WO1993008292A1

WO1993008292A1 - Particle-mediated transformation of animal somatic cells

Info

Publication number: WO1993008292A1
Application number: PCT/US1992/008848
Authority: WO
Inventors: William F. Swain; Ning-Sun Yang; Dennis E. Mccabe; Brian F. Martinell; Liang Cheng
Original assignee: Agracetus, Inc.
Priority date: 1991-10-16
Filing date: 1992-10-15
Publication date: 1993-04-29
Also published as: CA2098599A1; JPH06503479A

Abstract

A method is disclosed for the convenient transformation of the somatic cells of animals to introduce therapeutic proteins into the animal. Somatic cell transformation is useful for medical and veterinary care of genetic diseases, and other therapeutic or animal improvement purposes. Copies of an exogenous genetic construction can be coated on carrier particle and accelerated into the interior of cells of the animal in situ. If the exogenous genetic construct includes a secretory signal peptide, it has been found that circulating levels of the protein can be achieved by simple transdermal insertion of the carrier particles

Description

PARTICLE-MEDIATED TRANSFORMATION

OF ANIMAL SOMATIC CELLS

Cross-Reference to Related Applications This application is a continuation-in-part of Serial No. 07/494,933 filed March 14, 1990, which was a continuation-in-part of Serial No. 07/371,869 filed June 26, 1989, now abandoned.

Field of the Invention The present invention relates to the technologies of genetic transformation in general and relates, in particular, to strategies for the genetic transformation of the non-germ line cells of whole animals to achieve circulating levels of therapeutic proteins.

Background of the Invention Techniques have been developed for the genetic engineering of animals by which exogenous or foreign DNA can be either inserted into the genomic DNA of animals or incorporated into the nucleus of cells as functional plasmid DNA. Typically in the prior art such genetic transformation of animals is performed by microinjection or by the use of retroviral based transformation vectors the effect of which is to genetically transform an animal cell in vitro or in vivo with foreign DNA. If the insertion is into an embryonic cell, the foreign DNA is incorporated into the genome of the animal embryo and then becomes incorporated into the genome of each of the daughter cells which arise from that embryo. Such genetic transformations insert the incorporated DNA into all of the cells of the resulting whole organism including the germ line or sex cells- of the organism. This insures that the genetic trait is passed to the progeny of the transformed animal in a normal Mendelian fashion.

There are occasions in which it would be desirable to transform animal cells in situ so that the animal can be imbued with the gene product of a genetic construction without affecting the genetic makeup of the germ line of the animal. In particular, for human applications the use of such somatic cell transformation avoids many of the ethical and philosophical problems which would arise from human intervention with the germ lines of human beings. The genetically engineered somatic cells offer the ability to make genetic corrections for inherited genetic disorders which consist of inactive or deleted enzymes or structural proteins that are necessary for normal biological functioning. It is also possible that such genetic transformations of somatic cells, and not germ line cells, may be desirable for certain therapeutic applications. For example, certain proteins offering therapeutic utility to patients must be currently injected into patients on a periodic strict time-line basis. However, the periodic injection of large quantities of proteins, even if done frequently, can result in an over supply of the protein shortly after an injection and a diminished supply shortly before the next injection resulting in potentially toxic shock following the injection and an insufficient supply for therapeutic efficacy just prior to the subsequent injection. An alternative strategy might be to engineer the gene for the desired protein into somatic cells of the animal or human so that the transformed cells would produce the therapeutic protein at a consistent level while they are live. By introducing the transforming gene into somatic cells which have a pre-defined and ascertainable life expectancy, such as skin cells for example, it is possible to create such an in vivo therapeutic production system which is time limited in the administration of the protein dosage to the animal or person being treated. In veterinary applications it may be desirable to introduce hormones or other growth factors or proteins for animal improvement, therapeutic, or disease inhibiting purposes into somatic cell portions of the animal which are not transient but which stay with the animal for its life expectancy.

While the vast majority of efforts directed at transformation of animal organisms or animal cells in culture have been directed toward the use of microinjection techniques or retroviral transformation vectors, the apparatus used for the transformation technique in accordance with the present invention is based on a quite different methodology of transforming the foreign DNA into the genome of the transformed somatic cells. There is one suggestion in the prior art of an apparatus containing some of the features which allow the apparatus of the present invention to be particularly adapted for its present use. As disclosed by Klein et al.. Nature, 327: 70-73 (1987), an instrument for the acceleration of very small particles of metal carrying DNA thereon has been demonstrated to be effective for the transformation of plant cells in culture. The transforming DNA is coated onto very small particles which are physically accelerated by actually being shot on a ballistic projectile into the tissues to be transformed. While this apparatus has been demonstrated to have utility in transforming plant cells in culture, it suffers from a deficiency in that the adjustability of the force of impact of its particles is lacking making it a difficult apparatus to use for transformation of organisms over a wide range of kinetic energies of insertion of the particles into the transformed tissue.

