Therapeutic protein

 

(57) Abstract:

The invention relates to a new protein - human stem cell factor (FGC). DNA encodes FGC having the amino acid sequence shown in positions 1-39 in Fig. 2. Since the provisions of 40 FGC includes the following C-terminal amino acid sequence Glu Il Cys Ser Leu Leu Ile Cly Leu Thr Ala Tyr Lys Glu Leu Ser Leu Pro Lys Arg Lys Glu Thr Cys Arg Ala Ile Gln His Pro Arg Lys Asp. FGC provides the correct development of pre-implantation embryos in the processing of embryos. 2 S. and 1 C.p. f-crystals, 4 Il., table 1.

The invention relates to a new protein - human stem cell factor (FGC, SCP), DNA sequences coding for this protein, its use in therapy, in particular for in vitro fertilization, as well as to pharmaceutical compositions containing such protein.

Successful embryo implantation requires correct development of pre-implantation embryo, resulting in ripe the blastocyst, which is suitable for implantation in the receptive endometrium. Collected a significant amount of data that confirm the assumption that soluble growth factors, if they are secreted by the epithelium of the uterus directly affected by n, E. D. J. Cellular Biochem., 53; 280-287 (1993); and Schultz, G. A. and Hevner, S., Mutat. Res., 296; 17-31 (1992)).

In addition, it is shown that the developing embryos produce a number of cytokines that can act on the endometrium as autocrine (autocrine) and affect its susceptibility. Examples of these growth factors, which are produced by human embryos, include IL-1, IL-6, CSF-1 and TNF- (Zolti et al, Fertil. Steril., 56 (1991) 265-272, and Witkin et al, J. Reprod. Immunol., 19 (1991) 85-93).

It is shown that TNF - a is present in the culture medium of human embryos to the morula stage, but not in the environment of a blastocyst (Lachappelle et al, Human Reproduction, 8: 1032-1038 (1993)). The production of cytokines embryos can therefore be regulated stages-specific way.

Data about the possible direct effect of cytokines on the embryos were initially obtained in experiments with mice, when it was shown that many cytokines influence the development of pre-implantation embryos in vitro.

-Interferon and CSF-1 at physiological concentrations inhibit the development of many embryos to the blastocyst stage (Hill et al, J. Iimmunol., 139 (1987) 2250-2254). Also it is shown that TNF - has a less noticeable effect. Although TNF - a does not have an explicit action on the rate of formation of the blastocyst, this is, the result, with reduced blastocyst ICM (Pamplfer et al, Endocrinology, 134; 206-212 (1994)).

Other growth factors also have a specific impact on the ICM cells. For example, insulin-like growth factors 1 and 2 stimulate the proliferation of the ICM, while the factor inhibiting leukemia (PHIL, LIP) inhibits their differentiation (Harvey et al, Mol. Reprod. Dev., 31 (1992) 195-199).

In mouse models was observed that embryos cultured in vitro lag behind in development when compared to control in vivo and found a lower coefficient of pregnancy after transplantation of embryo (Bowman, P., and McLaren, A. , J. Embriol. Exp. Morphol., 24; 203-207 (1970)). Thus, a better understanding of the role of growth factors during development can lead to improved culture conditions in vitro and to enhance the effectiveness of programs of fertilization of human in vitro fertilisation (IVF).

The stem cell factor (FGC, SCF) is a growth factor, related in structure to CSF-1, and operates through tyrosinekinase receptor C-kit (C-kit). In the bone marrow FGC and CSF-1 act synergistically to promote proliferation and differentiation of stem cells in mikrofalowe colonies. In EP-A-0423980 described polynucleotide sequence of human FGC and discusses possible prima mice shows that C-kit is expressed during all the time of pre-implantation development (Arceci et al (1992)). The applicants have now found that the same is true for human embryos. At some stage human embryos also Express mRNA FGC, and it is assumed that this growth factor may act like autocrine. This is opposite to the case of mice, when expression of SFK is not detected in pre-implantation embryos (Arceci et al (1992)).

The primary transcript FGC consists of eight exons (Martin, F. H. et al, Cell, 63: 203-211 (1990), and in this work also describes a variant form of FGC. Also described splice variant of FGC, which appears due to the loss of exon 6 (Flanagan et al, Cell, 63:1025-1035 (1991)).

Now also found a different, a new splice variant, which appears due to the inclusion between exons 3 and 4 of the new exon, consisting of 155 base pairs. This also leads to a shift in the frame of the genetic code, encodes the type of FGC, contains 33 amino acids after exon 3, before the end when vnutriramochnym stop codon, which is now in exon 4 due to a shift in the reading frame.

