CA2098599A1 - Particle-mediated transformation of animal somatic cells - Google Patents

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

WO 93~082~ PC~ /~JS92/08848 PARTICLE-~EDIATED TRANSFORMATION
OF ANIMAL SO~ATIC CEI,LS

Cros~-Reference to Related A~lications This application i~ a continuation-in-par~ of Serial No. 07/494,933 filed March 14, 1990, which was a :::
continuation-in-part of Ssrial No. 07~3~1,86~ filed June 26, 1989, now abandoned.
Field of the Invention The pre~ent i~vention relates to the technologies of genetic transformation in g~neral and relat~s, in ~: .
particular, to qtrategies for the ge~etic transformation of the non-germ line cell~ of whole animal~ to achieve circulating levels of therapeuti.c protein~. ``
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ackcround of th.e Invention Techniqu~s hava been develop~d for the genetic `~::
engineering of animal~ by ~hich exogenous or foreign DNA ~ -can be ~ither inserted into the genomic DNA of animals or ::
incorporated into ~he nucleus of cells a~ functional plasmid DNA. Typically in the prior art such genetic tranQformation of animals is perform~d by microin~ection or b~ the use of retroviral based transformation vectors the efect of which i8 to genPtically transform an animal cell in vitro or in vi~o with foreign DN~. 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 ari~e from that embryo. Such genetic ;'~

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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 pa~sed to the progeny of the S transformed animal in a normal Mendelian fashion.
There are occasion~ in which it would be desirable to ~ransform animal cell~ in 3itu so ~hat 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 applica~ions the use of such somatic ~ell tran~formation avoids many of the ~ -e~hical and philo-qophical problems which would arise from human in~ervention with the germ lineq of human beingq.
The genetically engineersd somatic cells offer the ability to make genetic corrections for inherited genetic di~orders which con3ist of inactive ox deleted enzymes or ~ ~
structural proteins that ~re nece~Qary for normal ~ ~ -biological functioning. It i9 also pos3ible that such ~ -genetic tran~fo~mations of 30m8tic cells, and not germ -~
line cell~, may be desirable for cer~ain therapeutic ~ -applications. For example, certain proteins offering therapeutic utility to patients mu~t be currently in~ec~ed ~-into patientq on a periodic stri~t time-line ba is. -~
However, the period$c in~ection of large quantities of proteinY, even if done frequentl~y, can result in an over supply of t~e protein ~hortly after an in~ection and a diminished supply shortly before the next in~ection ;~
re ulting in potentially toxic shock following the in~ection and an insufficient 4upply for therapeutic efficacy ~ust prior to the ~ubsequent in~ection. An alternative ~trategy might be to engineer the gene for the de~ired protein into somatic cells of the animal or human so that the transformed cells would produce the therapeutic protein at a consistent le~el while they are 3~ live. By introducing the ~ransforming gene into somatic cells which have a pre-defined and a~certainable life expectancy, 3uch a~ skin cells for example, it is possible to create such an in vivo therapeutic production ~y~tem ' ' :: ., - :

w093/0~29~ 2 a ~ ~ r 1~ ~ PCT/US9~/Og~X

which is ~ime limited in the a~ninis~ration of the protein dosage to the anlmal 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 Rtay with the animal for its life expectancy.
While ~he vast ma~ority of efforts directed at tran~formation cf animal or~ani3ms or animal cells in culture have been directed toward the u~e of microin~ection techniq~le or retroviral ~xansformation vectors, th~ apparatus u~ed for the transformation :
technique in accordance wiSh the present invention is based on a quite different methodology of tr~nsforming the foreigr.l DNA into the genome of the transfoxmed somatic cells. There ii~ one ~uggestion in the prior art of an apparatu con~aining some of the features which allow the apparatus of the present invention to be particu~arly adapted for its present u~e. As disclosed by glein et al., Nature, 327: 70-73 tl987), im instrument for the ~'~
acceleration of very small particle~ of mstal ~arrying DNA
thereon has boen dQmon~trated to be effective for the transformation of plant cells in culture. The 2S tran~forming DNA ii~ coat~d onto very ~mall particles which are physically accel~rated by actually being shot on a ballistic pro~ectile into the ti'~ue~ to be transformed.
While thiq Apparatus ha~ been demoni~trated to ha~e u~ility :
i~ tr~nsforming plan~ cell~ in culture, it suffer~ from a :~ :
deiici~ncy in that the ad~u~tability of the force of ~ :
impact o~ it~ particle~ lacking making it a difficult apparatus to use for transfoxmation of organism3 over a wide range of kinetic energies of insertion of the particle~ into the transformed tissue.
Summarv of the Invention ~he present invention is directed toward a method of txansforming the somatic cells of animals in Yi~o in which ::: : ~