Summary of the Invention

The present invention is directed toward a method of transforming the somatic cells of animals in vivo in which the exogenous DNA construct including a sequence coding for the protein desired to be expressed in the somatic animal cells, .and linked to a promoter operative in animal cells, and including a signal peptide capable of causing secretion of the protein, is coated onto small microparticles being of sufficiently small size so as to be able to enter the cells of animals without disrupting their biological function, placing an animal at a target site, and then accelerating the particles at the target animal and into the cells of the target animal to thereby genetically transform a portion of the cells so treated so as to transform in vivo in the animal a number of cells to produce and secrete the protein coded by the exogenous gene. It is a further object of the present invention to provide animals which have been treated with foreign DNA so that their somatic cells contain therein both an expressing exogenous gene construct and a very small particle of metallic material which carried the gene construct into the animal cell.

It is yet another object of the present invention to provide a method of transforming somatic skin cells of animals so that proteins are produced in the animals for limited time periods before the skin cells are shed in a normal biological fashion.

Other objects, advantages, and features of the present invention will become apparent from the following specification when taken in conjunction with the accompanying drawings.

Brief Description of the Drawings

Fig. 1 is an exploded perspective view of apparatus used to perform the method of the present invention.

Fig. 2 is a top plan view of the discharge chamber of the apparatus of Fig. 1. Fig. 3 is a schematic illustration of a plasmid pWRG1601 used in one of the examples below. Description of the Preferred Embodiment The present invention is directed toward the transformation of the somatic cells of animals or human beings. By somatic cells as used herein it is meant to describe those cells of an animal or human being which when transformed do not change the genetic character or makeup of any of the germ or sex cells of the organism, so that when the animal or human reproduces through normal biological forms of reproduction, the introduced exogenous genetic material is not passed to the biological progeny of the organism. By genetically transforming somatic cells with a gene encoding a protein that includes a secretory signal peptide sequence, circulating levels of therapeutic proteins can be achieved for long periods of time.

The animal somatic cells transformed may be of any suitable tissue type in the target animal. Preferred target tissues include skin, muscle tissue and internal organ tissues, all of which may be transformed .in vivo. Somatic cells of tissues which are not normally exposed in the animal, i.e. internal organs, may be temporarily surgically exposed for the brief transformation procedure. Suitable target organs for somatic cell transformations also include the liver, spleen, pancreas, heart, kidney, brain, bone marrow, breast, sex organs, thyroid, organs of the gastro-intestinal tract and circulating cells such as leukocytes.

The invention is directed toward the introduction of exogenous, often chimeric, genetic constructions into animal somatic cells. Such exogenous genetic constructions consist of DNA from another organism, whether of the same or different species, which is introduced into the transformed organism through human manipulation, by the artificial introduction of genes into the cells of the transformed organism. The exogenous DNA construction would normally include a coding sequence for a transcription product or a protein of interest, together with flanking regulatory sequences effective to cause the expression of the protein or the transcription product coded for by the coding sequence in the transformed cells of an organism. Examples of flanking regulatory sequences are a promoter sequence sufficient to initiate transcription and a terminator sequence sufficient to terminate the gene product, coded for by the gene, whether by termination of transcription or translation. Suitable transcriptional enhancers or enhancers of translational efficiency can be included in the exogenous gene construct to further assist the efficiency of the overall transformation process and expression of the protein result in the transformed cells. Introns may also be included in the genetic construction to facilitate transcription and to provide for proper processing and transport of the transcribed RNA. Other gene products than proteins may also be expressed by the inserted genetic construction. For example, the inserted construction could express a negative RNA strand effective either to suppress the expression of a native gene or to inhibit a disease pathology. The inserted construction could itself be RNA, as an alterative to DNA, if only transient expression of the gene product was desired.