Thus, the present invention relates to FGC, which is Arg Ala Ile Gln His Pro Arg Lys Asp Ile Gln His Pro Arg Lys Asp

or a sequence that is essentially homologous to the sequence indicated.

Preferably, the new FGC invention contains the first 39 amino acids of the primary FGC (without the inclusion of any signal sequence), followed by the aforementioned new 33 amino acids. In one of the embodiments of the invention a new FGC invention has the sequence in positions 1-39 homologous essentially of the sequence shown in Fig. 2.

The amino acid sequence of the protein can be considered essentially as a homologous sequence of a different protein, if a significant number of constituent amino acids detect homology. Homology may be at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or even 99% amino acids, with preference in the ascending order.

Thus, the mechanism of alternative splicing can lead to the production of a new FGC in human embryos. Therefore, a new FGC according to the invention can be used in the processing of pre-implantation embryos to ensure proper differentiation and development before implantation of the subject.

In addition, sledovatelnot includes the sequence essentially homologous sequences

GAA ATC TGT TCA TTG TTG ATA GGG CTG ACG GCC TAT AAG GAA TTA TCA CTC CCT AAA AGG AAA GAA ACT TGC AGA GCA ATT CAG CAT CCA AGG AAA GAC TGA

and includes all other nucleotide sequence, which, due to degeneracy of the genetic code, also encode a given amino acid sequence, or which is homologous essentially the following sequence.

Sequences with significant homology can be viewed as a sequence, which will be hybridisierung with the nucleotide sequence shown in Fig. 2, in simple terms (for example, when 35-65oC in saline solution of about 0.9 M).

Design of DNA containing the DNA sequence of the invention, constitute another aspect of the present invention.

As described herein, the protein of the invention useful for the treatment of embryos to ensure proper development before implantation. It is shown that FGC acts by binding to C-kit transmembrane receptors. In addition, applicants have shown that human embryos Express C-kit during most stages of development of pre-implantation embryo.

Thus, in their others who spent embryo which includes a step of introduction of the FGC present invention the pre-implantation embryo (and, preferably, a human embryo); and

b) the method of ensuring the correct development of human pre-implantation embryo, which includes a step of introduction of the FGC human pre-implantation embryo. When this method is used FGC can be any naturally occurring form, including the previously described variants (Martin et al, CIT. above and Flangan et al, CIT. above), as well as a new variant described here.

In addition, the present invention also relates to the use of FGC in the production of medicines for use in ensuring the correct development of pre-implantation human embryos. And again, any form of FGC can be used to obtain a suitable medicines.

The drug is mainly the form of pharmaceutical compositions containing the protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical compositions comprise another aspect of the present invention. Such pharmaceutical compositions of the present the present invention will be described using the following examples, which should not be construed as limiting in any way the present invention. The examples refer to the figures briefly described below.

Fig. 1 represents a sequence of new exons and predicted amino acid sequence.

In Fig. 2 shows the sequence of human FGC.

In Fig. 3 shows an agarose gel with the products "alopecia" RT-PCR (reverse transcriptase - polymerase chain reaction) amplification of RNA from human embryos. Each bar shows the amplification products with blades that are specific to different cDNA target. Amplificatoare cDNA from different embryos load on each track. Track mark in accordance with designations of cDNA in the table (see below). Additional samples are: path p - trophoblast first trimester; path q-cDNA from 200 BeWO cells; lane r - 10 ng human genomic DNA; and lane s - without making cDNA as a negative control. The molecular weight marker DNA is a ladder (ladder) 123 base pairs loaded into track 1. The expected sizes of PCR products are shown in pairs of bases (p. O.).

Table

cDNA of human GM/BR> e 8 cell

f the morula

g blastocyst

h culture supernatant to a-g

j three United blastocyst

k culture supernatant for j

l 2x6 cells and g cells

m culture supernatant for l

n 1x4 cells and g cells

o culture supernatant for n;

samples a-h from a single donor.

In Fig. 4 shows the seed used for RT-PCR, the outer pair of A and B, the inner pair C and D.

