CA2126103C - Transfection of lung via aerosolized transgene delivery - Google Patents

Abstract

Methods and compositions for producing a mammal capable of expressing an exogenously supplied gene in cells of the airway are disclosed. Lipid carrier-nucleic acid complexes are prepared then delivered via aerosol to the lung airway. The inven-tion provides a direct method for transforming pulmonary cells as a means for treating disorders of the lung as for providing a means for delivering substances systematically following expression in the lung.

Description

'193/ 12756 PCT/US92/ 110118 TRANSFECTION OF LUNG VIA A~OSOLIZED
TRANSGENE II~ELiVF~tY
The present invention relates to methods and compositions for producing a transgenic mammal which comprises an exogenously supplied gene in lung tissue. The gene is supplied by aerosolliaed delivery, particularly to the airways and alveoli of the lung.
With tt~ advent of molecular cloning techniques, an expanding array of gems with mutations responsible for important human diseases have been identified and isolated. To date, attempts to replace absent or mutated genes in human pati~ts have relied ~ ex viva techniques. Ex viv~o dues include, but are not limited to, transformation of calls in vitm with either naked DNA or DNA
encapsulated in lipo~somes, followed by introduction into a host organ ("ex vivo"
gene therapy). The criteria for a suitable orb inclu~ that the target organ for implantation is the site of the relevant dig, the disease ia~ easily accessible, that it can be manipulated in vitro, that it is suso~ible to genetic modification methods and ideally, it should contain either non-replicating cells or cycling stem cells to perpetuate a genetic oorroction. It also should be pnssible to reimplant the genetically modified calls into the organism in a functional and stable form.
A
further requirement for ex viNV gene therapy, if for example a r~roviral vector is used, is that the cells be pro-mitotic; post mitotic cells are rrtfractory to infection with retroviral vectors. Exemplary of a target organ which meets the criteria for in vitro gene transfer is the mammalian bone marrow.
There are several drawbacks to ex vivio therapy. For example, if only differentiated, replicating cells are infectod, the newly introduced gene function will be lost as those cells mature and die. Ex view approaches also can be used to transfect only a limited number of cells and cannot be used to transfect cells which are not first removed from the body.
Retrovinises, adenoviruses and liposomes have been used in animal model studies in attempts to increase the efficiency of gene transfer; DNA has been introduced into animals by intratracheal (TIC, intravenc~s, intraperitoneal, intramuscular, and inttaarteiial injection. Expression of introduced genes, either complexed to cationic vectors or packaged in adenoviral voekors has been demonstratod in the lungs of raden~s after 1T instsliat~n. However, TT
injection is invasive and produces a iron-uniform distribution of the inst~ilod material;
it also is too invasive to be performed rOdly in h~nans. ~t the~for~e would be of interest to develop a non-invasive delivery te~hiriquo which also results in deeper penetration of material into the lung than other methods, and can be used to deposit material evenly throughout tl~e airways and alveoli. Such a delivery technique could be us~pd as a means of t for genetic disorders, particularly of the lung, via ga~ralized transga~e a~presin lung cps in viva.
1'Iazinslri, tt al., Am. .T. Re~pir: Cell Idol. Bird. (1991) 4:206-209, relates to liposome-e~diatod gene tra~fer of DNA into the intact rodent lung.
Three fusion gene ooruarwcta were ~pl~od to c li~ including ( 1 ) the chlorampheni~col aeetyltrtmaferaae ("CAT") gale linlaed to a Rous sarcoma virus ("RSV") promo; (2) the CAT gare licdlaed to a moue mammary tumor virus ("MM'fV") Praraotcr; and (3) a cytom~,a~ovirus-~-ga~ctusuda~se ("CM'V-~-gal") furion gene. The lipoaome/IDN ~A aom~mucs w~ instihed into the cervical trachea of rats and delectable l~ls of gate l~cpltssi~a~n absncvod.
Brigham et al., Am. J fed. 3~. (1989) ~~~78-281, describes the in vivo ~ansfection of marine lung with tix CAT gay using a liposome vehicle.
Transfection was ac~lnpli~d by intravenou, intzatracheal .~o~r intraperitrrneal injection. Both intraand inrianach~~al admiaist~tiorl resulted in the expression of the CAT gone in the lungs. H~tver, ~itor~cal administration did n~. ,~, ~ Wantherr, Cal Rtsto~h (1991) ~9:(l~lbstract).