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i Wos3/082~2 2 ~ ~ 8 ~ ~ ~ Pcr/us92/08~8 the exogenous DNA construct including a ~equence coding for the pro~ein de~ired to be expressed in the somatic animal cells, and linkad to a promoter operative in animal cells, and including a ignal peptide capable of causing secretion of the protein, is coated onto small microparticles being of sufficiently small size so as to be able ~o enter the cell~ of animals without disrupting their biological function, placing an animal at a target site, and then accelarating the particl33 at the target animal an~ into the call~ of the target animal ~o thereby genetically tran~form a portion of the cells so treated so ;
a3 to transform in vivo in the animal a number of cell~ to produce and secrete the protein coded by the exogenous gene.
It i9 a further object of the present invention to provide animal3 which have been treated with foreign DNA `
so that their ~omatic cells contain therein both an expres3ing exogenou~ gene construct and a very small particle of metallic material which carried the gene construct in~o th~ ani~al cell.
It i~ yet another ob~ect of the pre ant invention to provide a method o~ tran~forming somatic ~kin cells of animals ~o that proteins are procluced in the animalR for limitsd tims periods before tho S~kin cells are ~hed in a -~
normal biological fashion.
Other ob~cts, advantages, ~md features of the prQsent invention will b~come ap~arent from the following ~pecification when taken in con~unction with the accompanying drawings.
Brief Description of the Drawinqs Fig. 1 is an exploded perspective view of apparatus used to perform the method of the present invention.
Fig. 2 is a top pl~n view of the discharge chamber of the apparatus of Fig. 1.
Fig. 3 i~ a schematic illustration of a plasmid pWRG1601 used in one of the examples below.

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DescriPtion of the Preferred Emhodiment The presen~ invention is directed toward the transformation of. the somatic cells of ar.imals or human being~. By ~omatic cellR as u~ed herein it is meant to describe ~hose cell~ of an animal or human being which when transformed do not change the genetic character or makeup of any of the germ or 9ex cells of the organism, RO
~hat when the animal or human reproduces through normal biological forms of reproduction, the introduced exogenous genetic material is not pacRed to ~he biological progeny of the organi3m. By genetically transforming somatic cells with a gene encoding a protein that include~ a -sacretory signal peptide sequence, circulating level~ of ~-therapeutic protein~ can be achieved for long periods of time.
The animal somatic cells transforme~ may be of any suitable ti~ue type in the target animal. Preferred target tis~ueR include ,skin, muscle ti ue and internal organ tissue3, all of which may be transformed in vivo.
Somatic cell~ of tis~ue~ which are not normally expo~ed in the animal, i.e. i~ternal organs, may be t~m,porarily surgi,-ally exposed for the brief transformation procedure.
Suitable target organs for 30mati.c cell transformations ~`
also include the liver, spleen, pancreas, heart, kidney, brain, bone marrow, breaYt, ~ex organs, thyroid, organs of the ga~tro-intestinal tract and ~:irculating cell-~ 3uch as leuko~ytes.
The invention iR directed tow~rd ~he introduction of exogenouR, of~en chimeric, genetic con~truction~ into animal somatic cell~. 5uch exogenous genetic construction~ eonsi~t of DNA from another organi~m, whether of the same or different species, which i~
introduced into the transformed organism throu~h human manipulation, by the artificial introduction of gene~ into the cells of the tran~formed organi3m. The exogenous DN~
construction would normally include a coding sequence for ~-a tran~cription product or a protein of interes , together with flanking regula~ory sequences effe,tive to cause the , , , .