Another regulatory sequence of particular interest is a secretory signal peptide. A signal peptide sequence is a protein-coding DNA sequence located at the 5', or upstream, end of a protein-coding DNA sequence. The signal peptide itself is an amino terminal portion of the immature protein which directs sorting of the protein to various compartments in the cell, and directs co-translational and post-translational processing of the protein produced. This processing typically involves transport of the protein across cell membranes. A secretory signal peptide is a signal peptide which conditions secretion of the protein from the cell, through internal cell membrane compartments and ultimately into the extracellular fluid. Many secretory signal peptides effective in mammalian cells have been identified and the signal peptide can either be the one natively associated with the protein to be expressed or can be a heterologous signal peptide joined to a foreign protein. A suitable signal peptide from human growth hormone, HuGH, is set forth in SEQ ID No. : 1 below. There are two general types of secretory pathways, termed regulated secretion and constitutive secretion. In the former, the secretory proteins are stored in an intermediate vesicle called a secretory granule and are released by fusion of the secretory granule membrane and the plasma membrane in response to a secretogogue. In the constitutive pathway, secretory granules are not observed and although the secretory protein apparently transverses the various membranes to the outside of the cell, it is not clear how this happens. The secretory hormones insulin and growth hormone are normally secreted by the regulatory pathway when produced in their normal sites, islet cells and the anterior pituitary respectively. The regulatory pathway used is, however, generally dependent on cell type as much as the protein so it would be expected that human growth hormone secretion in skin cells would follow a constitutive pathway, since skin cells other than sebacious gland cells do not normally exhibit regulated secretion.

As used here, the term "transformation" is used to describe genetic transformation, or the process of insertion of foreign genes into living cells and the expression in the cells of proteins or other gene products encoded by the foreign genes. The term "transformation," as used here, is not intended to be used to describe the onset of malignant activity by a cell or cell line, which is also sometimes referred to as a "transformation. "

The present invention makes particular use of an apparatus for using an adjustable electric discharge to create a gaseous shock wave to physically accelerate DNA coated onto small particles into the genetic material of somatic animal cells. A suitable apparatus for use within the present invention is illustrated in Fig. 1. The apparatus consists of a spark discharge chamber 12 into which are inserted two electrodes 14 which are spaced apart by a distance of approximately 1 - 2 mm. The spark discharge chamber is a horizontally extended rectangle having two openings 16 and 18 out its upward end. One opening 18 is covered by an access plate 20. The other opening, located opposite from the electrodes 14 is intended to be covered by a carrier sheet 22. The electrodes 14 are connected to a suitable adjustable source of electric discharge voltage. Such a source of electric discharge voltage would preferably include suitable electric switching connected to a capacitor of the 1 to 2 micro farad size range, with the amount of the voltage of the charge introduced into the capacitor being adjustable, such as through the use of an autotransformer, through a range of, for example, 1 to 50,000 volts.

Suitable switching is provided so that the capacitor can be discharged through the electrodes 14 safely and conveniently by a user.

The carrier sheet 22 intended to be placed upon the opening 18 on the spark discharge chamber 12 is preferably a sheet of aluminized saran coated mylar although any other light, strong, durable sheet material may also be used. Above the opening in the discharge chamber, placed approximately 5 - 10 millimeters above it, is a retaining screen 24. Placed approximately 5 - 25 millimeters above the retaining screen is a target surface 26. In its use, the exogenous foreign gene construct intended to be transformed into the animal somatic cells is prepared by suitable DNA preparation techniques well known to one of ordinary skill in the art and it is coated onto small particles of a durable dense material such as gold, the particles typically being 1 to 3 microns in size. The carrier particles with the DNA coated thereon is then placed upon the carrier sheet 22 which is inserted on top of the spark discharge chamber 12. A target tissue, such as a live and anesthetized animal, is then placed adjacent to the target surface 26. Then a small droplet of water, approximately 2 - 4 microliters in size, is placed bridging between the ends of the electrodes 14. The access plate cover 20 is then placed over the top of the discharge chamber 12. At this point, the atmosphere between the carrier sheet 22 and the target is largely replaced with helium, by enclosing the apparatus and target and introducing helium in the enclosure in sufficient quantity to largely displace the atmospheric gases.

At this point the initiation of the spark discharge between the electrodes may be initiated by means of the use of the appropriate electronic switching. The force of the electric discharge bridges the spark discharge cap between the electrodes 14 instantly vaporizing the small droplet of water placed therebetween. The force of the vaporization of that water creates a gaseous shock wave within the spark discharge chamber 12 which radiates outward in all directions. The impact of the shock wave upon the carrier sheet 22 propels the carrier sheet 22 upwards with great velocity. The upwardly traveling carrier sheet 22 accelerates upward in direction until contacting the retaining screen 24. The presence of the helium provides less drag on the flight of the carrier sheet and on the carrier particles as well as less force for the shock wave to propagate to the target. At the retaining screen 24, the carrier sheet 22 is retained, and the DNA-coated particles previously applied thereto fly off of the carrier sheet and travel freely on toward the target surface. The particles therefor proceed into the target surface and enter the cells thereof. The momentum of the particles as they impact the surface of the target organism or tissue is adjustable based on the voltage of the initial electric discharge applied to the electrodes 14. Thus by variations in the amount of the electric energy discharged through the electrodes 14, the velocity by which the particles impact the target can be adjusted, and thus the depth of penetration of the particles into the tissue of a target, can be continuously adjusted over the range of adjustment of the electric discharge throughout the electrodes 14. The rates of application of DNA onto the carrier particles and of application of coated carrier particles onto the carrier sheet can also be adjusted to optimize performance of the device with different cell and tissue types.

