Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-07-25
2003-07-15
Priebe, Scott D. (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C424S093200, C435S455000, C435S320100
Reexamination Certificate
active
06593304
ABSTRACT:
TECHNICAL FIELD
The present invention relates to recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA, a microorganism carrying the recombinant DNA and an agent for treatment of arteriosclerosis comprising the recombinant DNA which are used in gene therapy.
BACKGROUND ART
Smooth muscle-type myosin heavy chain SM1 isoform protein is responsible for contraction and relaxation of smooth muscles, and is one of the myosin heavy chain isoform proteins expressed specifically in smooth muscle cells. As the DNA coding for the protein, the nucleotide sequence of cDNA coding for rabbit SM1 isoform is known (P. Babij et al.: Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), but there is not known any homology among nucleotide sequences for such DNAs. Further, there is not known any recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA which recombinant DNA can be injected into animal cells.
It has been reported that when cDNA coding for a protein called calponin which is a protein existing in smooth muscle cells is introduced and expressed in a vascular smooth muscle cell line derived from rat pulmonary arteries, the time required for doubling the cells is prolonged by about 4 hours (Takahashi et al.: Circulation, 88, I-174 (1993)) and that when cDNA for human calponin is injected into a rabbit topically at a site where the carotid artery has been abraded with a balloon, thickening of the intima is inhibited (Takahashi et al.: Circulation, 88, I-656 (1993)). It is not known that recombinant DNA comprising DNA coding for myosin heavy chain SM1 isoform protein inserted into vector DNA has pharmacological effect and is used for in gene therapy.
For treatment of arteriosclerosis, percutaneous transluminal coronary angioplasty (PTCA) in which a stenosed site is enlarged by a balloon catheter is extensively conducted. The treatment method has the advantages of easier operation and higher degree of success than the bypass surgery etc., while the treatment method has the disadvantage that 30 to 40% of the patients after the treatment suffer from restenosis of blood vessels due to abnormal proliferation of vascular cells. Therefore, there is demand for developments in an agent for treatment of arteriosclerosis effectively used for preventing such restenosis.
DISCLOSURE OF THE INVENTION
The present invention includes:
(1) A recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA.
(2) The recombinant DNA according to (1) wherein the vector DNA is a retrovirus vector, adenovirus vector, adeno-associated virus vector or a plasmid capable of being expressed in an animal.
(3) The recombinant DNA according to (2) wherein the plasmid capable of being expressed in an animal is pCXN2 or PAGE208.
(4) The recombinant DNA according to any one of (1) to (3) wherein the smooth muscle-type myosin heavy chain SM1 isoform protein is of a human smooth muscle type, rabbit muscle type or mouse muscle type.
(5) The recombinant DNA according to any one of (1) to (3) wherein the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.
(6) DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, which is the nucleotide sequence shown in SEQ ID NO:1, or the nucleotide sequence shown in SEQ ID NO:1 in which at least one nucleotide is added, deleted or replaced.
(7) A microorganism carrying the recombinant DNA of (5).
(8) The microorganism according to (7) which belongs to the genus Escherichia.
(9) An agent for treatment of arteriosclerosis which comprises the recombinant DNA of any one of (1) to (5).
The DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes cDNA or genomic DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein, preferably DNA derived from humans, rabbits, mice, etc.
The nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein includes the nucleotide sequence of cDNA (SEQ ID NO:5) coding for rabbit SM1 isoform protein (SEQ ID NO:4) (Proc. Natl. Acad. Sci. USA, 88, 10676 (1991)), the nucleotide sequence of the DNA coding for mouse myosin heavy chain SM1 isoform protein as shown in SEQ ID NO:1, and the nucleotide sequence as shown in SEQ ID NO:2 which is specified by homologous regions between the above nucleotide sequences (In the Sequence Listing, Y represents T, U or C; S represents G or C; V represents A, G or C; B represents B, C, T or U; W represents A, T, or U; N represents A, C, G, T, U or a single bond; and the codon YAS (388-390) represents TAC or CAG.). Accordingly, the nucleotide sequence shown in SEQ ID NO:2 contains the nucleotide sequence of SEQ ID NO:1 and the nucleotide sequence of the cDNA coding for rabbit SM1 isoform protein. A partial nucleotide sequence of cDNA coding for human smooth muscle-type myosin heavy chain SM1 isoform protein is also known (Amer. J. of Medical Genetics, 46, 61-67 (1993)), and a nucleotide sequence of this partial nucleotide sequence combined with a partial nucleotide sequence of SEQ ID NO:2 is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.
Further, the nucleotide sequence of SEQ ID NO:2 or the combination of a partial nucleotide sequence of SEQ ID NO:2 and the nucleotide sequence of the DNA derived from human smooth muscles, in which one or more nucleotides have been added, deleted or replaced by means of site-directed mutagenesis (Nucleic Acid Research, 10, 6487-6508 (1982)), is also contained in the nucleotide sequence of the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein according to the present invention.
The vector DNA that can be used includes virus vectors such as retrovirus vector, adenovirus vector and adeno-associated virus vector and plasmids capable of being expressed in an animal such as pCXN2 (Gene, 108, 193-200 (1991)) and PAGE207 (Japanese Patent Laid-Open Publication No. 46841/1994) as well as their modified vectors.
The agent for treatment of arteriosclerosis according to the present invention can be produced by compounding the recombinant DNA comprising DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein inserted into vector DNA as an active ingredient together with a base used in agents for gene therapy. Just before administration, the pharmaceutical preparation can be used in gene therapy for arteriosclerosis, if necessary after encapsulation in liposomes etc. (Proc. Natl. Acad. Sci. USA., 90, 11307 (1993)).
Where the DNA coding for smooth muscle-type myosin heavy chain SM1 isoform protein is inserted into a virus vector, a therapeutic agent can be produced by preparing virus particles containing the recombinant DNA and then compounding them together with a base used in agents for gene therapy (Nature Genet., 8, 42(1994)).
The base used in agents for gene therapy may be any base generally used in injections. The base includes, for example, distilled water, a salt solution of sodium chloride, a mixture of sodium chloride and an inorganic salt, or the like, solutions of mannitol, lactose, dextran, glucose, etc., solutions of amino acid such as glycine, arginine, etc., a mixed solution of an organic acid solution or a salt solution and glucose solution, and the like. Further, injections may be prepared in a usual manner as a solution, suspension or dispersion by adding adjuvant such as an osmotic pressure controlling agent, pH adjusting agent, vegetable oils such as sesame oil, soybean oil, etc. or surface active agents such as lecithin, non-ionic surface active agent, etc. to the above base. These injections can also be powdered, lyophilized, etc. to be dissolved just before use.
The agent for treatment of arteriosclerosis
Arakawa Emi
Hasegawa Kazuhide
Ishiyama Haruo
Matsuda Yuzuru
Oda Shoji
Priebe Scott D.
Vessell Research Laboratory Co. Ltd.
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