W093/08292 2 ~ 9 ~ PCT/US92/0~8 expression of the protein or the transcription product coded for by the coding sequence in the transformed cells of an organism. Example~ of flanking regulatory sequences are a promoter sequence ~ufficient to initiate transcription and a terminator s2quence sufficien~ to terminate the gene product, coded for by the gene, whether by termination of tran~cription or translation. Suitable ' transcriptional enhancerR or enhancers of tranYlational efficiency can be included in the exogenou~ gene con~truct -~
to further a~ t the ef~iciency of the overall transformation proces~ and expression of the protein result in the transformed cells. Intron~ may also be included in the genetic con~truction to facilitate transcription and to provide for proper processing and transport of the tran~cribed R~A. Other gene products than proteins may also ~e expressed by the inser~ed genetic construction. For example, the inserted construction could expres~ a negativs R~A strand effec~ive either to 6uppra~s the expre~sion of a native gene or to inhibit a disea~e pathology. The inser~ed construction could itself be RNA, as an alterative to DN~, if only tranqient expression of the gene product was desired.
Another regulatory sequence of particular interest is a secretory signal peptide. A si.gnal peptide seque~ce is a protein-coding ~NA sequence loc:ated at the 5', or -~
up~tream, end of a protein-coding DNA ~equence. The signal peptide it~elf i8 an amino terminal portion of the imma~ure protein which directs sorting of th~ protein ~o variou~ compar~ment in the cell, and direct~
co-tran~lational and post-translational proces~in~ of the protein produced. Thi~ procensing typically in~olves transport of the protein acro~s cell membrane~. A
secretory signal peptide is a signal peptide which conditions secretion of the protein from the cell, through internal cell membrane compartmentq and ultimately into the extracellular fluid. Many ~ecretory ignal peptides effective in mammalian cells have been identified and the signal peptide can eithar be the one natively associated W0~3/~8292 2 0 9 ~ a r,l ,) PCT/U5~2/~8~8 with the proteLn to be expressed or can be a heterologous signal peptide joined to a foreign protein. A ~uitable signal peptide from human growth hormone, HuGH, i~ set forth in SEQ ID No.: 1 below.
There are two general types of secretory pathways, termed regulated ~ecretion and constitutive secretion. In the former, the secretory proteins are stored in an intermediate vesicle called a secretory granule and are -relea~ed by fu~ion of the ~ecre~ory granule membrane and the pla~ma membran~ in respon~e to a secretogogue. In the constitutive pathway, ~e~retory gsanules are not observed and although the Yecretory protein apparently tran~verses the variou~ membranes to the outside of the cell, it is not clear how thi~ happens. The secretory hormones insulin and growth hormone are normally secreted by the regulatory pathway when produced in their normal sites, islet cell~ and the anterior pituitary rs~pectively. The regulatory pathway u~ed is, howaver, generally dependent on cell typo a~ much as the protein so it would be expected that human gxowth hormone secretion in skin cells would follow a con~titutive pathway, since skin cells other than ~ebacious gland cell~ do not normally exhibit regulated s~cretion.
As used here, the term ~transformationl' is u~ed to describe genetic transformation, or the process of insertion of foreign genes into li~ing cells and the expre~sion in the cells of proteins or other gene produc~s encod~d by the foreign genes. The term "transform2tion,~
a~ u~ed here, is not intended to be u~ed to deYcribe the ~
onset of ~alignant activity by a call or cell line, which ~-is also somatimes referred to as a 'transforma~ion.' The present i~vention makes particular use of an;~
apparatus for using an ad~ustable electric discharge to create a ~aseou~ shock wave to physically accelerate DNA
coated onto small particles into the genetic material of somatic animal cell~. A uitable apparatus for use within ` i the pre~ent invention is illus~rated in Fig. 1. The apparatua con~ists of a spark discharge chamber 12 into ..'~
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w~93/ox292 2~9~3~ PCT/~S92/08~8 which are inserced two elect_ode~s 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 i9 covere~ by an access plate 20. The other opening, located oppo~ite from the electrodes 14 is intended to be covered by a carrier sheet 22. The electrodes 14 are connected to a suitable ad~ustable source of electric discharge voltage. Such a source of electric di3charge voltage would preferably include suitable electric ~witching 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, l to 50,000 volt~. .
Suitable ~witching is provided so that the capacitor can be discharged through the electrodeR 14 safely and conveniently by a us~r. ~:~
The carrier sh~et 22 intended to be placed npon the opening 18 on the ~p2rk di~charge cham~er 12 is preferably a sheet of aluminized saran coated mylar although any ~ ;
other light, strong, durable sheet material may al~o be usedO Above ths opening in the d.ischarge chamber, placed approximately 5 - 10 millimeters labove lt, i~ a retaining screen 24. Placsd approximately 5 - 25 millimeters above the retaining zcreen is a target surface 26. In its u~e, the exogenou3 foraign gene construct intended to ba ~:
transformed into ~he animal ~omatic cells i~ prepared by ~uit~bl~ DNA preparation techniqse~ well known to one of ordinary 3kill in the art and it i~ coated onto small :.
particle3 of a duxable den~e ma~erial such a~ gold, the : :
particles typically being 1 to 3 miGron~ in ~lize. The carrier particle~ with the DNA coated thereon i9 then placed upon the carrier sheet 22 which is in~erted on top of the ~p~rk di~charge chamber 12. A target ti~ue, such as a live and ane~thetized animal, i~ then placed adjacent to the target ~urface 26. Then a ~mall droplet o~ water, approximately 2 - ~ microliters in ~ize, i~ placed .

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W093/0829' 2 ~ 9 ~ PCT/US~2/08 bridging between the ends of the electrodes 14. The acces~ plate cover 20 is then placed over the top of the discharge chamber 12. At this point, the atmosphere between the car~ier sheet 22 and the target is largely S 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 di~charge ~etween the alectrodes may be initiated by meanY of the use of the appropria~e electronic switching The force of the electric discharge bridges the ~p~rk discharge cap between the ele~trodes 14 instantly ~aporizing the small droplet of water plac~d therebetween. The force of the vaporization of that water creates a g~seou~ shock wave within the spark di~charge chamber 12 which radiate-~outward in all directions. The impact of the ~hock wave upon the carrie~ sheet 22 propels the carrier sheet 22 ~-upwards with greAt velocity. The upwardly ~raveling carrier sheet 22 accelerates upward i~ direction until contacting the retaining 3creen 24. The presence of the helium provides less drag on the flight of the carrier sheet and on the carrier particl~as as well as less force for the shoc~ wave to propagate to the target. At the retaining scr~en 24, the carrier ~heet 22 i~ retained, and th2 DN~-coated particles previously applied thereto fly o~f o~ the carrier sheet and tra~el freely on toward the target sur~ace. Ths particle~ therefor procoed into the ~arge~ surface and ~nter the cells thereof. The momentum o~ the particle~ as they impact the surface of the target organism or ti~sue i8 ad~ustable based on the voltage of the initial electric discharge applied to the electrodes 14. Thus by variation~ in the amount of the electric energy diRcharged through the electrodes 14, the velocity by which the particles impact the target can be adjusted, and thu~ the depth of penetration of the particles into the tissue of a target, can be continuously ad~u~ted over the range of ad~ustment of the electric discharge .. .. . . . . .

