Vector-mediated delivery of polynucleotides encoding soluble VEGF receptors
The present invention provides vector compositions for expression of a soluble form of VEGFR3 and methods for their use in the inhibition of one or more of lymphangiogenesis, lymphatic metastasis and angiogenesis, as a therapeutic strategy for treatment of cancer.
1 . A recombinant viral vector comprising:
a protein coding nucleic acid that comprises a nucleotide sequence that encodes a polypeptide that comprises a soluble, ligand binding extracellular domain fragment of a Vascular Endothelial Growth Factor Receptor 3 (VEGFR3); and
a promoter operably linked to the protein coding nucleic acid, wherein said promoter is capable of promoting expression of the protein coding nucleic acid in mammalian cells, said promoter selected from the group consisting of: (a) a cytomegalovirus enhancer/chicken beta-actin/Rabbit β-globin promoter (CAG) promoter; and (b) an EF-1 alpha promoter.
2 . The vector of claim 1 , wherein the protein coding nucleic acid sequence comprises a polynucleotide sequence at least 90% identical to nucleotides 1-987 of SEQ ID NO: 1.
3 . The vector of claim 1 , wherein the protein coding nucleic acid sequence comprises a polynucleotide sequence corresponding to nucleotides 1-987 of SEQ ID NO: 1.
4 . The vector of claim 1 , wherein the fragment comprises the first three (1-3) Ig-like domains of human VEGFR-3.
5 . The vector of claim 4 , wherein the fragment comprises an amino acid sequence at least 90% identical to residues 1-328 of SEQ ID NO: 2.
6 . The vector of claim 4 , wherein the fragment comprises an amino acid sequence corresponding to residues 1-328 of SEQ ID NO: 2.
7 . The vector of claim 4 , wherein the polypeptide further includes a signal peptide.
8 . The vector of claim 7 , wherein the polypeptide further comprises a human IgG Fc amino acid sequence attached to the fragment of VEGFR-3.
9 . The vector of claim 8 , wherein the human IgG Fc amino acid sequence comprises an amino acid sequence at least 90% identical residues 342-572 of SEQ ID NO: 2.
10 . The vector of claim 8 , wherein the human IgG Fc amino acid sequence comprises residues 342-572 of SEQ ID NO: 2.
11 . The vector of claim 8 , wherein the polypeptide comprises an amino acid sequence at least 90% identical to SEQ ID NO: 2.
12 . The vector of claim 8 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 2.
13 . The vector of claim 1 , wherein said promoter is a cytomegalovirus enhancer/chicken beta-actin/Rabbit β-globin promoter (CAG) promoter.
14 . The vector of claim 8 , further comprising at least one nucleotide sequence selected from the group consisting of:
(a) functional viral 5′ and 3′ inverted terminal repeat (ITR) sequences;
(b) a Woodchuck Hepatitis Virus Post-transcriptional regulatory element (WPRE); and
(c) a bovine growth hormone polyadenylation sequence (bGHpA).
15 . The vector of claim 8 , that is a recombinant adeno-associated virus (rAAV) vector.
16 . The vector of claim 14 , wherein said vector comprises in the 5′ to 3′ direction, the 5′ ITR, the promoter, the protein coding nucleic acid, the WPRE, the poly A sequence; and the 3′ ITR.
17 . The vector of claim 16 , wherein the vector comprises an amino acid sequence at least 90% identical to SEQ ID NO: 6.
18 . The vector of claim 16 , wherein the vector comprises the amino acid sequence of SEQ ID NO: 6.
19 . A method of inhibiting one or more of lymphangiogenesis, lymphatic metastasis and angiogenesis in a mammalian subject, comprising:
administering a replication-deficient vector according to claim 1 to a mammalian subject in need of treatment to inhibit one or more of lymphangiogenesis, lymphatic metastasis, and angiogenesis.
20 . A method of inhibiting one or more of lymphangiogenesis, lymphatic metastasis and angiogenesis in a mammalian subject, comprising:
administering a replication-deficient vector according to claim 16 to a mammalian subject in need of treatment to inhibit one or more of lymphangiogenesis, lymphatic metastasis, and angiogenesis.
21 . The method of claim 20 , wherein vector is administered by at least one route selected from the group consisting of intravenous, intramuscular, and into the portal vasculature of said mammal.
22 . The method of claim 20 , wherein the mammal has cancer.
23 . A method of treating cancer in a mammal, comprising:
administering a replication-deficient vector according to claim 1 to a mammal in need of treatment for cancer, in an amount effective to cause said vector to infect mammalian cells and cause production in the mammal of the soluble VEGFR3 polypeptide in a therapeutically effective amount.
24 . A method of treating cancer in a mammal, comprising:
administering a replication-deficient vector according to claim 16 to a mammal in need of treatment for cancer, in an amount effective to cause said vector to infect mammalian cells and cause production in the mammal of the soluble VEGFR3 polypeptide in a therapeutically effective amount.
25 . The method of claim 24 , wherein vector is administered by at least one route selected from the group consisting of intravenous, intramuscular, and into the portal vasculature of said mammal.
26 . The method according to claim 24 , wherein the mammal has at least one cancer selected from the group consisting of prostate, kidney, melanoma and lung cancers.
27 . A method of expressing sVEGFR3 in a mammalian cell, comprising:
administering to a mammalian cell a vector according to claim 1 , to cause the cell to express the polypeptide.