Polynucleotide encoding a polypeptide having heparanase activity and expression of same in genetically modified cells

• US 20020102560A1
• Filed: 02/06/2001
• Published: 08/01/2002
• Est. Priority Date: 08/31/1998
• Status: Active Grant

• Current Assignee: Insight Biopharmaceuticals Limited
• Original Assignee: Insight Strategy & Marketing Ltd.
• Inventors: Feinstein, Elena, Pecker, Iris, Vlodavsky, Israel
• Granted Patent: US 7,666,651 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12Y 302/01166 Heparanase (3.2.1.166)

Abstract

A polynucleotide (hpa) encoding a polypeptide having heparanase activity, vectors including same, genetically modified cells expressing heparanase, a recombinant protein having heparanase activity and antisense oligonucleotides and constructs for modulating heparanase expression.

Claims

1. An isolated nucleic acid comprising a genomic, complementary or composite polynucleotide sequence encoding a polypeptide having heparanase catalytic activity.

2. The isolated nucleic acid of claim 1, wherein said polynucleotide or a portion thereof is hybridizable with SEQ ID NOs:

9, 13, 42, 43 or a portion thereof at 68°

C. in 6×

SSC, 1% SDS, 5×

Denharts, 10 % dextran sulfate, 100 μ

g/ml salmon sperm DNA, and ³²p labeled probe and wash at 68°

C. with 3×

SSC and 0.1% SDS.

3. The isolated nucleic acid of claim 1, wherein said polynucleotide or a portion thereof is at least 60% identical with SEQ ID NOs:

9, 13, 42, 43 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty—

12, gap extension penalty—

4).

4. The isolated nucleic acid of claim 1, wherein said polypeptide is as set forth in SEQ ID NOs:

10, 14, 44 or portions thereof.

5. The isolated nucleic acid of claim 1, wherein said polypeptide is at least 60% homologous to SEQ ID NOs:

10, 14, 44 or portions thereof as determined with the Smith-Waterman algorithm, using the Bioaccelerator platform developed by Compugene (gapop;

10.0, gapext;

0.5, matrix;

blosum62).

6. A nucleic acid construct comprising the isolated nucleic acid of claim 1.

7. A host cell comprising the nucleic acid construct of claim 6.

8. A recombinant protein comprising a polypeptide having heparanase catalytic activity.

9. The recombinant protein of claim 8, wherein said polypeptide includes at least a portion of SEQ ID NOs:

10, 14 or 44.

10. The recombinant protein of claim 8, wherein the protein is encoded by a polynucleotide hybridizable with SEQ ID NOs:

9, 13, 42, 43 or a portion thereof at 68°

C. in 6×

SSC, 1% SDS, 5×

Denharts, 10% dextran sulfate, 100 μ

g/ml salmon sperm DNA, and ³²p labeled probe and wash at 68°

C. with 3×

SSC and 0.1% SDS.

11. The recombinant protein of claim 8, wherein the protein is encoded by a polynucleotide at least 60% identical with SEQ ID NOs:

9, 13, 42, 43 or portions thereof as determined using the Bestfit procedure of the DNA sequence analysis software package developed by the Genetic Computer Group (GCG) at the university of Wisconsin (gap creation penalty—

12, gap extension penalty—

4).

12. A pharmaceutical composition comprising, as an active ingredient, the recombinant protein of claim 8.

13. A method of identifying a chromosome region harboring a heparanase gene in a chromosome spread comprising the steps of:

(a) hybridizing the chromosome spread with a tagged polynucleotide probe encoding heparanase;

(b) washing the chromosome spread, thereby removing excess of non-hybridized probe; and

(c) searching for signals associated with said hybridized tagged polynucleotide probe, wherein detected signals being indicative of a chromosome region harboring a heparanase gene.