, . ,. , : ~ ,.. , .: , w093/0~2~2 ~ PCT/US92/0~8 throughout the electrodes 14. The rates o application of DNA onto the carrier particles and of application of coated carrier particl~s on~o the carrier Rhee~ can also be adjustad to optimiz~ performance of the deYice with different cell and tissue type~.
The apparatus of Fig. 1 has been previously demons~rated to be useful for the transformation of differentiated or undifferentiated tissue in a variety of form~ including cellular masse~ in culture and whole growing organisms. It has been found through the work discu4sed herein that the apparatu~ i~ equally ~uitable for the tran~formation of either animal cell~ in culture or for the transformation of cellR of various animal somatic ti~sues. I~ i8 also po~sible to ~ran~form portion~ o~ whole animals in vivo by ~ne~thetizing the animal, as appropriate for the species and type of animal, and then placing the anesthetized ~nimal over a hole cut in a planar surface which will act as the target surfaceO
The portion of the animal expo~ed through the hole in the target 3urface 26 will therefore be the tre~ed target tissue transformed by the transfoxmation process.
If the prooe~s i~ directed, as intended here, toward achieving tharapeutically significant level-~ of circulating protein, the e~ogenou~3 gene cons~ruction includes a protein coding sequence which include~, at its 5' end, a ~ecretory ~ignal peptid~s sequ3nce. The copie of the gene construction can ~hen be carried by the carrier pa~ticles into the ti~sueR o~ the patient animal.
The tissue can be a surface ti~ue or a internal ti3sue or organ temporarily expo~ed by surgery. Surprisingly, it haq baen found that ~i~nificant levels of circulating protein in the bloodstream of a patient anim~l can be achieved by a par~icle acceleration treatment to the intact epidermi~ of the animal. Such treatment to the epidermi3 rssults in protein production and circulation for an extended, though perhaps not pe~m nent, period of time. To achieve potentially permanent, or at least long term, expression of circulating protein, the skin tissue .. ..
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w093~0829' ~ 9 8 ~ ~ ~ PCT/US92/OX~B

layer may be temporarily unco~er.ed and the transformation bla~t may be applied to the underside skLn layer, the dermi~. To uncover the skin tissue layer, the skin is surgically separated from underlying mu~cle layers, a relatively simple procedure. This separation expoRes 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 xesult in at least long term gene expre~sion.
Exam~les a) Vec~ors used The first examples make use of a pair of chimeric expression vector~ con3tructad so as to expres~ in animals the enzym2 chloramphenicol aeetyltran~fera~e (CA~), which confers r~si~tan~e to the antibiotic chlor3mphenicol.
Bo~h chimeric gene expres~ion pla~mids have been previou~ly described and d~monstrated to be effective in animal tran~fection ~tudi~s. The plasmid pSV2cat was described by Gorman et al., MoI. Cell Biol., 2:1044-1051 (1932) and the expression vector pRSVcat was described by Walker et al., Nature, 306:5~7-561 (1~3). The plasmid pSV2cat is a chimeric cat gene construction including the Simian viru~ 40 ~SV40) early promoter, the chloramphenicol ace~yltransferase coding region from the plasmid pBR322-Tn9, the SY40 t-antigen intron, and the SV40 early polyadenylation region carried in the pBR322 vector. The ~;
plasmid doe~ not contain a complete SV40 ~iral genome and i~ not i~fectiou8. The plasmid pR5Vcat ic also a pBR32~ ` ~
ba~e plasmid that include6 a chimeric ~ous Sarcoma virus `
(RSV) long t~rmlnal repeat and promoter fragment, the rat coding region from Tn9, an intron from the mouse beta-~lobulin gRne and the polyadenylation region from the SY40 early transcription unit. Thi~ plasmid also does not contain a complote viral ~eno~e and i not infectious. A
related plasmld also u3ed i8 de~ignated pRSVNPTII and includes the Rouse Sarcoma Virus promoter, the coding region for tne neo~ycin phosphotran~ferase-II gene, coding for resistance to the antibiotics kanamycin and G418, and : .. - : . , , ~ . : .- ... . ...
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a polyadenylation region from SV40. This plasmid as well does not contain a complete viral genome and i5 not infectious.
Another vector u~ed in the examples described below is referred to as pWRG1601 and is illustrated i~ Fig. 3.
The vector pWRG1601 include~ a segment formed from pGEM3 (Promega) including oppoRitely oriented phage promoters, and, in ~n expres~ion cassette, the cytomegalovirus immediate-~arly promoter (pCMVieP) followed by the tran~cribed and 3~ flanking regions of the human growth hormone (HuGH) gPno aB 8at forth in Saldon, et al., Mo~lec.
Cell ~iol., 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 3equence includes, at its 5'end, a 3equence en~oding a ~ecretory 3ignal peptide. A
DNA ~equence of 337 nucleotide~ is ~et forth as SEQ ID
NO.: 1 below, which includes ~wo exon~ which together code for a 26 amino acid ~ignal peptide and al~o intron A of the HuGH gene.
pWRG1602 waq derived from pNRG1601 by deletion of the Hind III fra~ment~ that contain the EBV regions of the plasmid a~ follows. pWRG1601 was digected with re~triction endonuclea~e Hind III and the endq of the fragments produc~d made blunt by treatment wi~h ~lenow DNA
polymeras~ and all four deoxynucleotide triphosphates.
Synthetic Sal I oligonucleotide linkers were added to the endY of the moleculas and these were ~ubsequently digested with Sal I and the ra~ments circularized by ligation with T4 DNA ligase. The resulti~g plasmids were recovered by transformatlon into E~cherichia coli and ~election of transformant~ ~or xesistance to ampicillin. The structure of pWRG1602 i~ shown cchematically in Figure 4. pWRG1602 includ~ the CMV-HuGH gene and pGEN3 regions from pWRGl601 r but delete3 the EBV regions.
b) Nammalian Somatic Cell~ In Vi~o Nice were anesthetized with chloroform. On each mouqe, an arQa of approximately 1 cm2 on its side was ,, , . , . . :