The apparatus of Fig. 1 has been previously demonstrated to be useful for the transformation of differentiated or undifferentiated tissue in a variety of forms including cellular masses in culture and whole growing organisms. It has been found through the work discussed herein that the apparatus is equally suitable for the transformation of either animal cells in culture or for the transformation of cells of various animal somatic tissues. It is also possible to transform portions of whole animals in vivo by anesthetizing the animal, as appropriate for the species and type of animal, and then placing the anesthetized animal over a hole cut in a planar surface which will act as the target surface. The portion of the animal exposed through the hole in the target surface 26 will therefore be the treated target tissue transformed by the transformation process.

If the process is directed, as intended here, toward achieving therapeutically significant levels of circulating protein, the exogenous gene construction includes a protein coding sequence which includes, at its 5' end, a secretory signal peptide sequence. The copies of the gene construction can then be carried by the carrier particles into the tissues of the patient animal. The tissue can be a surface tissue or a internal tissue or organ temporarily exposed by surgery. Surprisingly, it has been found that significant levels of circulating protein in the bloodstream of a patient animal can be achieved by a particle acceleration treatment to the intact epidermis of the animal. Such treatment to the epidermis results in protein production and circulation for an extended, though perhaps not permanent, period of time. To achieve potentially permanent, or at least long term, expression of circulating protein, the skin tissue layer may be temporarily uncovered and the transformation blast may be applied to the underside skin layer, the dermis. To uncover the skin tissue layer, the skin is surgically separated from underlying muscle layers, a relatively simple procedure. This separation exposes the underside of the skin tissue layer, i.e. the dermis, which may then be treated by particle acceleration. Such treatment has been found to result in at least long term gene expression. Examples a) Vectors used

The first examples make use of a pair of chimeric expression vectors constructed so as to express in animals the enzyme chloramphenicol acetyltransferase (CAT) , which confers resistance to the antibiotic chloramphenicol. Both chimeric gene expression plasmids have been previously described and demonstrated to be effective in animal transfection studies. The plasmid pSV2cat was described by Gorman et al., Mol. Cell Biol. , 2:1044-1051 (1982) and the expression vector pRSVcat was described by Walker et al., Nature, 306:557-561 (1983). The plasmid pSV2cat is a chimeric cat gene construction including the Simian virus 40 (SV40) early promoter, the chloramphenicol acetyltransferase coding region from the plasmid pBR322-Tn9, the SV40 t-antigen intron, and the SV40 early polyadenylation region carried in the pBR322 vector. The plasmid does not contain a complete SV40 viral genome and is not infectious. The plasmid pRSVcat is also a pBR322 base plasmid that includes a chimeric Rous Sarcoma virus (RSV) long terminal repeat and promoter fragment, the cat coding region from Tn9, an intron from the mouse beta-globulin gene and the polyadenylation region from the SV40 early transcription unit. This plasmid also does not contain a complete viral genome and is not infectious. A related plasmid also used is designated pRSVNPTII and includes the Rouse Sarcoma Virus promoter, the coding region for the neomycin phosphotransferase-II gene, coding for resistance to the antibiotics kanamycin and G418, and a polyadenylation region from SV40. This plasmid as well does not contain a complete viral genome and is not infectious.

Another vector used in the examples described below is referred to as pWRG1601 and is illustrated in Fig. 3. The vector pWRG1601 includes a segment formed from pGEM3 (Promega) including oppositely oriented phage promoters, and, in an expression cassette, the cytomegalovirus immediate-early promoter (pCMVieP) followed by the transcribed and 3' flanking regions of the human growth hormone (HuGH) gene as set forth in Seldon, et al., Molec. Cell Biol.. 6:3173-3179 (1986); DeNoto, et al., Nucl. Acid Res.. 9:3719-3730 (1981); and Seeburg, DNA , 1:239-249 (1982). The HuGH protein coding sequence includes, at its 5'end, a sequence encoding a secretory signal peptide. A DNA sequence of 337 nucleotides is set forth as SEQ ID NO.: 1 below, which includes two exons which together code for a 26 amino acid signal peptide and also intron A of the HuGH gene. pWRG1602 was derived from pWRG1601 by deletion of the Hind III fragments that contain the EBV regions of the plasmid as follows. pWRG1601 was digested with restriction endonuclease Hind III and the ends of the fragments produced made blunt by treatment with Klenow DNA polymerase and all four deoxynucleotide triphosphates.

