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The invention relates to novel heparanases, heparanase splice variants, and to polynucleotides encoding them. Particularly, the invention relates to Spalax heparanases, and to Spalax and human heparanase splice variants. Heparanase splice variants can be used, for example, to modulate the activity of heparanase in diseases disorders or conditions caused by or associated with the enzymatic activity of heparanase. For instance, a splice variant capable of down regulating the activity of heparanase can be used to treat primary tumors and/or to prevent or treat metastasis.

   

Inventors:Nasser Nicola, Avivi Aaron, Vlodavsky Israel, Nevo Eviatar

 

FIELD OF THE INVENTION

The present invention relates to heparanases and heparanase splice variants, particularly to Spalax heparanase and human and Spalax heparanase splice variants, to polynucleotides encoding them, and to pharmaceutical compositions and methods comprising said heparanases or polynucleotides.

Abbreviations: ECM: extracellular matrix; HS: Heparan sulfate; HSPGs: Heparan sulfate proteoglycans; SH: Spalax heparanase; VEGF: vascular endothelial growth factor.

BACKGROUND OF THE INVENTION

Heparan sulfate proteoglycans (HSPGs) are macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues. Heparan sulfate (HS) binds to and assembles ECM proteins and plays important roles in the structural integrity of the ECM and in cell-cell and cell-ECM interactions. HS chains sequester a multitude of proteins and bioactive molecules and thereby function in the control of a large number of normal and pathological processes . Apart from sequestration of bioactive molecules, HSPGs have a coreceptor role in which the proteoglycan, in concert with the other cell surface molecule, comprises a functional receptor complex that binds the ligand and mediates its action .

Enzymatic degradation of HS by heparanase, a mammalian endoglucuronidase, affects the integrity and functional state of tissues and is involved in fundamental biological phenomena, ranging from pregnancy, morphogenesis and development to inflammation, angiogenesis and cancer metastasis. Heparanase elicits an indirect angiogenic response by releasing HS-bound angiogenic growth factors (e.g., basic fibroblast growth factor--bFGF and vascular endothelial growth factor--VEGF) from the ECM and by generating HS fragments that potentiate bFGF receptor binding, dimerization and signaling.

By degradating HS of cell surface and ECM, heparanase facilitates locomotion of inflammatory and tumor cells, release growth factors bound to the ECM, and induce new blood vessels formation (angiogenesis). Heparanase expression in tumor cells is correlated with worse prognosis, and its expression in experimental tumor models resulted in increased tumor growth and metastasis formation. Moreover, elevated levels of heparanase have been detected in sera of animals and human cancer patients bearing metastatic tumors, and in the urine of some patients with aggressive metastatic disease. Regulation of heparanase activity in normal tissues is poorly understood.

Despite earlier reports on existence of several distinct mammalian HS-degrading endoglycosidases (heparanases), the cloning of the same single gene (SEQ ID NO: 41) by several groups  suggests that mammalian cells express primarily a single dominant functional heparanase enzyme. Since the cloning of human heparanase, no splice variants were described.

Human heparanase is synthesized as a latent 65-kDa precursor whose processing involves proteolytic cleavage and formation of an active enzyme composed of two 50-kDa and 8-kDa subunits.

Heparanase exhibits endoglycosidase activity at acidic pH , which exists in nonvascularized core of tumor masses. Heparanase mRNA is increased in human breast, colon, lung, prostate, ovary and pancreas tumors compared with the corresponding normal tissues. In human normal tissues, heparanase mRNA expression is limited to the placenta and lymphoid organs.

Because heparanase promotes angiogenesis and cancer progression, the present inventors found of interest to investigate the evolution of this unique enzyme in a wild mammal that was exposed to underground hypoxic stress throughout the family Spalacidae evolutionary history .


Among the strategies used by Spalax to tolerate hypoxia are: higher myocardial maximal oxygen consumption , structural adaptations in tissues that result in a decreased diffusion distance of oxygen to the mitochondria , increase in the lung diffusion capacity , specific differences in myoglobin which augment oxygen delivery at low oxygen tensions , and increased density of blood vessels, correlated with a unique VEGF expression pattern . Hemoglobin and hematocrit are higher in the northern species which survive more hypoxia than the southern ones.

The present inventors have recently cloned and elucidated the expression of p53 and VEGF in Spalax. p53 gene in healthy Spalax individuals possesses two amino acid substitutions in its DNA binding domain, identical to mutations found in human tumors. These adaptive substitutions endow Spalax p53 with several-fold higher activation of cell arrest and DNA repair genes compared to human p53, and they also favor activation of DNA repair genes over apoptotic genes. Expression of VEGF was constitutively high in Spalax muscles, regardless of the oxygen levels, similar to its expression in highly metastatic tumor cells and unlike its levels in rat muscle.

SUMMARY OF THE INVENTION

In accordance with the present invention, novel heparanases were found and isolated from the subterranean blind mole rat of the genus Spalax (hereinafter "Spalax"). The high rate of alternative splicing of the heparanase gene in Spalax enabled the identification of Spalax heparanase splice variants that until now could not be detected in other species. Based on the these Spalax variants, also human heparanase splice variants were isolated and identified.

Thus, in one aspect, the present invention relates to an isolated
In another aspect, the present invention relates to an isolated polynucleotide encoding a polypeptide of the invention or a fragment thereof, as defined above.


The invention further includes polynucleotides of a nucleic acid sequence having at least about 60% identity, for example, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or at least 99% sequence identity with a nucleic acid sequence identified above as well as polynucleotides encoding the polypeptides of the invention but comprising degenerate codons.

The invention also provides a vector, preferably an expression vector, comprising a polynucleotide of the invention, a host cell comprising said expression vector and a process of producing a polypeptide of the invention comprising culturing said host cell under suitable conditions to express said polypeptide, and isolating the polypeptide from the culture.

The invention further relates to pharmaceutical compositions comprising a polypeptide or a polynucleotide or a vector comprising said polynucleotide of the invention, and a pharmaceutically acceptable carrier.

In one embodiment of the invention, the pharmaceutical composition comprises a polypeptide/heparanase splice variant of the invention capable of downregulating the enzymatic activity of heparanase and is useful for treatment of diseases, disorders and conditions such as, for example, primary tumors and/or prevention or treatment of metastasis.

In another embodiment of the invention, the pharmaceutical composition comprises a Spalax heparanase and/or a polypeptide/heparanase splice variant of the invention capable of pro-angiogenic activity and is useful for treatment of diseases, disorders and conditions such as, for example, vascular diseases.

The invention also provides a method for the treatment of a subject suffering from a disease, disorder or condition caused by or associated with the enzymatic activity of heparanase comprising administering to said subject an effective amount of a polypeptide according to the invention, or a polynucleotide encoding said polypeptide or a vector comprising said polynucleotide.