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W093/08292 2 ~ 8 ~ ~ ~ PCT/US92/08~8 shaved. The mour~e was then placed on a petri di~h having a window cut in it with the shaved patch over the window.
DNA of pRSVcat waR 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.
3,00Q) with the addition of CaC12 to a final concentration of 0.6 N. The DNA coated gold beads were then rinsed in lOU~ ethanol and applied to the carrier Rheet a8 an ethanolic su3pension at a concen~ration of dried gold coated be~ds of 0.05 mg/cm2 of the carrier ~heet.
The petri dish with the mou e wa~ placed over the apparatus of Figs. 1 and 2 as the target surface. Prior to the electxic 3park di~charge, the area between the carrier sheet and the target wa~ flushed with helium (4 liter3/min) for 15 seconds to reduce atmospheric drag on the carrier sheet and any poQsible shoGk wave damage to the animal.
After the tran~formation event, the animal~ all appeared unharmed and they seemed to recover completely.
No bruising or bleeding wa~ observed in the test animals.
After 24 hour3 the mice were sacrificed and the ~kin patch was removed and assayed for CAT ac:tivity. The assay was performed by te3ting for acetylation activity with a radio-labeled chloramphenicol. Radioactive decay of the acetylated product could then b~ used as a mea~ure of transformed enzyme activity.
For the various electric discharqe lev21s and controls used, the result3 are ~ummarized in the following table.

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Conditions CPM p~r Total Protein Co~mt~ pe~
50 microliter Mic~ogram/~l 50 Microgram Protein .
12 KV voltage16,686 4.4 3792 ~ 1 micron 16 KV voltage6,281 5.6 1121 & 1 micron 12 RV voltage15,937 5.6 2854 & 1 micron 12 KV voltaye14,969 3.5 4276 & 1 micron :
DNA + Kaolin 123 4.3 28 (DNA soak control) -DNA I DMSO 117 2.3 50 (DNA soak con~-rol) No DNA (control) 119 5.6 21 Theqe rssults indicata cat activity of at least 100 times background levels. ~hus a foreign gene was delivered and expre~ed in ~oma~ic cells without evidsnce of harm or damage to the animal. : :
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c) Amphibian Somatic Cell~ In ViYo A (X~nopu~) fro~ was anesthetized by chilling to 4 C. The chilled frog wa~ al80 placed over a window cut in a pe~ri di~h lid and pla~ed in the transformation apparatus of Figs. 1 and 2 in thl~ 3ame fashion as with the -~
mice.
The conditions and procedu~e used ~or the mice were repeated for the frog exc~pt for the following. The DNA
used was pSV2cat. The DNA coated gold beads were loaded onto the carrier sheet at a den~ity of 0.1 mg/cm2. :~
Again after the trsn formation proc~s, the animal appeared entirely unharmed. Again no bruising or bleeding of the animal wa3 detected. After 24 hours, the frog was-sacrificed and the treated 1 cm2 patch o skin was removed and a~sayed for CAT activity. The results are tabulated ~.
in the following table.

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W~93/0829' 2 ~ ~ 8 ~ 9 ~ PC~/US92/0~8 Conditions CPM per Total Protein Counts per 50 microliter Microgram/~l 50 microgram Protein 12 KV (belly)13,149 2.16261 16 KV (bac~)17,570 4.04392 Control (belly) 153 1.4 109 Control (bac~) 145 4.1 32 Thus, in this example levelq of CAT activity were observed great.er than 50 timeq background. Thus delivery and expres~ion of a foreign gene was achieved in somatic cells without any iden~ifiable damage or injury to the animal.
d ) Amphibian Somatic Cell3 In Vivo - 5yBtemic Product : `:
In a second experiment on Xenopu~, one animal was ~::
treated under similar condltionq, aR above, but twice on the same frog (16 ~V on itY back, 12 RV on its belly). In this ea~e only 0.05 mg~cm2 instead of 0.1 mgJcm2 DNA ;~
coated bead~ were used. The fro~ waC sacrificed after 20 `~`
hour , and the transformed ~kin patcheq ~mpled. In addition, a portion of non-transf.ormed skin (shielded at the time of bla~ing) was ~ampledl for CAT activity. The resul~s are summarized in the ~ol.lowing table.
Total Protein Count~ Per Re~ults~PM/SOul us~l 50 uq Prot~in 12 ~V (belly)2,085 7.5 278 16 XV (back)9,343 8.61,Oa6 Untreated skin1,301 5.1 255 ~rom el~ewh~re on the same frog Total acti~ity in the transfonmed ~kin patches was reduced due to ths lower bead loading rate, but the non-transformed ski~ sample clearly shows at least a 2 fold elevation above a non-transformed animal's skin, a~ l in the previou experiment, thus showing a 3ys~emic : `' .. . . . .