Synthetic Sal I oligonucleotide linkers were added to the ends of the molecules and these were subsequently digested with Sal I and the fragments circularized by ligation with T4 DNA ligase. The resulting plasmids were recovered by transformation into Escherichia coli and selection of transformants for resistance to ampicillin. The structure of pWRG1602 is shown schematically in Figure 4. pWRG1602 includes the CMV-HuGH gene and pGEM3 regions from pWRG1601, but deletes the EBV regions.

b) Mammalian Somatic Cells In Vivo

Mice were anesthetized with chloroform. On each mouse, an area of approximately 1 cπr on its side was shaved. The mouse was then placed on a petri dish having a window cut in it with the shaved patch over the window. DNA of pRSVcat was then coated onto 1-3 micron gold particles at a rate of 0.1 microgram of DNA per milligram of gold. The DNA was applied to the gold by precipitation with 25mM spermidine with 6% polyethylene glycol (m.w. 3,000) with the addition of CaCl2 to a final concentration of 0.6 M. The DNA coated gold beads were then rinsed in 100% ethanol and applied to the carrier sheet as an ethanolic suspension at a concentration of dried gold coated beads of 0.05 mg/cm² of the carrier sheet.

The petri dish with the mouse was placed over the apparatus of Figs. 1 and 2 as the target surface. Prior to the electric spark discharge, the area between the carrier sheet and the target was flushed with helium (4 liters/min) for 15 seconds to reduce atmospheric drag on the carrier sheet and any possible shock wave damage to the animal.

After the transformation event, the animals all appeared unharmed and they seemed to recover completely. No bruising or bleeding was observed in the test animals. After 24 hours the mice were sacrificed and the skin patch was removed and assayed for CAT activity. The assay was performed by testing for acetylation activity with a radio-labeled chloramphenicol. Radioactive decay of the acetylated product could then be used as a measure of transformed enzyme activity.

For the various electric discharge levels and controls used, the results are summarized in the following table. Conditions CPM per Total Protein Counts per

50 icroliter Microgram/μl 50 Microgram Protein

12 KV voltage 16,686 4.4 3792 & 1 micron

16 KV voltage 6,281 5.6 1121

& 1 micron

12 KV voltage 15,937 5.6 2854

& 1 micron 12 KV voltage 14,969 3.5 4276

& 1 micron

DNA + Kaolin 123 4.3 28

(DNA soak control)

DNA + DMSO 117 2.3 50 (DNA soak control)

No DNA (control) 119 5.6 21

These results indicate cat activity of at least 100 times background levels. Thus a foreign gene was delivered and expressed in somatic cells without evidence of harm or damage to the animal.

c) Amphibian Somatic Cells In Vivo

A (Xenopus) frog was anesthetized by chilling to 4° C. The chilled frog was also placed over a window cut in a petri dish lid and placed in the transformation apparatus of Figs. 1 and 2 in the same fashion as with the mice.

The conditions and procedure used for the mice were repeated for the frog except for the following. The DNA used was pSV2cat. The DNA coated gold beads were loaded onto the carrier sheet at a density of 0.1 mg/cm .

Again after the transformation process, the animal appeared entirely unharmed. Again no bruising or bleeding of the animal was detected. After 24 hours, the frog was sacrificed and the treated 1 car patch of skin was removed and assayed for CAT activity. The results are tabulated in the following table. Conditions CPM per Total Protein Counts per

50 microliter Microgram/μl 50 microgram

Protein

12 KV (belly) 13,149 2.1 6261 16 KV (back) 17,570 4.0 4392

Control (belly) 153 1.4 109

Control (back) 145 4.1 32

Thus, in this example levels of CAT activity were observed greater than 50 times background. Thus delivery and expression of a foreign gene was achieved in somatic cells without any identifiable damage or injury to the animal.

d) Amphibian Somatic Cells In Vivo - Systemic Product In a second experiment on Xenopus, one animal was treated under similar conditions, as above, but twice on the same frog (16 KV on its back, 12 KV on its belly). In this case only 0.05 mg/cm² instead of 0.1 mg/cm DNA coated beads were used. The frog was sacrificed after 20 hours, and the transformed skin patches sampled. In addition, a portion of non-transformed skin (shielded at the time of blasting) was sampled for CAT activity. The results are summarized in the following table.

