Use of hyaluronan in gene therapy

Details Use of hyaluronan in gene therapy Use of hyaluronan in gene therapy

1997-06-16

2002-11-05

McKelvey, Terry (Department: 1636)

Chemistry: molecular biology and microbiology

Process of mutation, cell fusion, or genetic modification

Introduction of a polynucleotide molecule into or...

C435S455000, C530S395000, C536S024500

Reexamination Certificate

active

06475795

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates to the use of forms of hyaluronic acid (hyaluronan) in gene therapy. This invention also relates to the use of hyaluronan as a targeting agent in gene therapy to target active agents which ablate the function of targeted genes in the control or treatment of disease and/or conditions.
Hyaluronic acid is a large, complex oligosaccharide consisting of up to 50,000 pairs of the basic disaccharide glucuronic acid-&bgr;(1-3) N-acetylglucos-amine &bgr;(1-4). It is found in vivo as a major component of the extracellular matrix. Its tertiary structure is a random coil of about 50 nm in diameter. Hyaluronic acid appears in nature in its sodium salt form. Hyaluronic acid and its pharmaceutically tolerable or acceptable salts (such as sodium hyaluronate) are identified herein as HA.
HA has the ability to bind a large amount of water, which in vivo makes it a viscous hydrated gel with viscoelastic properties. It is found in this form in the mammalian eye, both in the vitreous and in the extracellular matrix.
HA has been used in the treatment of diseases and conditions of the human body both systemically and topically because of its ability to target an active agent to sites where the disease or condition is located (International Patent Publications No. WO 91/04058 and No. WO 93/16733). It has been shown that HA forms depots for example, at the injured carotid artery (relative to uninjured contralateral arteries) and in colorectal tumours growing in experimental animals, and is retained in the skin of humans and animals. In all these cases, the sites of the deposits are areas of high HA receptor expression, indicating that HA targets specifically to areas for example, of underperfused and pathological tissues that are expressing high levels of these receptors, particularly to tissues undergoing unusual proliferation and migration, including tissues responding to injury, inflammation, development, and tumorigenesis.
Cells of the immune system, including macrophages, neutrophils, T-cells and natural killer cells play a significant role in the conditions described above. These cells rely upon HA receptors to mature into reactive cells that are able to release growth factors and cytokines, as well as oxidizing agents that then set in motion a repair response that contributes to tissue damage. In addition, these cells rely upon HA receptors to adhere to cells such as those of the endothelium and tumour cells and to locomote (migrate) into the site of injury. Tumour cells also require HA receptors to migrate, proliferate and metastasize. Thus, it is possible to interfere with white cell infiltration, for instance into injured rat carotid artery, by adding high concentrations of HA, to inhibit tumour cell proliferation and to block smooth muscle locomotion. Reagents such as function blocking antibodies and peptides mimicking critical regions of the HA receptors are also able to exert the same effect.
Collectively, these results show that HA specifically interacts with unique receptors that occur for example, on the surface of a variety of injured or tumorigenic cells.
Cell surface receptors specific for HA have been identified, including the histocompatibility antigen CD44 and receptor for hyaluronan-mediated motility (RHAMM).
In all of the diseases and conditions discussed above, as well as others, researchers are now attempting to treat the diseases and conditions (for example, restenosis) by gene therapy. The rationale is to ablate a function of a specific gene by either blocking messenger RNA translation to proteins with “antisense ” oligonucleotides or gene expression with antisense cDNA. To date, this has largely involved use of these reagents processed within liposomes and by directly applying antisense cDNA's or oligonucleotides to the site of disease. Viruses (for example, adenovirus) have also been proposed but it is difficult to target the viruses to specific sites of disease evidenced by minimal uptake. For example, the process of using viruses which adhere to cells and are taken up by them to target them to DNA is inefficient and the risk of using viruses is to be frowned on and is, for the most part, unacceptable in man. Where gene therapy involves the use of liposomes, it is difficult to target liposomes efficiently and uptake may be even lower than with viruses.
Directly applying the oligonucleotides to the site of injury such as a stenotic plaque has produced beneficial effects in animals but large amounts of oligonucleotides are required and the process prolongs for example, the balloon angioplasty operation which increases the danger to the patient. These results indicate that while gene therapy is a theoretically viable approach to treat disease and conditions, the technical difficulties of efficient targeting and uptake need to be improved.
In an article appearing at page 8 of “The Toronto Star”, a daily newspaper published in Toronto, Ontario, Canada on Sep. 22, 1996, there appeared an article discussing the use of gene therapy for the treatment of cancer. The article stated:
“Gene therapy for cancer tries to address the problem of tumors arising because they contain faulty genes. There are several mutant genes that have been implicated in a variety of tumors, but one of the most important is a gene called p53.
In normal cells, p53 helps keep the cell's genetic material in good working order. If there are breaks in the DNA double helix, p53 switches on and initiates DNA repair. If things get out of hand and repair isn't possible, p53 triggers the death of the cell. If such genetic problems go unrepaired, abnormal cell growth—tumors—result.
That is exactly what happens when mutant or nonfunctional versions of p53 arise. The statistics tell the story. At least half of all malignant tumors contain cells with problems in their p53 genes. This includes 80 per cent of colon cancers, 50 per cent of lung cancers, and 40 per cent of breast cancers.
In a small-scale trial reporting in this month's Nature Medicine, a team at the University of Texas treated nine lung cancer patients who had failed all conventional treatment. All had multiple tumors, some of which had metastasized to other organs such as the brain. The gene therapy consisted of injecting good copies of p53 into selected tumor sites.
The technology of delivering genes to tumors is amazing. The genes are placed inside inactivated viruses, tens of millions of viruses per milliliter are injected into the tumor, and some of them invade cancer cells and presumably integrate their genetic package—including the “good ” p53—into those cells.
In the Texas trial, two of the patients either couldn't complete the trial or died before its completion. In three of the remaining seven, the tumor treated with gene therapy actually shrank. In three others, the tumor, while not shrinking, showed no signs of growth. One patient showed complete regression of the tumor. Programmed cell death inside the tumor, a process known to be initiated by active p53 genes, was seen in six of the patients.
Remember, these were patients for whom conventional cancer therapy held no hope. In fact, many of them died within months of the trial from the growth of other tumors that had not been targeted by the gene therapy.”
The suppression of expression of genes encoding proteins which mediate undesirable activity has been achieved in a variety of situations by the introduction of ‘anti-sense’ DNA sequences into the DNA of target cells. These anti-sense sequences are DNA sequences which, when transcribed, result in synthesis of RNA whose sequence is antiparallel to the sequence encoding the protein. Such anti-sense sequences have been tested in a number of viral diseases. Alternatively, anti-sense oligodeoxynucleotides can be introduced into target cells; such short sequences are not themselves transcribed, but inhibit transcription and/or subsequent translation of the corresponding sense DNA sequence in the target cell.
Until recently it was widely thought that the minimum sequence len

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