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Wo93~082s2 2 ~ 9 ~ P~T/US92/08~8 accumulation of the enzyme produced in the tran~formed skin patches.
e) _ vivo transformation Holtzman rat~ were anesthetized. The abdominal cavities of the anesthetized rat3 were ~hen opened surgically to expose the liver of the animal. The attached liver~ were never removed, only exposed. The abdominal cavity of the rats was then sutured closed. The ~-animals recovered from tha sur~ery and the transformation procedur~. No bleeding was observed from the animal's liver post-treatmen~. The living animals, with the liver thus exposed, were then subjected to a particle-mediated transformation procedure with the animal being placed on the apparatu~ of Figs 1 and 2, 80 that it~ exposed liver .
was at the target Rurface 26.
The DNA u~ed in the rat liver transformation procedure3 was pRSVcat, coated at a rate of 1 microgram per milligram onto gold particle~. Thiq wa~ done by combining 20 mi~rogram~ DNA, 100 microliter of buffer (1~0 mM NaCl, 10 m~ Tri~ 8.0), 50 microliter~ of CaC12 (2.5 ~) and 20 milligram~ o~ 1 mi~ron gold powder. The mixture wa~ then spun down, dried, and r~suspended in ethanol prior to loading onto the carrier sheet. The loading rate on the carrier ~heet wa~ 0.05 milligrams of dried coated gold per sguare centimeter. ~ -After the carrier Rheet wa3 in place, and the rat properly loca~sd with its organ exposed, the space of the particle travel wa~ flooded wi~h 2 liters per minu~e helium at atmospheric prss~ure. No vacuum containment was used. The rat livers were sub~ected to transformation events with spark discharge voltages of 10 or 14 ~ -kilovslts.
Two day~ latert the animal~ were ~acrificed and the ~;
livers were exciced. The excised liver ~issues were .
analyzed for CAT activity. The gold particle~ were found to have penetr~ted up to 300 microns into the liver ti~sue.

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. . : .
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W093/OB292 2 0 9 ~3 9 ~ PCT/US92/08~g The following i~ 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 ,Conversi,on ActivityLma Protein -Control (no 0.21% 0.09 transformation) Liver at lOKV 2.6% 1.38 Liver at 14~V 2.2~ 1.06 1 Unit CAT 37.6% 1 unit ~dafined) -,~:
Other than liver, mou~e abdominal muscle tissues were similarly treated for gene transfer as de cribed above for ' ,~
liver, and the reYult.~ are shown in the following.
Percent Sample Conver~ion CAT unit/m~ Protein Control (non-blasted ~u~cle)0.03~ 0.014 .-'' Muscle-l (15~V) 1.5% 0.7 Muscle-2 (15XV) O.94~ 0.34 1 Unit 33~ 1 unit (defined) These examples thus demonstrate t:hat it is feasible to ~,~
per~orm in vi~o and in situ transformations of 30matic cells preY~nt a~ part of internal organ~ with thi~
tran~formation technique. Transient ~ctivity of the transformant gene can be expectsd for at least one to four ,':
week~ and a lesser level of stable expre~sion may be achieved for months. ", f) Transfection o~ Skin With a Gene Encoding a Secreted Protein Thi~ example w. 3 intended to demon3trate circulating levels of a po~sntially therapeutic protein in blood achieved by gene transfer to ~kin. BALB/c mice, 7 to 8 ,~, wv93/o82s2 PCT/US92/~8~8 2098~9~

w~eks old, (approx. 20 grams) were treated. The human growth hormone expresqion plasmicl pWRGlÇ01 described above was used to express HuGH in the mice.
The mice were anesthetized using a Ketamime and S Rompun 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 microgram~ per milligram. The DNA wa3 precipitated on the carrier particleY with calcium chloxide and spermidine a~ described above. The coated carrier particle~ were then loaded onto oarrier sheets at a rate of 0.5 mg/cm2. The electric di~charge apparatus of Figs.
1 and 2 wa~ set for 23 kV d$scharge.
A taxget surface wa~ formed by an invarted cup~shaped targe~ support with a hole cu~ in it~ top to correspond to the t~rget area, and which was adlusted to keep the target animal~ at a con~tant height abov~ the retaining screen.
The animal wa~ placed on the target ~urface with the treatment area position2d over the hole. A vacuum (15 mm of mercury) wa~ drawn on the inside of the cup-shaped ~ ;
support, th~n tha particle bombart~ment was performed.
After txea~ment, the animals were ex~mined. Some redns38 and occ~cional fragments of mylar were observed, b~t the ani~als otherwise ~eemed healthy.
Th~ anim~l~ were sa~rificed a~ 2, 4, 5 and 7 hour~
aftQr tr~a~mentO After ~acr~fice, the treated area of Rkin wa~ remo~ed, weighed and placed in 1 ~olume (g/ml) of TES buffer and minced until fine. The solids were concantrated and frozen until as~ayed. Whole blood was collected from each animal by cardiac puncture re3ulting -in 750 microlitsrs of blood psr mouse. The blood was pl~ced at 4C overnight to clot and spun down the ~ext day. The serum was frozen for analy is.
The tissue~ were l~ter analyzed using the Allegro (T~) ~uman growth hormone radioimmunoa ~ay (RIA) system :: ` :: : ` : `

wVs3/0829~ 9~ PCT/US92/08~8 --19 ~, from Nichols Ins~itute. To calibrate the assay, control samples spiked with known amounts of human growth hormone were prepared.
Control sample~ were prepared from non-transformed mouse skin which were treated in parallel to the experimental samples. The control samples averaged 1755.5 co~nts, an un~sual background level. Control~ of mouse serum samples averaged 219 counts.
T18-RUeg and serum from ~e~en ~reated mice were analyzed at variou~ time3 after treatment. The resul~s are given below in Table 2.

