Human SCF, a splice variant thereof, its pharmaceutical use

Details Human SCF, a splice variant thereof, its pharmaceutical use Human SCF, a splice variant thereof, its pharmaceutical use

1997-07-31

2001-01-23

Draper, Garnette D. (Department: 1647)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S252300, C435S069500, C530S351000, C514S04400A, C514S002600, C424S085100

Reexamination Certificate

active

06177556

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a novel human stem cell factor (SCF) protein, DNA sequences coding for this protein, its use in therapy, particularly in in vitro fertilisation, as well as pharmaceutical formulations comprising such a protein.
BACKGROUND OF THE INVENTION
Successful embryo implantation requires correct development of the pre-implantation embryo, resulting in a hatched blastocyst which is able to implant into receptive endometrium. A considerable body of data has been collected which supports the idea that soluble growth factors, if secreted by the uterine epithelium, act directly on the embryo to control this process (Pampfer, S. et al, Bioessays, 13: 535-540 (1991); Tartakousky, B., and Ben Yair, E.,
Development Biology
, 146: 345-352 (1991); Anderson, E. D.,
J. Cellular Biochem
., 53: 280-287 (1993); and Schultz, G. A. and Hevner, S.,
Mutat. Res
., 296: 17-31 (1992)).
In addition, developing embryos have been shown to produce a variety of cytokines which may act in an autocrine fashion on the endometrium to influence its receptivity. Examples of growth factors shown to be produced by human embryos include IL-1, IL-6, CSF-1 and TNF-&agr; (Zolti et al,
Fertil. Steril
., 56 (1991) 265-272 and Witkin et al,
J. Reprod. Immunol
., 19 (1991) 85-93). TNF-&agr; has been shown to be present in culture medium of human embryos up to the morula-st-age, but not that from the blastocyst (Lachappelle et al,
Human Reproduction
, 8: 1032-1038 (1993)). Production of cytokines by the embryo may therefore be regulated in a stage-specific manner.
Data on the possible direct effects of cytokines on embryos have come primarily from experiments in mice where many cytokines have been shown to affect the development of pre-implantation embryos in vitro. IFN-&ggr; and CSF-1, at physiological concentrations, inhibit the number of embryos developing to the blastocyst stage (Hill et al,
J. Immunol
., 139 (1987) 2250-2254). TNF-&agr; has also been shown to have more subtle effects. Although TNF-&agr; has no apparent effect on rates of blastocyst formation, it appears to specifically inhibit proliferation of cells contributing to the inner cell mass (ICM), which results in blastocysts with a reduced ICM (Pampfer et al,
Endocrinology
, 134: 206-212 (1994)).
Other growth factors also have specific effects on ICM cells. For instance, insulin-like growth factors 1 and 2 stimulate ICM proliferation, whereas leukaemia inhibitory factor (LIF) inhibits their differentiation (Harvey et al,
Mol. Reprod. Dev
., 31 (1992) 195-199).
It has been observed, in mouse systems, that embryos cultured in vitro lag in development compared to in vivo controls, and exhibit lower pregnancy rates after embryo transfer (Bowman, P. and McLaren, A.,
J. Embryol. Exp. Morphol
., 24: 203-207 (1970)). Thus, a better understanding of the role of growth factors in development could lead to improved in vitro culture conditions and enhance the outcome in human IVF programs.
Stem cell factor (SCF) is a growth factor related in structure to CSF-1, and acts through the c-kit tyrosine kinase receptor. In bone marrow, SCF and CSF-1 act synergistically to promote proliferation and differentiation of stem cells into macrophage colonies.
EP-A-0423980 discloses the nucleic acid sequence of human SCF, and discusses potential uses of SCF in conditions requiring stimulation of cell proliferation, particularly blood cells.
In mouse, c-kit has been shown to be expressed throughout pre-implantation development (Arceci et al (1992)). We have now shown that the same is true in human embryos. At certain stages the human embryos also express SCF mRNA, suggesting that this growth factor may act in an autocrine fashion. This is in contrast to mouse, where no expression of SCF was detected in pre-implantation embryos (Arceci et al (1992)).
The full length SCF transcript consists of eight exons (Martin, F. H. et al,
Cell
, 63: 203-211 (1990)), which paper also discloses a variant form of SCF. A splice-variant of SCF has also been described which arises by virtue of the loss of exon 6 (Flanagan et al,
Cell
, 63: 1025-1035 (1991)).
SUMMARY OF THE INVENTION
There has now been found a further, novel, splice-variant which appears to arise due to the inclusion of a novel exon consisting of 155 base pairs between exons 3 and 4. This also results in a frameshift, and codes for a species of SCF comprising 33 novel amino acids following exon 3, before terminating at an in frame stop codon which now appears in exon 4 due to the frameshift.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Thus, the present invention provides SCF which includes the following C-terminal sequence (SEQ ID NO:1):
Glu Ile Cys Ser Leu Leu Ile Gly 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
or a sequence which is substantially homologous thereto.
Preferably, the novel SCF of the invention comprises the first 39 amino acids of full length SCF (not including any signal sequence) followed by the above-noted 33 new amino acids. In one embodiment the novel SCF of the invention has a sequence at positions 1-39 substantially homologous to that shown in FIG.
2
.
At the amino acid level, a protein sequence may be regarded as substantially homologous to another protein sequence if a significant number of the constituent amino acids exhibit homology. At least 40%, 50%, 60%, 70%, 80% 90%, 95% or even 99%, in increasing order of preference, of the amino acids may be homologous.
Thus, the alternative splicing mechanism can result in the production of a novel SCF in human embryos. Therefore, the novel SCF of the invention can be used in the treatment of pre-implantation embryos to ensure correct differentiation and development prior to implantation in a subject.
In addition, the invention also provides a DNA sequence coding for a protein of the invention which sequence includes a sequence Substantially homologous to (SEQ ID NO:2):
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 nucleic acid sequences which, by virtue of the degeneracy of the genetic code, also code for the given amino acid sequence or which are substantially homologous to such a sequence.
Sequences having substantial homology may be regarded as those which will hybridise to the nucleic acid sequence shown in
FIG. 2
under stringent conditions (for example, at 35 to 65° C. in a salt solution of about 0.9M).
DNA constructs comprising DNA sequences of the invention form another aspect of the present invention.
As discussed herein, the protein of the invention is useful in treating embryos to ensure correct development prior to implantation. SCF has been shown to act by binding to the transmembrane receptor c-kit. Furthermore, we have shown that human embryos express c-kit throughout most stages of pre-implantation embryo development.
Thus, in further aspects, the present invention provides:
(a) a method for ensuring the correct development of a pre-implantation embryo which comprises the step of administering the SCF of the present invention to a pre-implantation embryo (and preferably a human embryo); and
(b) a method for ensuring the correct development of a human pre-implantation embryo which comprises the step of administering SCF to a human pre-implantation embryo. In this method, the SCF used can be any of the naturally occurring forms, including previously described variants (Martin et al, supra and Flanagan et al, supra), as well as the novel variant described herein.
In addition, the invention also provides the use of SCF in the manufacture of a medicament for use in ensuring correct development in human pre-implantation embryos. Again, any form of SCF can be used to produce a suitable medicament.
The medicament is preferably presented in the form of a pharmaceutical formulation comprising the protein of the invention together with one or more pharmaceutically acceptable carriers and/or excipients. Such pharmaceutical formulati

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