incubated in the presence of at least one homeobox peptide, optionally linked to another peptide sequence.

13. Cellular composition for use in neuron graft techniques, said composition being characterised so that it contains a group of neurons which it is desired to graft, and tranformed cells suitable for synthesising and secreting a homeobox peptide or a fusion peptide comprising a homeobox sequence.

14. Pharmaceutical composition, characterised in that it comprises as active principle, at least one polypeptide such as defined in any of claims 1 to 5.

15. Polyteptide according to Claim 1, substantially as described herein by way of example and with reference to the accompanying drawings.

16. Use of a polypeptide according to Claim 15, substantially as described herein by way of example.

17. Neuron process treatment, substantially as described herein by way of example and with reference to the accompanying drawings.

18. Cellular compositions, substantially as described herein by way of example and with reference to the accompanying drawings.

19. Pharmaceutical composition, comprising a polypeptide according to Claim 15.