Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
1997-04-11
2001-02-27
Crouch, Deborah (Department: 1632)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C435S459000
Reexamination Certificate
active
06194389
ABSTRACT:
SUMMARY OF THE INVENTION
This invention relates to the transformation of animal cells and tissue with heterologous DNA by microprojectile bombardment.
BACKGROUND OF THE INVENTION
The transformation of living cells by propelling microprojectiles at those cells at high velocity, with the microprojectiles carrying exogenous DNA or RNA, was originally proposed by T. Klein, E. Wolf, R. Wu and J. Sanford,
Nature
327, 70 (1987). See also J. Sanford et al.,
Particulate Science and Technology
5, 27 (1987). The original work involved the tranformation of onion epidermal cells with RNA derived from tobacco mosaic virus. The findings with onion epidermal cells have been extended to other plants. For example, the transformation of soybean callus by particle bombardment is described by P. Christou et al.,
Plant Physiol.
87, 671 (1988), and the transformation of soybean meristem is described by D. McCabe et al.,
Bio/Technology
6, 923 (1988). European Patent Application Publication No. 0 301 749 to P. Christou et al. The transformation of embryonic maize callus cells by particle bombardment is described by T. Klein et al.,
Proc. Natl. Acad. Sci. USA
85, 4305 (1988), and the production of transformed maize seed by the particle bombardment of maize pollen is described in European Patent Application Publication No. 0 270 357 to D. McCabe et al.
In addition to the transformation of plants, microprojectile bombardment has been used to transform cellular organelles. Mitochondrial transformation in yeast by particle bombardment is described by S. Johnston et al.,
Science
240, 1538 (1988), and chloroplast transformation in
Chlamydomonas
by particle bombardment is described by J. Boynton et al.,
Science
240, 1534 (1988).
The use of particle bombardment for the transformation of animal tissue or cells has received comparatively little attention. Sanford et al.,
Particulate Science and Technology
5, 27, 35-36 (1987), suggest the use of particle bombardment for human gene therapy, but do not suggest the tissue type or the developmental stage of tissue useful for carrying out such therapy. U.S. patent application Ser. No. 06/877,619, titled “Method for Transporting Substances Into Living Cells and Tissues and Apparatus Therefor,” concerns the introduction of biological materials into cells by microprojectile bombardment. Suggested biological substances are stains such as fluorescent or radiolabled probes, viruses, organelles, vesicles, proteins such as enzymes or hormones, and nucleic acids such as DNA and RNA. Suggested procedures include: (a) the particle bombardment of animal cells such as eggs, bone marrow cells, muscle cells, and epiderman cells at page 16, lines 5-6; (b) the particle bombardment of human tissue or other animal tissue such as epidermal tissue, organ tissue, and tumor tissue at page 16, lines 13-14; and (c) human gene therapy for sickle cell anemia by the particle-mediated transformation of bone marrow tissue at page 22, lines 8-9.
W. Brill,
Particle Propulsion by Electric Discharge
(Tape of Speech at AAAS meeting on Plant Molecular Biology/Genetic Engineering for Agriculture (VI) (January 1989), discusses the transformation of nematodes to correct a missing body wall myosin gene by particle bombardment. The utility of transforming nematodes is, however, comparatively limited.
In view of the foregoing, an object of this invention is to provide new uses for the treatment of animals, particularly vertebrates, and their tissues and cells, by microprojectile bombardment.
A more particular object of this invention is to use microprojectile bombardment as a means for administering proteins or peptides to an animal subject.
SUMMARY OF THE INVENTION
A first aspect of the present invention is a method of transferring a gene to preselected vertebrate cells. The method comprises the steps of, first, providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the vertebrate cells and a heterologous gene positioned downstream of the regulatory sequence and under the transcriptional control thereof. The microprojectiles are then accelerated at the preselected cells, with the microprojectiles contacting the cells at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein ( as used herein, the plural form of terms such as “cell,” “microparticle,” and “polynucleic acid sequence” is intended to encompass the singular).
A second aspect of the present invention is a method of transferring a gene to preselected vertebrate tissue. The method comprises the steps of, first, providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the vertebrate tissue and a heterologous gene positioned downstream of the regulatory sequence and under the transcriptional control thereof. The microprojectiles are then accelerated at the preselected tissue, with the microprojectiles contacting the cells of the tissue at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein.
A third aspect of the present invention is a method of transferring a gene to a preselected tissue in situ in a vertebrate subject. The method comprises the steps of, first, providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the vertebrate tissue and a heterologous gene positioned downstream of the regulatory sequence and under the transcriptional control thereof. The microprojectiles are then accelerated at the animal subject, with the subject positioned so that the microprojectiles contact the preselected tissue, with the microprojectiles contacting the cells of the tissue at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein.
The data disclosed herein provide the first demonstration of particle-mediated transformation of (a) vertebrate cells, (b) vertebrate tissue, and (c) vertebrate tissue in situ of which these applicants are aware.
Also disclosed herein is a method of administering a protein or peptide to a vertebrate subject. This method is based in part on our finding that vertebrate tissue transformed by particle bombardment is surprisingly free of callus formation, inflammation, and other defensive responses. Thus, proteins and peptides released from the transformed cells (by virtue of their being transformed) can circulate throughout the animal subject in which the cells reside, and cells which circulate in the animal subject (e.g., lymphocytes) have access to the transformed cells. In this method, target vertebrate tissue (preferably dermis or hypodermis tissue) is selected and microprojectiles provided. The microprojectiles carry polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the selected tissue and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof. The gene codes for a protein or peptide. The microprojectiles are then accelerated at the selected target tissue, with the microprojectiles contacting the cells of the tissue at a speed sufficient to penetrate the tissue cells and deposit the polynucleic acid sequences therein to provide transformed tissue cells. The transformed tissue cells are then maintained in the animal subject, with the transformed tissue cells present in the subject in a number sufficient to produce a physiological response (e.g., an endocrine response, an immune response) to the protein or peptide coded for by the gene in the subject upon expression of the gene.
REFERENCES:
patent: 4798786 (1989-01-01), Tice et al.
patent: 4870009 (1989-09-01), Evans et al.
patent: 4944942 (1990-07-01), Brown et al.
patent: 4945050 (1990-07-01), Sanford et al.
patent: 5100792 (1992-03-01), Sandor
Johnston Stephen A.
McElligott Sandra G.
Sanford John C.
Williams R. Sanders
Crouch Deborah
Duke University
Jenkins & Wilson, P.A.
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