Chemistry: molecular biology and microbiology – Vector – per se
Patent
1994-08-22
1999-12-14
Crouch, Deborah
Chemistry: molecular biology and microbiology
Vector, per se
435458, 514 44, 128200, 128 14, 128 18, 128 23, 128 24, 424450, C12N 1500, C12N 1563
Patent
active
060016442
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
The present invention relates to methods and compositions for producing a transgenic mammal which comprises exogenously supplied nucleic acid coding for a molecule having cystic fibrosis transmembrane conductance regulator activity. The nucleic acid is supplied by aerosolized delivery, particularly to the airways and alveoli of the lung, or by systemic delivery.
BACKGROUND
Many genetic diseases are caused by the absence or mutation of the appropriate protein, for example as a result of deletions within the corresponding gene. One of the most common fatal genetic diseases in humans is cystic fibrosis (CF). Cystic fibrosis (CF), a spectrum of exocrine tissue dysfunction, which eventually leads to respiratory failure and death results from a mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The CFTR gene has now been to chromosome 7q31, and cloned. A 3 bp deletion, resulting in the loss of a phenylalanine residue at amino acid position 508, is present in approximately 70% of CF chromosomes, but is not seen on normal chromosomes. The other 30% of CF mutations are heterogeneous and include deletion, missense, and splice-site mutations. Transfection of even a single normal copy of the CFTR gene abolishes the CF secretory defect in CF cell lines, an observation which supports the feasibility of gene therapy for CF. These results demonstrate that expression of a wild-type CFTR transgene can exert a dominant positive effect in CF cells which concurrently express an endogenous mutant CFTR gene. Thus, expression of the wild-type CFTR transgene in the lungs of CF patients can correct the CF phenotype. However, to date, the inability to produce high level expression of transgenes in the lung by either aerosol or intravenous (iv) administration has precluded the use of gene therapy for the treatment of CF. Expression of a wild-type CFTR transgene in cells from CF patients corrects the chloride secretory defect, the primary biochemical lesion of CF. Chloride secretion is normalized in cells of CF patients despite the presence of the mutant CFTR protein, indicating that when wild-type and mutant CFTR proteins are coexpressed in cells, the wild-type CFTR is dominant.
To date, attempts to replace absent or mutated genes in human patients have relied on ex vivo techniques. Ex vivo techniques include, but are not limited to, transformation of cells in vitro with either naked DNA or DNA encapsulated in liposomes, followed by introduction into a suitable host organ ("ex vivo" gene therapy). The criteria for a suitable organ include that the target organ for implantation is the site of the relevant disease, the disease is easily accessible, that it can be manipulated in vitro, that it is susceptible to genetic modification methods and ideally, it should contain either non-replicating cells or cycling stem cells to perpetuate a genetic correction. It also should be possible to reimplant the genetically modified cells into the organism in a functional and stable form. A further requirement for ex vivo gene therapy, if for example a retroviral vector is used, is that the cells be pre-mitotic; post-mitotic cells are refractory to infection with retroviral vectors. There are several drawbacks to ex vivo therapy. For example, if only differentiated, replicating cells are infected, the newly introduced gene function will be lost as those cells mature and die. Ex vivo approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not first removed from the body. Exemplary of a target organ which meets the criteria of in vivo gene transfer is mammalian bone marrow; mammalian lung is not a good candidate for ex vivo therapy.
Retroviruses, adenoviruses and liposomes have been used in animal model studies in attempts to increase the efficiency of gene transfer. Liposomes have been used effectively to introduce drugs, radiotherapeutic agents, enzymes, uses, transcription factors and other cellular effect into a variety of cultured cell
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Debs Robert J.
Zhu Ning
Crouch Deborah
Martin Jill D.
The Regents of the University of California
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