~'"''~ 93/12756 PCT/US92/11(1~8 Canonico et al., Clip. Res. (1991) ~Q:219A describes the expression of the human a-1 antitrypsin gene, driven by the CM~~ prmnoter, in cultured bovine lung epithelial cells. The gene was at~dci to cClls irl culture using cationic liposomes. The experimenters also defected the presence of a-1 antitzypsin in histological sections of the lung of New Znd whites rabbi s following the intravenous delivery of gene constsvcts com~ie~ted to hpos. Yoshimura et al.
disclose expression of the human cystic fibrc~~sis e;nlne conductance regulator gate in mouse lung after intratrachi~tal liposome-DLV'A gene transfer.
Wolff et al., Science (1990) ?~Z:1465-1468 to direct transfer of the CAT, ~B-gal attd luciferase gicnes into mouse skeletal muscle tn viwiv. Gene acpression was observed in all three case. Nobel et al., Science (1990 x:1285-1288, pertains to in viv~n infra-arterial transfection of pigs with liposomes ~ntaining a ~_ gal expression plasmid. SilOO-specific gene cm was observed in the arterial wall. None of the above cued art, however; praatioes or teaches the use of aerosol administration to grazes diieedy to the rung. An e~eample of a review article of human gene the~py pzooedurcs is: A;ndaainn, Science (1992) x:808-813.
PCT/US90101515, having International 'Publ~ation No. WO
90/ 11092, describes a method for introdnaked TINA hhhto muscle tissue.
Dabs et al. disclose pentamidine uptxk~e in the hung by ion and delivery in lipc~omes. Am Rev Respfr Dis (1987) 13~: 731-737.

SUMMARY
Methods and compositions are provided for producing a mammal which comprises am exogenously supplied nucleic acid in its lung cells. The method includes the ;steps of preparing a lip~.d carrier-nucleic acid mixture suitable for nebulization, nebulizing the mixture, and depositing the resulting nebulized mixture in the lung of a mammalian host of intere;~t in an amount sufficient to transform cells contacted by the deposited nebulized mixture. The exogenously supplied nucleic acid generally is provided in an expression cassette and includes a coding sequence operably joined to transcript~_onal and translational regulatory sequences functional in the mammal.
The methods and compositions find use particularly for in vivo gene therapy of pulmonary disorders.
In a preferred aspect, the invention provides a composition comprising: a nebuli.zed t:ransfection agent, prepared by nebulizing a. mixture comprising complexes between transcription cassettes or expression cassettes and cationic lipid carriers in a pharmaceutically acceptable carrier or diluent, wherein said mixture is substantially free of macroaggregates of. said complexes and said lipid carriers have a diameter_ from 0.:1 microns to 10 microns and the ratio of DNA to lipid carrier is in a ratio of 4:1 to 1:10 micrograms DNA to nanomoles cationic lipid and wherein said transcription cassettes or expression cassettes comprise a DNA sequence r_,apable of producing a transcription product in a mammalian cell transfected by said nebulized transfection agent.
BRIEF DE;.~CRIPTION OF THE FIGURES
Figure 1 demonstrates that aerosol administration 4a cf pRSV-CAT-DOTMA: cholesterol complexes resulted in expression of the CA.T gene in mouse lungs. Lanes 1-3 were derived from mice receiving no treatment.; lanes 4-6 represent mice administered 0.5 mg pRSV-CAT with 1.0 mole DOTMA-cholesterol liposomes; lanes 7-9 were derived from mice receiving 2.0 mg pRSV-CAT alone; and lanes 10-12 represent mice given 2.0 mg pRSV-CAT with 4.0 ~mol DOTMA-cholesterol liposomes in a 2 to 1 molar ratio. The CAT gene is not normally present :in mammalian cells; lanes 10-12 show spots indicative of CAT activity (the positive spots in lanes 10-11 are faint and do riot reproduce well in the figure). The results thus indicate that the lung was successfully transfected by the pRSV-CAT DOTMA-cholesterol:liposome aerosol. The results also show that neither aerosol administration of the pRSV-CAT alone, nor a lower aerosol dose of pRSV-CAT: DOTMA-cholesterol complexes produce detectable expression of the CAT gene in mouse lungs. Thus, both the cationic liposome carrier, and a sufficient dose of DOTMA.:liposome-RSV-CAT DNA complexes are required to produce trarusgene expression in the lung after aerosol administration. Maximum transgene expression is achieved by complexing the l.iposomes and DNA together at an appropriate ratio and in an appropriate diluent.