Total activity in the transformed skin patches was reduced due to the lower bead loading rate, but the non-transformed skin sample clearly shows at least a 2 fold elevation above a non-transformed animal's skin, as in the previous experiment, thus showing a systemic accumulation of the enzyme produced in the transformed skin patches.

e) .In vivo transformation

Holtzman rats were anesthetized. The abdominal cavities of the anesthetized rats were then opened surgically to expose the liver of the animal. The attached livers were never removed, only exposed. The abdominal cavity of the rats was then sutured closed. The animals recovered from the surgery and the transformation procedure. No bleeding was observed from the animal's liver post-treatment. The living animals, with the liver thus exposed, were then subjected to a particle-mediated transformation procedure with the animal being placed on the apparatus of Figs 1 and 2, so that its exposed liver was at the target surface 26.

The DNA used in the rat liver transformation procedures was pRSVcat, coated at a rate of 1 microgram per milligram onto gold particles. This was done by combining 20 micrograms DNA, 100 microliter of buffer (150 mM NaCl, 10 mM Tris 8.0), 50 microliters of CaCl2 (2.5 M) and 20 milligrams of 1 micron gold powder. The mixture was then spun down, dried, and resuspended in ethanol prior to loading onto the carrier sheet. The loading rate on the carrier sheet was 0.05 milligrams of dried coated gold per square centimeter.

After the carrier sheet was in place, and the rat properly located with its organ exposed, the space of the particle travel was flooded with 2 liters per minute helium at atmospheric pressure. No vacuum containment was used. The rat livers were subjected to transformation events with spark discharge voltages of 10 or 14 kilovolts.

Two days later, the animals were sacrificed and the livers were excised. The excised liver tissues were analyzed for CAT activity. The gold particles were found to have penetrated up to 300 microns into the liver tissue. The following is a summary of the results of the procedure, with the level of CAT activity indicated by percentage of substrate catalyzed, and also indicated as a percentage of a defined standard unit of CAT activity.

Percent CAT Unit

Sample Conversion Activitv/mc Protein

Control (no 0.21% 0.09 transformation)

Other than liver, mouse abdominal muscle tissues were similarly treated for gene transfer as described above for liver, and the results are shown in the following.

These examples thus demonstrate that it is feasible to perform jLn vivo and .in situ transformations of somatic cells present as part of internal organs with this transformation technique. Transient activity of the transformant gene can be expected for at least one to four weeks and a lesser level of stable expression may be achieved for months.

f) Transfection of Skin With a Gene Encoding a Secreted Protein

This example was intended to demonstrate circulating levels of a potentially therapeutic protein in blood achieved by gene transfer to skin. BALB/c mice, 7 to 8 weeks old, (approx. 20 grams) were treated. The human growth hormone expression plasmid pWRGlδOl described above was used to express HuGH in the mice.

The mice were anesthetized using a Ketamime and Ro pun mixture (10 ml and 2ml, respectively) by intraperitoneal injection of .05 ml. The lower half of the animals were shaved. Nair hair remover was used to remove remaining hair in the treatment area.

The copies of DNA of plasmid pWRG1601 were loaded on amorphous gold (Engehard 1740) carrier particles at a rate of 0.5 micrograms per milligram. The DNA was precipitated on the carrier particles with calcium chloride and spermidine as described above. The coated carrier particles were then loaded onto carrier sheets at a rate of 0.5 mg/cm². The electric discharge apparatus of Figs. 1 and 2 was set for 23 kV discharge.

A target surface was formed by an inverted cup-shaped target support with a hole cut in its top to correspond to the target area, and which was adjusted to keep the target animals at a constant height above the retaining screen. The animal was placed on the target surface with the treatment area positioned over the hole. A vacuum (15 mm of mercury) was drawn on the inside of the cup-shaped support, then the particle bombardment was performed. After treatment, the animals were examined. Some redness and occasional fragments of mylar were observed, but the animals otherwise seemed healthy.

The animals were sacrificed at 2, 4, 5 and 7 hours after treatment. After sacrifice, the treated area of skin, was removed, weighed and placed in 1 volume (g/ml) of TES buffer and minced until fine. The solids were concentrated and frozen until assayed. Whole blood was collected from each animal by cardiac puncture resulting in 750 microliters of blood per mouse. The blood was placed at 4°C overnight to clot and spun down the next day. The serum was frozen for analysis.

The tissues were later analyzed using the Allegro (TM) Human growth hormone radioimmunoassay (RIA) system from Nichols Institute. To calibrate the assay, control samples spiked with known amounts of human growth hormone were prepared.

Control samples were prepared from non-transformed mouse skin which were treated in parallel to the experimental samples. The control samples averaged 1755.5 counts, an unusual background level. Controls of mouse serum samples averaged 219 counts.

