Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues
Reexamination Certificate
1998-08-26
2002-01-15
McGarry, Sean (Department: 1635)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
C435S320100
Reexamination Certificate
active
06339139
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a gene transfer system comprising a ligand oligopeptide for growth factor receptor, an endosome-releasing oligopeptide and a polycationic polypeptide to form a complex with exogenous gene either in the form of free DNA of a recombinant eukaryotic expression vector or in the form of a recombinant viral vector. Via receptor mediated endocytosis, exogenous DNA was targetably transduced into the tumor cells for the purpose of tumor gene therapy.
BACKGROUND INFORMATION
Gene therapy is to transduce the exogenous DNA into certain type(s) of human cells for the treatment of human diseases. For the therapeutic purpose, it is of prime importance to generate a gene transfer system with safety and high efficiency and targetability. So far as it is concerned. there are two types of systems currently used to transfer exogenous genes into human cells. The first type is the viral vector system and the second is the non-viral vector techniques. However, no effective systems are available. Referring to the non-viral system, it was reported in recent years that exogenous DNA could be transduced into cells by receptor-mediated gene transfer techniques. The exogenous DNA can form a complex with a ligand associated polypeptide. After the binding of ligand and cell receptor, the DNA/polypeptide can be endocytosed thereby transferring the exogenous gene into cells enriched with the relevant receptors. Wu G. Y. et al (J. Biol. Chem. 263:14621, 1988) described that asiologlycoprotein conjugated polylysine can mediate uptake of exogenous gene by hepatocytes. Transferrin was also used as a ligand to transduce exogenous genes into cells by binding with cell surface transferrin receptor and subsequent endocytosis (Birnstiel M. L. et al, PNAS, USA. 87:3410-3414, 1990).
However, the gene transduction by these receptor-mediated gene transfer system was limited to non-tumorous cells, such as hepatocytes, but not described to target malignant cells. One of the major limitations of these vectors or other non-viral systems is the enzymatic degradation of the endocytosed DNA due to the fusion of lysosome and endosome vesicles, thereby reducing the transduction efficiency and expression of exogenous genes. However, it is well known that some envelope domains of virus, such as adenovirus or influenza virus, can cause endosomolysis and release the endocytosed virus, to protect from the lysosomal fusion and degradation. It was reported that defective or chemically inactivated adenovirus can be used to disrupt the endosome so as to increase the transfer efficiency of the receptor-mediated gene transfer system (Birnstiel M. L., PNAS, USA. 89:6094-6098, 1992).
SUMMARY OF INVENTION
The present invention is a novel targeting gene transfer system for tumor gene therapy. The present system comprises a ligand oligopeptide for receptor recognition (LOP), a polycationic polypeptide(PCP) for DNA binding and an endosome-releasing oligopeptide (EROP) for endosome disrupting. This system includes the 3-element composite polypeptide vector LOP/PCP/EROP which can bind DNA to form a 4-element complex gene transfer system and 2-element composite polypeptide vector LOP/PCP which can bind DNA to form a 3-element complex gene transfer system. The LOP includes E5(14 amino acid) for IGF-I and IGF-II receptor, GE7(16 amino acid) for EGF receptor and GV1(32 amino acid), GV2(36 amino acid) for VEGF receptor recognition. The PCP includes protamine, polylysine and histone. The EROP is a synthetic 20 amino acid oligopeptide homologous to haemagglutinin domain of Influenza viral envelope (HA20). To take advantage of the recognition capability of LOP for receptor overexpressed on surface membrane of cancer cells, therapeutic genes are to be transduced into cells via endocytosis and endosomolysis for tumor gene therapy.
