Particle-mediated transformation of animal tissue cells

Abstract

A method of transferring a gene to vertebrate cells is disclosed. The method comprises the steps of: (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the tissue cells and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and (b) accelerating the microprojectiles at the cells, with the microprojectiles contacting the cells at a speed sufficient to penetrate the cells and deposit the polynucleic acid sequences therein. Preferably, the target cells reside in situ in the animal subject when they are transformed. Preferred target cells are dermis or hypodermis cells, and preferred genes for insertion into the target cells are genes which code for proteins or peptides which produce a physiological response in the animal subject.

1 . A method of transferring a gene to preselected vertebrate cells, said method comprising the steps of:
 (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the cells and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and   (b) accelerating the microprojectiles at the preselected cells, with the microprojectiles contacting the cells at a speed sufficient to penetrate the vertebrate cells and deposit the polynucleic acid sequences therein.   
     
     
         2 . A method according to  claim 1 , wherein the preselected cells are skin cells. 
     
     
         3 . A method according to  claim 2 , wherein the skin cells are selected from the group consisting of basal cells, dermis cells, and hypodermis cells. 
     
     
         4 . A method according to  claim 1 , wherein the preselected cells are muscle cells. 
     
     
         5 . A method according to  claim 1 , wherein the cells are transformed in vitro. 
     
     
         6 . A method according to  claim 1 , wherein said microprojectiles have diameters of from about 1 micron to about 3 microns. 
     
     
         7 . A method of transferring a gene to a preselected vertebrate tissue, said method comprising the steps of:
 (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the tissue and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and   (b) accelerating the microprojectiles at the preselected 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.   
     
     
         8 . A method according to  claim 7 , wherein the preselected tissue is skin tissue. 
     
     
         9 . A method according to  claim 7 , wherein the skin tissue is selected from the group consisting of basal cell layer tissue, dermis tissue, and hypodermis tissue. 
     
     
         10 . A method according to  claim 7 , wherein the preselected tissue is muscle tissue. 
     
     
         11 . A method according to  claim 7 , wherein the tissue is transformed in vitro. 
     
     
         12 . A method according to  claim 7 , wherein said microprojectiles have diameters of from about 1 micron to about 3 microns. 
     
     
         13 . A method of transferring a gene to a preselected vertebrate tissue in situ in a vertebrate subject, said method comprising the steps of:
 (a) providing microprojectiles, the microprojectiles carrying polynucleic acid sequences, the sequences comprising, in the 5′ to 3′ direction, a regulatory sequence operable in the tissue and a gene positioned downstream of the regulatory sequence and under the transcriptional control thereof; and   (b) accelerating the microprojectiles at the vertebrate subject, with the subject positioned so that the microprojectiles contact the preselected tissue, and 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.   
     
     
         14 . A method according to  claim 13 , wherein the preselected tissue is skin tissue. 
     
     
         15 . A method according to  claim 13 , wherein the skin tissue is selected from the group consisting of basal cell layer tissue, dermis tissue, and hypodermis tissue. 
     
     
         16 . A method according to  claim 13 , wherein the preselected tissue is muscle tissue. 
     
     
         17 . A method according to  claim 13 , wherein said microprojectiles have diameters of from about 1 micron to about 3 microns. 
     
     
         18 . A method of administering a protein or peptide to a vertebrate subject, said method comprising the steps of:
 (a) selecting a target vertebrate tissue;   (b) providing microprojectiles, the microprojectiles carrying 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 coding for a protein or peptide;   (c) accelerating the microprojectiles 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; and   (d) maintaining the transformed tissue in the animal subject, with the transformed tissue cells present in the subject in a number sufficient to produce a physiological response to the protein or peptide coded for by said gene in the subject upon expression of said gene.   
     
     
         19 . A method according to  claim 18 , wherein the transformed tissue cells produce an immune response to the protein or peptide coded for by said gene in the animal subject upon expression of said gene therein. 
     
     
         20 . A method according to  claim 18 , wherein the transformed tissue cells increase the systemic concentration of the protein or peptide coded for by said gene in the animal subject upon expression of said gene therein. 
     
     
         21 . A method according to  claim 20 , wherein said gene codes for the production of growth hormone. 
     
     
         22 . A method according to  claim 18 , wherein the selected target tissue is selected from the group consisting of dermis tissue and hypodermis tissue. 
     
     
         23 . A method according to  claim 18 , wherein the tissue is transformed in vitro, and the transformed tissue cells are thereafter transferred to the animal subject. 
     
     
         24 . A method according to  claim 18 , wherein the tissue cells are transformed in situ in the animal subject. 
     
     
         25 . A method according to  claim 18 , wherein said microprojectiles have diameters of from about 1 micron to about 3 microns. 
     
     
         26 . A method of administering a protein or peptide to a vertebrate subject by in situ microprojectile bombardment, said method comprising the steps of:
 (a) selecting a target vertebrate tissue residing in the subject, the tissue selected from the group consisting of dermis tissue and hypodermis tissue;   (b) providing microprojectiles, the microprojectiles carrying 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 coding for a protein or peptide;   (c) accelerating the microprojectiles at the subject, with the subject positioned so that the microprojectiles contact the selected target tissue, and with the microprojectiles contacting the cells of the target tissue at a speed sufficient to penetrate the tissue cells and deposit the polynucleic acid sequences therein to provide transformed tissue cells; and   (d) maintaining the transformed tissue cells in the subject, with the transformed tissue cells present in the subject in a number sufficient to produce a physiological response to the protein or peptide coded for by said gene in the subject upon expression of said gene.   
     
     
         27 . A method according to  claim 26 , wherein the transformed tissue cells produce an immune response to the protein or peptide coded for by the gene in the animal subject upon expression of said gene therein. 
     
     
         28 . A method according to  claim 26 , wherein the transformed tissue cells increase the systemic concentration of the protein or peptide coded for by said gene in the animal subject upon expression of said gene therein. 
     
     
         29 . A method according to  claim 28 , wherein said gene codes for the production of growth hormone. 
     
     
         30 . A method according to  claim 26 , wherein the microparticles are propelled through the epidermis of the animal subject. 
     
     
         31 . A method according to  claim 26 , further comprising the step of surgically exposing the tissue cells, and wherein the microparticles are propelled into the tissue cells without passing through the epidermis. 
     
     
         32 . A method according to  claim 26 , wherein said microprojectiles have diameters of from about 1 micron to about 3 microns.