Tissues and serum from seven treated mice were analyzed at various times after treatment. The results are given below in Table 2.

TABLE 2

Time Post Skin Blood Skin HGH Blood HGH Mouse Blast Counts Counts ng/ml ng/ml

2 7 hr 10,943 793.5 .4 .030 3 7 hr 2,097 25 .13 0 5 5 hr 700 0 .030 0

As can be seen from these results, the HGH protein is first expressed in situ at measurable levels approximately 5 hours post-blast, and continues to rise thereafter.

Blood levels of circulating protein are detectable 7 hours post-treatment.

g) Transfection of Skin - Multiple Blast

Mice were bombarded with single or multiple blasts of pWRG1602. The plasmid DNA was loaded onto carrier particles at 2u_g DNA/mg carrier particles. The coated carrier particles were layered on the carrier sheet at 0.5 mg/cm². The particle bombardment was conducted at 22 kV either once or four times on the same animal at separate sites. The skin and blood of the animals was tested 24 hours post blast. The values of HuGH given in Table 2 below are net after subtraction of background. TABLE 2

Number of blasts HuGH ng/ml Blood HuGH n in skin

1 0.074 1 0.10 1 0.115

1 0.13

Avera e of sin le blast 0.10 ± 0.02

5 .7 + .

h) Transfection of Skin - Time Study

These transformations were performed with pWRG160l loaded onto carrier particles at 2u_g DNA per mg carrier particles. The coated carrier particles were loaded onto carrier sheets at 0.4 mg/cm². Mice were bombarded through the skin at 22 kB, four blasts per animal.. The animals were sacrificed at the times indicated and the level of HuGH measured. The results are set forth in the following Table 3, again with the results being net above background. TABLE 3

i) Liver Transfection of Secretable Protein Gene

This experiment was also performed using pWRG1601. Plasmid DNA was loaded onto carrier particles at 2.5 u_g DNA/mg gold particles, and the coated particles were loaded onto the carrier sheet at a rate of 0.1 mg/cm .

Blasting was done at discharge voltages between 16 and 18 kV.

Rats were anesthetized and the abdominal cavity was surgically opened. The liver of the animals was treated with a particle acceleration apparatus aimed at the exposed liver. After treatment, the incisions were sutured, and the rats maintained under standard animal care procedures. One day later, the animals were sacrificed, and serum and liver samples were collected. In repeated experiments on over 20 rats, levels of circulating growth hormone were achieved over background levels. The results of three replicates are summarized in Table 5.

TABLE 5 Number of Animals

6 3

2 6 2

6

2

The best two animals had circulating levels of HGH of 0.11 and 0.12 ng/ml of serum. j) Transfection into Dermal Tissue

This example was also done with plasmid pWRG1601.

Rats were, anesthetized and abdominal skin hair removed by a hair clipper and Nair treatment. The abdomen was opened by scalpel incision and the tissues teased open to separate skin from muscle. The fascia lining was removed from the underneath side of the skin tissue. The particle acceleration procedure was performed on the underside of the skin layer (dermis) . After the transformation procedure phosphate buffered saline was added and the skin was sutured. Controls were bombarded with uncoated carrier particles. For each data point, 1 ml of fluid was collected from the subcutaneous tissue space, and 100 microliter of this sample was assayed for human growth hormone by RIA. The results of two replicates are set forth in Table 6.

TABLE 6

Replicate 1

Time after Blast Animal Body fluid HGH Level (Averaged

8 hrs -HGH (2 animals)

+HGH (7 animals) 8 ng/ml 24 hrs -HGH (2 animals)

+HGH (7 animals) 17 ng/ml

Re licate 2 one animal)

Body fluid HGH Level

0.3 ng/ml

0.6 ng/ml

0.4 ng/ml

0.37 ng/ml

0.3 ng/ml

These results indicate long-term expression and secretion of the inserted gene. Analogous in situ transformation experiments performed with a non-secreted gene (luciferase) indicated that an inserted gene transfected into dermal tissue exhibited persistent significant levels of expression (at least 10%, sometimes between 40% and 80%, of initial level) over 6 months. Thus secretory levels of therapeutic proteins can be sustained over this time period by dermal treatment.