It is an object of the present invention to provide a new gene transfer system that can targetably transduce exogenous genes into certain type of cancer cells in vitro and in vivo with high efficiency. This system consists of following elements:
i. A receptor-specific ligand oligopeptide(LOP) that is designed to recognize the cells with relevant expressed receptor.
ii. A polycationic polypeptide (PCP) for forming a complex with DNA from plasmid containing exogenous gene. The PCP is polylysine, protamine or histone.
iii. An endosome-releasing oligopeptide (EROP) for the release of exogenous DNA from endosome after endocytosis.
iv. The polypeptide comprising of the above elements to form a complex with DNA from recombinant eukaryotic expression vector containing exogenous gene as a novel gene transfer system (GDS).
The other object of the present invention is to provide the system that comprises composite polypeptide vector:
i. LOP and EROP are together linked with PCP to form LOP-PCP-EROP 3-element composite polypeptide vector
ii. LOP and EROP are independently linked with PCP to form LOP-PCP and EROP-PCP 3-element composite polypeptide vector.
The other object of the present invention is to design and prepare the LOP E5, GE7, GV1 and GV2:
i. E5 is to recognize and bind specifically to IGF-I and IGF-II receptors that are highly expressed in human hepatic or breast cancer.
ii. GE7 is to recognize and bind specifically to EGF receptor or indirectly to c-erbB2, that are highly expressed in human hepatic, breast, gastric, esophageal, lung cancer and brain glioblastoma.
iii. GV1 and GV2 are to recognize and bind to vascular endothelial growth factor receptor(VEGF R), which are highly expressed in angiogenetic vessels in most solid malignant tumors.
The other object of the present invention is to use protamine, polylysine and histone as a backbone of the GDS to form a complex with DNA.
The other object of the present invention is to use the composite polypeptide LOP-PCP and EROP-PCP to bind DNA and form a LOP-PCP/EROP-PCP/DNA complex. DNA of exogenous genes include:
1. Antisense sequence of protooncogenes ras
H
, ras
K
, ras
N
, c-myc, bcl-2 and growth factor receptor. The antisense sequence is either in a form of double stranded DNA or in a form of oligoribonucleotides or oligodeoxyribonucleotides.
2. Cancer suppressor gene p53 and Rb.
3. Suicide gene HSV-TK and CD.
4. Apoptosis-inducing gene p15, p16 and p21
WAF−1
.
5. Cytokine gene GM-CSF, Interferon &agr; and &ggr;, Interleukin 2,3,4,12 and 15.
The other object of the present invention is to use the haemagglutinin domain oligopeptide HA20 as an element in GDS to break the endosome and prevent lysosomal degradation of transduced gene after endocytosis.
The HA20 is to be used either as a part of the covalently bound composite polypeptide, LOP-PCP/HA20-PCP to form a complex with DNA or as a free element used in conjunction with LOP-PCP/DNA complex.
The another object of the present invention is to utilize the GDS to transduce genes into cells either in vitro or in vivo for human tumor gene therapy.
DETAILED DESCRIPTION OF THE INVENTION
The present invention principally relates to 4 parts: providing ligand oligopeptides (LOP) for specific recognition and binding to cell growth factor receptors; providing a 2-element composite polypeptide vector LOP-PCP to form a 3-element gene transfer system composed of a complex of DNA from exogenous gene and LOP-PCP; providing a 3-element composite polypeptide vector LOP-PCP-EROP to form a 4-element gene transfer system composed of a complex of DNA from exogenous gene and LOP-PCP-EROP or LOP-PCP plus EROP-PCP; providing methods and techniques to transduce gene(s) into cells both in vitro and in vivo for tumor gene therapy.
1. The first part of the present invention is to provide the LOP E5, GE7, GV1 and GV2 for specific recognition and binding to insulin growth factor I and II receptor(IGF-I R and IGF-II R), epidermal growth factor receptor(EGF R) and vascular endothelial growth factor receptor(VEGF R) respectively.
It has been demonstrated that IGF-I R and IGF-II R are over-expressed in human hepatic cancer and other malignancies. EGF R is highly expressed in human hepatic, mammary, ovarian,
Gu Jianren
Tian Peikun
McGarry Sean
Nath & Associates PLLC
Novick Harold L.
Shanghai Cancer Institute
Shibuya Mark L.
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