Time Po~t Skin Blood Skin HGX Blood HGH
Mouse Bla~t Count~ Counts n~/ml naL~l 2 '7 hr 10,943 793.5 .4 .030 3 7 hr 2,097 25 .13 0 5 5 hr 700 0 .030 A can be ~een from ~heRe re~ult3, the HGH pro~ein i5 ~:
first expre~sed in ~itu at measurable levels approximately 5 hour~ post-blast, and continue~ to rise thereafter.
Blood level~ of circulating protein are detectable 7 hours po~t-treatment. , g) Transfection of Skin - Multiple Bla~t Mice were bom~rded with single or multipla bla3ts of pWRG1602. Tha pla~mid DMA wa3 loaded onto carrier particle at 2~g DNA~g carrier particle~. The coated carrier particl~R were layered on the ~arrier ~heet at 0.5 mg/c~2. ~ho particle bo~bardment was conducted at 22 kV ;~
either once or four time on the 3ame animal at separate sites. The skin and blood of the animals was te~ted 24 hours post blast. The value3 of HuGH given in Table 2 below are net after sub~rac~ion of background.

- . : : , -:

W093/0~9~ PCT/US92/08~
2~

TABLE ~
N ~ er of blasts HuGH nq/ml ~lood HuGH na in skin 5 1 0.074 57.5 1 0.10 26.3 1 0.115 46.3 1 0.13 55.0 Average of single blast 0.10 + 0.02 46.3 + 14 4 .43 58.8 4 .45 62.5 4 .43 55.0 4 .40 ~2.5 Average of four bla~ts 0.43 ~ 0.02 59.7 + 3.6 15 h) Tran-Rfection of Skin - ~ime Study The~e tr~n~ ormations were performed with pWRG1601 loaded on~o carrier p~rticleR at 2ug DNA per mg carrier particle30 The coated carriar particles were loaded on~o carrier ~heets at 0.4 mg/cm2. ~ice were bombarded through 20 the skin at 22 k~, four blast~ per animal. The animal~
were sacrificed at the time~ indicated and the level of HuGH measured. The results are ~et forth in the following Table 3, again with the result~ being net above :
background.
T~BLE 3 Time of SacrificeHuGH in Skin HuÇH in Blood (ng) (ng/ml) lOh 7.25 0.115 lOh 5-75 0-03 lOh 2.5 0.07 lOh 8.75 0.135 lOh ~verage 6.1 + 2.7 0.1 + 0.03 .1 day 90 0 . 33 1 day 65 0 . 25 1 day 35 0 . 22 da~ 6 2 . 5 0 . 3 2 1day average 63.1 + 22.5 0.28 + 0.05 40 ~ 2 day3 52 count~/min 40 2 days 48 above background 2 days 36 " - ~
2 days average48.5 + 9. 3 : . .
3 day~ 10.8 counts/min 30%
3 days 4.4 above background 3 day3 20 . 0 3 day avera~e12 . 7 + 6 ~ 7 . .
': . .: ' . . , ' .

W093/08292 2 ~ 9 8 ~ ~ ~ PCT/~S92/08~8 i) Liver TransfeCtion of Secretable Protein Gen~
This experiment was also performed u~ing pW~G1601.
Plasmid DNA was loaded onto carrier particles at 2.5 ug DNA/mg gold particles, and the coated particles were loaded onto the carrier sheet at a rate of 0.1 mglcm2.
~lasting was done at discharge ~oltage~ be~ween 16 and 18 kV.
Ra~ w~re ane3thetized and the abdominal cavity was ~;
~urgically open~d. The liver of the anLmals was treated with a particl~ acc~leration apparatus aimed at ~h~
expo~ed liver. After tr~atme~t, the inci~ions were sutured, and the rats maintained under standard animal care pro~edures. One day later, tha anLmals were sacrificed, and ~erum and liver samples were ~ollected.
In repeated expariments on over 20 rats, lavel3 of circulating growth hormone were achieved over baGkground levels. The reRult~ of three replicat~R are ~ummarized in ; ;
T2ble 5. - ;

Blood Count~ in RIA Num~er of Animal~
(16 hours) Group 1 HGH 349 + 76 6 "
no HGH 200 + 29 3 Group 2 ~G~ 394 + 29 2 HGH I lux 318 + 57 6 no HGH 220 ~ 23 2 Group 3 no HGH 242 ~ 21 2 ;
The best two animals had circulating le~els of HGH of ;~
O.11 and 0.12 ng/~l of serum.