Example 1

Embryo culture and RNA extraction

In this study, use of cryopreserved human embryos that are fertilized as part of the IVF program. These embryos were given to parents for research purposes, this study was conducted according to the requirements of the Commission on embryology and fertilization of human rights and the local ethics Committee. Frozen embryos are thawed and cultured in a balanced salts medium Earl with the addition of 0.4% human serum albumin (Armour Pharma icals UK) to the desired stage of development, and then quickly frozen in liquid nitrogen in 5 μl of culture fluid (and, thus, are lysed by ice crystals). Identical volume of culture supernatant freeze as the To from first trimester trophoblast allocate method Chomsczynski and Sacchi, Anal. Biochem., 162; 156-159 (1987), in which frozen tissue is homogenized in 5 ml of buffer solution containing 4 M thiocyanate guanidine (Gibko BRL Livingston, Scotland), 25 mm sodium citrate, pH 7.0, 0.5% of sarkosyl and 0.1 M 2-mercaptoethanol. The lysate is acidified by adding 0.5 ml of 2 M sodium acetate, pH 4, and extracted with phenolchloroform using 5 ml of buffer saturated phenol, and 1 ml of the mixture with chloroform isoamyl alcohol (49: 1, o/o). The suspension was placed on ice for 15 minutes and centrifuged at 10,000 g for 20 minutes at 4oC. the Aqueous phase containing RNA, precipitious, washed twice in 70% ethanol, dried and resuspended in TE (10 mm Tris-HCl, pH 7.4 and 1 mm etc). The RNA concentration determined spectrophotometrically at 260 nm.

RNA derived from embryos of the individual using the Protocol proportional reduction based on the procedure described above. To facilitate precipitation of the RNA, at the stage of homogenization add 100 μg yeast tRNA carrier (Gibco BRL, Livingston, Scotland). Other details are the same as those described above, except that all volumes are 50 times less, and the entire procedure is performed in a 400-ál Eppendorf tubes.

Example 2

Polymerase chain reaction, using AMV reverse transcriptase (Super RT, HT Biotech, Cambridge, Conn. Cor.). Primesouth 305 µg RNA oligo-dT (Pharmacia) according to the manufacturer's instructions, for 60 minutes at 42oC. PCR amplification of cDNA preparations carried out as described previously (Snarkey, A. et al, Molecular Endocrinol., 6: 1235-1241 (1992)), with thermoacetica for (Hybaid) omnigenous DNA in a final volume of 30 μl using 1-E DNA Taq polymerase (Cetus, Emeryville, CA) and 10 μm of each of the pairs of outer blades (see Fig. 4) the manufacturer's recommended buffer. Use the following circular profile, 30 s at 95oC, 30 s at XoC, 30 s at 72oC for 30 cycles, where X is the annealing temperature for each pair of cytokine blades, as shown below.

The outer seed (oC)

FGC - 54

GIS-tRNA - 52

s-set - 56

The inner seed (oC)

FGC - 54

GIS-tRNA - 59

s-set - 56

Oligonucleotide priming

Oligonucleotide priming for FGC, C-kit, histidyl-tRNA synthetase synthesized on a DNA synthesizer PS250 Cruachem. Sequence nucleating design from the published nucleotide sequences (see Fig. 4) so that the amplification of any contaminating genomic DNA will result in sportsouth two pairs of blades for each cDNA target, Protocol-nest PCR. One thirtieth of cDNA products amplified using Amplitaq (Cetus) recommended by the manufacturers of the buffer. After 30 cycles of PCR using the outer pair of blades one fiftieth of the reaction mixture of the first cycle is transferred to another test tube containing an inner pair of blades, and subjected to amplification for 30 cycles. As a negative control, an equal volume of the culture fluid in which grow the embryos extracted and subjected to RT-PCR according to the same scheme. In addition, as a positive control extracted with 200 cells cell line BeWo (ESAS N 86082803).

The seed used in this study is shown in Fig. 4 together with the size of the expected product. The identity of each product is confirmed by cloning and sequencing as described previously (Sharkey et al, Mol. Endocrinol. (1992)). To verify that the detected product is the result of the amplification of cDNA, and not contaminating genomic DNA, the chosen seed, to cover the intron-exon joints. 10 ng of genomic DNA is subjected to PCR at the same time as cDNA to verify the absence of the product of the expected size generated from genomic DNA.

by in vitro fertilization. The embryos are grown to an appropriate stage, then quickly frozen, and extracted with whole RNA. In order to obtain a detectable product FROM RT-PCR from total RNA extracted from a single embryo, using the Protocol nested PCR, in which the cDNA is subjected to two series of PCR amplification with the outer pair of blades, and then with the inner pair. Priming is based on published cDNA sequences and constructed for overlapping intron-exon junctions to the amplification of contaminating genomic DNA could be easily separated from the products of the cDNA.