The present invention is not to be limited to the particular embodiment or examples disclosed above, but embraces all such modified forms thereof as come within the scope of the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Swain, William F Yang, Ning-Sun McCabe, Dennis E Martinell, Brian F Cheng, Liang

(ii) TITLE OF INVENTION: Particle-Mediated Transformation of

Animal Somatic Cells

(iii) NUMBER OF SEQUENCES: 1

(iv CORRESPONDENCE ADDRESS:

A) ADDRESSEE: Quarles & Brady

B) STREET: P.O. Box 2113

C) CITY: Madison

D) STATE: Wisconsin

E) COUNTRY: USA

F) ZIP: 53701-2113

(v COMPUTER READABLE FORM:

A) MEDIUM TYPE: Floppy disk

B) COMPUTER: IBM PC compatible

C) OPERATING SYSTEM: PC-DOS/MS-DOS

D) SOFTWARE: Patentin Release #1.0, Version #1.25

(vi CURRENT APPLICATION DATA:

A) APPLICATION NUMBER: US

B) FILING DATE: 16-OCT-1991

C) CLASSIFICATION:

(viii ATTORNEY/AGENT INFORMATION:

A) NAME: Seay, Nicholas J.

B) REGISTRATION NUMBER: 27,386

C) REFERENCE/DOCKET NUMBER: 1122990695

(i TELECOMMUNICATION INFORMATION:

A) TELEPHONE: (608) 251-5000

B) TELEFAX: (608)251-9166

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 336 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO ( iv) ANTI -SENSE : NO

(v) FRAGMENT TYPE: N-terminal

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens

(ix) FEATURE: (A) NAME/KEY: intron

(B) LOCATION: 11..266

(ix) FEATURE:

(A) NAME/KEY: sig_peptide (B) LOCATION: 1..336

(x) PUBLICATION INFORMATION: (A) AUTHORS: Seldon,

(C) JOURNAL: Mol. Cell. Biol (E) ISSUE: 6

(F) PAGES: 3173-3179

(G) DATE: 1986

(K) RELEVANT RESIDUES IN SEQ ID NOilt FROM 1 TO 337

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGGCTACAG GTAAGCGCCC CTAAAATCCC TTTGGCACAA TGTGTCCTGA GGGGAGAGGC 60

AGCGACCTGT AGATGGGACG GGGGCACTAA CCCTCAGGGT TTGGGGTTCT GAATGTGAGT 120

ATCGCCATCT AAGCCCAGTA TTTGGCCAAT CTCAGAAAGC TCCTGGCTCC CTGGAGGATG 180

GAGAGAGAAA AACAAACAGC TCCTGGAGCA GGGAGAGTGT TGGCCTCTTG CTCTCCGGCT 240 CCCTCTGTTG CCCTCTGGTT TCTCCCAGGC TCCCGGACGT CCCTGCTCCT GGCTTTTGGC 300

CTGCTCTGCC TGCCCTGGCT TCAAGAGGGC AGTGCC 336

Claims

1. A method of achieving circulating levels of a protein in an animal comprising the steps of: coating copies of an exogenous genetic construction, the construction constructed so as to be able to express the protein in the cells of the animal, onto carrier particles of dense material of a size very small in relation to the size of the animal cells, the exogenous genetic construction including sequence coding for a secretory signal peptide; layering the coated carrier particles onto a planar carrier sheet; placing the animal cells in the direction of travel of the carrier sheet; and accelerating the carrier sheet toward the animal cells, the carrier sheet being restrained from hitting the animal cells but the carrier particles traveling into the animals cells so that the exogenous genetic construction is introduced into the animal cells with minimal damage to the cells.

2. The method of Claim 1 wherein the exogenous genetic construction includes a protein coding DNA sequence and effective flanking regulatory sequences effective to express the protein in the animal cells.

3. The method of Claim 1 wherein the animal cells are .in vivo in the living animal and wherein the entire live animal is placed in the direction of travel of the carrier sheet.

4. The method of Claim 3 wherein the animal cells that are transformed are in the skin of the animal.

5. The method of Claim 3 wherein the animal cells that are transformed are liver cells.

6. The method of Claim 1 wherein there is a retaining screen placed between the initial location of the carrier sheet and the animal cells to retain the carrier sheet after it is accelerated toward the animal cells.

7. The method of Claim 1 wherein the carrier particles are 1-3 micron gold particles.

8. The method of Claim 1 wherein the carrier sheet is accelerated by a gaseous shock wave.

9. The method of Claim 7 wherein the shock wave is generated by an electric voltage discharge.

10. A method of introducing a protein into the body fluids of an animal comprising the steps of preparing an exogenous genetic construction coding for expression of the protein in animal cells and including a sequence coding for a secretory signal peptide; coating copies of the exogenous genetic construction onto inert carrier particles; and accelerating the coated carrier particles into the skin of the animal in such a fashion that some cells of the animal express the exogenous genetic construction to cause secretion of the protein.

11. A method as claimed in Claim 10 further including surgically exposing the skin of the animal and accelerating the carrier particles into the underside of the exposed skin.

12. A method as claimed in Claim 10 wherein the signal peptide is from human growth hormone.