Hybrid hepatocyte growth factor gene having high expression efficiency of two heterotypes of hepatocyte growth factor

Abstract

The present invention relates to a hybrid Hepatocyte Growth Factor (hgf) gene which is prepared by inserting an inherent or foreign intron between exons 4 and 5 in hgf cdna, which has a base sequence of SEQ ID NO: 2. The gene has high expression efficiency and simultaneously expresses two heterotypes of hgf and dHGF (deleted variant hgf). Further the gene may be used for treating or preventing ischemic or liver diseases.

Description

enterior anterior tibial muscle of the hind limb of mice with an insulin syringe. After 5 days, the mice were sacrificed and the muscles around the injection spot were removed and smashed in a protein extraction buffer (25 mM Tris-HCl (pH 7.4), 50 mM NaCl, 0.5% Na-deoxycholate, 2% NP-40, 0.2% SDS) to separate total proteins. The amount of the total proteins was measured with a DC protein analysis kit (Bio-Rad Laboratories, CA, USA) and the amount of expressed HGF was determined with an ELISA kit (R&D System) according to the manufacturer's instruction.

As can be seen from the result shown in FIG. 10, the amount of HGF expressed from HGF-X7 is 250-fold higher than that from HGF gene cDNA or dHGF gene cDNA.

Together with the result of the experiment of Example 2 (in vivo), this result demonstrates that the expression efficiency of HGF-X gene is much superior to those of HGF gene cDNA or dHGF gene cDNA.

EXAMPLE 4

Gene Therapy Employing HGF-X7 in a Rabbit Ischemic Hind Limb Model

In order to examine whether HGF-X7 gene is effective in the treatment of ischemic hind limb disease, gene therapy was carried out on a rabbit ischemic hind limb model as follows.

A rabbit ischemic hind limb model, which is a standard animal model for the ischemic limb disease, was prepared by the method described by Takeshita et al., Journal of Clinical Investigation 93:662 (1994). At the day before operation (day 0), each of 30 white rabbits from New Zealand (male, from 3.8 to 4.2 kg) was intramuscularly injected with 5 mg/kg of xylazine and, then, anesthetized by an intramuscular injection of 50 mg/kg of ketamine. Subsequently, the left femoral region of the rabbit was incised and all branches of the femoral artery were separated and tied. The region from the proximal part to the branching point of the saphenous and popliteal arteries was incised to prepare the model. After the operation, 15 mg/kg/day of cefazolin was injected intramuscularly for 5 days and 0.3 mg/day of morphine, for 10 days. 10 days after the operation (day 10), angiography was carried out for the left hind limb where the ischemia was induced, and the degree of arteriogenesis was recorded as a basal level. The rabbits were randomly divided into two groups and injected at four sites in the femoral muscle with 500 μg of plasmid pCP-HGF-X7 (experimental group) or 500 μg of plasmid pCP (control), respectively. 40 days after the operation (day 40), angiography was carried out again for the left hind limb and the degree of arteriogenesis at the arteriole level was determined and compared to that of day 10.

As can be seen from the result in FIG. 11, the degree of angiogenesis was significantly enhanced in the experimental group administered with pCP-HGF-X7 as compared with the pCP-administered control group.

This result demonstrates that HGF-X7 gene can be effectively used in the gene therapy of an ischemic disease.

While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

1. A method for co-expressing two heterotypes of Hepatocyte Growth Factor (hgf) in vivo in a mammalian subject with ischemic disease in order to treat the disease, comprising:

transforming or transfecting to a dna construct into a cell of a mammalian subject with ischemic disease by administering a dna construct to the subject, the dna construct comprising:

(a) a promoter,

(b) a first cdna which has the same sequence as exons 1-4 of the human hgf gene wherein said exons 1-4 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said first cdna,

(c) a polynucleotide that has the same sequence as intron 4 of the hgf gene or a functional fragment thereof, and

(d) a second cdna which has the same sequence as exons 5-18 of the human hgf gene wherein said exons 5-18 are arranged in sequential order without an intron therebetween, or degenerates thereof which do not alter the amino acid sequence encoded by said second cdna;

wherein (c) is located between (b) and (d); and the hgf construct simultaneously encodes two heterotypes of human hgf, and

whereby two heterotypes of human hgf are co-expressed within the mammalian subject, thereby treating the ischemic disease.

2. The method of claim 1, wherein said intron has the same sequence as a fragment of intron 4 of the hgf gene.

3. The method of claim 2, wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 19.

4. The method of claim 3, wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 19.

5. The method of claim 4, wherein the construct comprises the sequence of SEQ ID NO: 19.

6. The method of claim 2, wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 20.

7. The method of claim 6, wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 20.

8. The method of claim 7, wherein the construct comprises the sequence of SEQ ID NO: 20.

9. The method of claim 2, wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 21.

10. The method of claim 9, wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 21.

11. The method of claim 10, wherein the construct comprises the sequence of SEQ ID NO: 21.

12. The method of claim 1, wherein the one intron has the same sequence as the full intron 4 of the hgf gene.

13. The method of claim 1, wherein the construct comprises a nucleotide sequence not less than 90% identical to SEQ ID NO: 2.

14. The method of claim 13, wherein the construct comprises a nucleotide sequence not less than 95% identical to SEQ ID NO: 2.

15. The method of claim 14, wherein the construct comprises the sequence of SEQ ID NO: 2.

16. The method of claim 1, wherein the construct further comprises a terminator sequence, a self-replication sequence, or a secretory signal.

17. The method of claim 1, wherein the expression efficiency of the construct is higher than the expression efficiency of hgf cdna or deleted variant hgf (dHGF) cdna.

18. The method of claim 1, wherein the expression level of the construct is about 20- to 100-fold higher than the expression level of the hgf cdna or dHGF cdna.

19. The method of claim 1, wherein the cell is a mammalian cell, a bacterial cell or a yeast cell.

20. The method of claim 19, wherein the cell is a mammalian cell.

21. The method of claim 20, wherein the transformation of said mammalian cell is in vivo.