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.. ... . . . . .
.

w093/0~9~ PCT/US92/08~
~0~3~5~

;) Tran~fec~ion into Dermal Tissue This example wa~ al~o done with plasmid pWRG1601.
Rats wer~ ane~thetized and abdominal skin hair removed by a hair clipper and Nair treatment. The abdomen was opened ~y scalpel inci~ion and the ~i~sues ~ea~ed 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 tranRformation procedure pho~phate buffered saline was added and the skin was su~ured. Controls were bombarded with uncoated carrier particles. For each data point, 1 ml of fluid was collected from the subcutaneous tis3ue space, and 100 microliter of thi3 sample was assayed for human growth hormone by RIA. The results of two replicates are set forth in Table 6.

Replicate 1 Time after Blast Animal Body fluid ~Averaqe~
8 hrs -HGH ~2 animals) 0 ~HGH (7 animals) 8 ng/ml 25 24 hr~ -HG~ t2 animals) 0 +HGH (7 animals) 17 ng/ml RePlicate 2 tone animal) ~ime ater 81ast Bodv fluid HGH Level 30 16 hr~ 0.3 ng/ml 24 hrq 0.6 ng/ml 3 days 0.4 ng/ml 4 dayR 0.37 ng/ml -5 days 0.3 ng/ml The~e re ult~ indicate long-term expression and secretion of the inserted gene. Analogous in situ transformation experiment~ performed with a non-secreted gene ~luciferase) indicated that an inserted gene :: : . ~ ~ . , .

w093/0829~ 2 ~ 9 PCT/US921~884~ :

transfected into dermal tissue exhibited persistent significant levels of expression (at least 10~, ~ometimes . ~-.
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 ~he particular embodiment or examples disclosed above, but ::~
embraces all such modified forms thereof as come within :
~he scope of the following claims. ~:

. .

w093/08~9~ 2 ~ PCT/US92/08~4X

-2~-SEQUENCE LISTING

(1) GENERAh INFORMATION:
(i) APPLICANT: Swain, William F
Yang, Ning-Sun McCabe, Dennis E
~artinell, Brian F
Cheng, Liang (ii) TITLE OF INVENTION: Particle-Mediated Transformation of Animal Somatic Cells (iii) NU~BER OF SEQUENCES: 1 (i~) CORRESPONDENCE ~D~RESS: :
(A) ADDRESSEEs Quarle~ & Brady (B) STREET: P.O. Box 2113 (C) CITY: Madison (D) STATE: Wiscon3in ~::
(E) COUNTRY: USA
(F) ZIP: 53701-2113 (v) CO~PUTE~ R~ADABLE FORM:
(A) NEDIUN TYP~: Floppy disk (B) COMPUTER: IB~ PC compatlble (C) OPER~TI~G SY5TEMs PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Releiase #1.0, Ver~ion #1.25 (vi) CURRENT APPLICATION DATA~
(A) APPLICATION NUM~ER: US
(B) EILI~G DATE: 16-OCT-1991 (C) CLA~SIFIcATIoN:
(~iii) ATTORNEY/AGE~T INFORMATION:
(A~ NAM~: Seay, Nicholas J.
(B) REGI5TRA~ION NUMBER: 2'7,386 (C) R~FERENCE~DOCg~T NUNBER: 1122990695 (ix) TEL~CO~NUNXCA~ION I~FORMATION:
(A) TE~EP~ONE: (608) 251-5000 :~ :
(B) TELEFAX: (608)251-9166 : :
(2) INFORNATION FOR SEQ ID NO:l:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGT~: 336 ba3e pairs (B) TYPE. nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: D~t~ (genomic) (iii) HYPOTHETI~AL: ~tO ~; -:... :` i , . . : , ' , ' ~93/0829~ 2 ~ ~ ~ 3 ~ 9 PCT/~IS92/Og~

~25-(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/REY: sig peptide (B) LOCATION: 1... 336 (x) PUBLICATION INFOXMATION~
(A) AUTHORS: Seldon, ..
(C) JOURNAL: Mol. Cell. Biol ;
(E) ISSUE: 6 ~ :
(F) PAGES: 3173-3179 ~: :
(G) D~TE: ~986 :
(R) RELEVANT RESIDUES IN SEQ ID NO:l: FRON 1 TO 337 (xi) SEQUEN OE DESCRIPTION: SEQ ID NOal:
ATGGC$AC'AG GTAAGCGCCC CTAAAATCCC TTTGGCA~AA TGTGTCCTGA
GGGG~G~GGC 60 ::~
~GCGACCTGT AGATGGGACG G~&GCA~TAA CCC~CAGGÇT TTGGGGTTCT :~

ATCGCCATCT ~AGCCCAGTA T~TGGCCAAT CTCAGAAAGC TCCTGGCTCC
35 CTGGAG~ATG 180 ~;
GA ~ ~AA AACAAACAGC TCCTGG~GCA G&GAGAGTGT TGGCCTC~TG
CTCTCCGGCT 240 . :
40 CCCTCTGTTG CCCTCTG&TT TCTCCCAGGC TCCCGEACGT CCCTGCTCCT .. ~:-CTGCTCTGCC TGCCCTGGCT TC~AGAGÇGC AGTGCC

`,.,'; '' '; ~

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.. ..

Claims (12)

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 axe 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 cell 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.