First, cDNA from each embryo check with primers for histidyl-tRNA synthetase (gisters) to confirm the successful isolation of RNA and reverse transcription. Used seed give rise to unstable products more than 400 p. O. from genomic DNA and 110 p. O. from cDNA derived from mRNA gilrs. Transcripts for gilrs detected in mRNA from embryos at all stages of development, as well as falling in the lining of the uterus and choriocarcinoma cell line BeWo, used as a positive control (Fig. 3, the paths p and q, respectively). The product is not detected in an equal volume of supernatant of culture of embryos, extrana cDNA or RNA.

Examples of such RT-PCR analysis with seed for FGC and the set shown in Fig. 3. Stocks cDNA back Transcriber of each sample RNA in two separate events, and PCR analysis was repeated twice on each of the original cDNA. The results are shown in Fig. 3, where the selected samples in the expression of C-kit and FGC during pre-implantation development. The identity of PCR fragments of the correct size confirmed by sequencing cloned PCR products. When found products new size, clone and is sequenced.

For FGC predicted fragment is 966 p. O. However, it appears that the transcript FGC show stages-specific differences in size. After cloning and sequencing, it turns out that due to alternative splicing events, which embeds a new exon between exons 3 and 4, there is a new product. The predicted sequence of the new transcript is shown in Fig. 1. Picture of the new splicing also includes a shift in the reading frame, giving up 33 new amino acids before vnutriramochnym stop codon in exon 4.

When a similar analysis using seed-specific C-kit, the receptor for F. embryo. This suggests that the embryo has the ability to answer FGC during this period.

Discussion

It is shown that many growth factors influence the development of cultivated pre-implantation embryos of mammals (for review see Anderson, E. D., J. Cellular Biochem., 53; 280-287 (1993), and Schultz, G. A. and Hevner, S., Mutat. Res., 296; 17-31 (1992).

However, there is strong evidence of differences from sample to sample in the expression of receptors of growth factors during pre-implantation development. For example, mRNA EGF (epidermal growth factor) is expressed in the embryo of a pig, but not detected at any stage with the mouse pre-implantation embryos (Vaughan et al, Development, 116; 663-669 (1992); Rapolee et al. Science, 241; 1823-1825 (1988): Watson, A. J. et al, Biol. Reprod., 50; 725-733 (1994)).

Therefore, the usefulness of these studies for the search in the direction of the factors governing the development of human pre-implantation embryo is limited. In addition, specific growth factors and receptors were investigated in these works, was often chosen in this case (ad hoc). As for moral and practical reasons, this approach is not suitable for use in the case of human embryos. Therefore, the applicants used the method of "the plantation embryos individual. This method is widely used in the last few years for other species, as it is reliable, sensitive and cost-effective when working with embryonic material.

RT-PCR with seed for histidyl-tRNA synthetase was used for the cDNA samples to confirm that the cDNA was successfully obtained from a sample of RNA from each embryo. Specific to this binding gene cDNA was successfully detected in the cDNA samples obtained even from a single 2-cell embryo, which indicates that the method is sensitive enough for this stage.

FGC expressed at the 2-cell stage and then appeared again at 6-cell stage. It is compatible with maternal expression with subsequent reexpressing of the genome of the embryo on the 6-cell stage (Braude, P., et al, Nature, 332; 459-461 (1988)). It turned out that the transcript FGC find stage-dependent differences in the amount of transcript. They discovered after cloning and sequencing due to alternative splicing of the primary transcript. Two of these options similar to the options previously published (Martin et al, CIT. above and Sharkey et al, Mol. Endocrinol., 6: 1235-1241 (1992)), and one option is a new form, which predicts the sample FGC 33 new amino acids at the governmental and bioactive. Varieties, downregulation of pre-implantation embryos, include samples of known that they are bioactive, and it is shown that various forms of FGC can act through C-kit expressed by the embryo, and can affect the development of the embryo during this period.

The list of sequences is given at the end of the description.

1. DNA encoding the stem cell factor (FGC), which in position 1 - 39 has the amino acid sequence shown in positions 1 to 39 in Fig.2, but starting from position 40 has the following C-terminal amino acid sequence:

Glu Ile Cys Ser Leu Leu Ile Gly Leu Thr Ala Tyr Lys Glu Leu Ser Leu Pro Les Arg Lys Glu Thr Cys Arg Ala Ile Gln His Pro Arg Lys Asp

2. DNA under item 1, characterized in that it comprises the following sequence:

GAA ATC TGT TCA TTG TTG ATA GGG CTG ACG GCC TAT AAG GAA TTA TCA CTC CCT AAA AGG AAA GAA ACT TGC AGA GCA ATT CAG CAT CCA AGG AAA GAC TGA.

3. Human stem cell factor (FSK) encoded by the DNA under item 1 or 2.

 

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