S
Figure 2 shows the results of an experiment where mice were administered 12 mg of pCIS-CAT compleoed to 24 ~cmoles of DOTMA/DOPE 1:1 liposomes. Lanes 1-3 show the results from animals administered the aerosol in an Intox-designed nose-only aerosol exposure chamber; lance 4-7 are derived from S mice exposed to the a~troBOl in a modified mouse cage; and lanes 8-10 show the results from animals placed in a s~t~aller nwdii~~fied cage after being put in restrainers originally constructed for use in the Intox chamber.
Figure 3 shows construction of pZNl3.
Figure 4 shows construction of pZN29.
Figure 5 shows construction of pZN32.
Figure 6 shows the results of i~nmunostaining for intracellular CAT
protein in lung sections from mica sacrificed 72 hours after receiving an aerosol containing 12 mg of pCIB-CAT plasmi~d oohed to 24 ~c~pnols of D((ITMA:DOPE liposmm~es (A,B,C,D), or from untreated mice (E,F). The section shown in d was treated with normal rabbit swum in place of anti-CAT antibody.
Magnification: A,D (x 50); B,C,E (x 250).
Figure 7 shows CAT activity in lung extracts from mice sacrificed 72 hours after receiving an aemeol containing either 12 mg of CMY-CAT plasmid alone or 12 mg of CMV-CAT plasmid comps to 24 ~cmols of DOTMA:DOPE
(1:1) liposomes. Unmin were also assayed.
Figure 8 shows (A) CAT activity in lung extracts from mice sacrificeEi from one to tw~ty-acne days after receiving an aerosol containing 12 mg of pCIS-CAT plasmid compkxed to 24 ~cmols of DOT~A:DOPE liposomes; and (B) shows CAT activity in several differa~t tissue extracts Born mice and indicates that expression of the transgene is lung-specific after aerosalization of DNA-liposome complexes into normal mice sacrificed at the three day time point in Fig.
8A. Control extract amtains purified CAT ~nzyrne.
Figure 9 shows Southern blot hybridization of genomic DNA from the lungs of mice sacrificxd immediately after receiving an aerosol containing mg of pCIS-CAT plas~ni~d complexed to'24 ~rmols of DOTMA:DOPE liposomes (lanes 1-4, 6-9) and from an untreated corrtrc~l mouse (lane S). Samples were digested with the restriction auyme Hir~diB and probed wit~r a 1.6 kb CAT

WO 93/12756 PGT/US92/I~OIa, ~~2s~o~

fragment (upper panel). Tiu same membrane was hybridi~ with a 1.1 kb BSU
36-1 single copy probe from a mouse factor ~.A genomic clone (lower panel).
Figure 10 shows ph~microgtaphs of fm~a~ actions from lungs of control mice (Flgure5 108 and 10D) and n~i~ treated with ;gZ1~T32 complexed to DDAB:cholesterol (1:1) lipo~somes (Figures 10A, IOC; and 10E).
Figure 11 shows a full re~icdov map for HMV (Towne) of the immediate early e~nca~r and pmn~er negipn of HClluIV (Towne) in Figure 11A
and HCMV(AD169) in Figure 11C. Figure 118 shapws a xquence comparison of the 2 HCMV promc~rs. The position of tht NcoI site is indicated by an asterisk.
In awaordancx with d~ abject imentimn, ~ruic acid constructs together with methods of p~pazaml use are prodded which allow for in vivo modulaation of phenotype andlor of cells in the re~pintc~ry react of a mammalian host following deliver of a ~t dose of a lipid carrier-nucleic acid aerosol to the halt mammal 1b provide for transfaction ~of boat lung cells.
The lipoid carrier-nucleic add is obta~ad by nebuli~tion of a lipid carrier-nucleitc said sample ntixturc prod in a b~logi~lly 1e fluid that minimi~tes aggragadom of the lipid carria~:nuic acid oon~llaces. The methods and compositions can be used to producx a ~mnsal aompri~sing ~ eaogenously supplied gene in lung tissue, pla~ly alvaarlar and airway passage cells.
Central to du preemt invention is the disoovaly that genes can be delivered to the lung win aerosol edministtam, and subsequently dressed in vivo. The instant invlmtioa takes advantage ~f the ua~a of lipid carriers as a delivery mecharnism. Lipid s are able io atably bind h charge interacl~ons or entrap arid retain ~clac acid and parmnt a ~ystam amenable to nebulization, whereb~r igams can be ddivared b c pulmonary tissues.
Lipid carriers include but are not linnibed to liposorna and micelles, as well as biod~sdable cationic oompoimds aisiapg ma~di~Od phaaphoglyoerides particularly allcyl.hogl~ Pat~i~ar sites ~t the hmg are targeted by va~rrying the sire of ~e ~,ra~eol pad~inialaeed, as dilsa~ssed snore fully below. Targetitng a8~ts, such as imp ctirec~Dod ~t surfaae antigens _~..~",._.,~.~.,..,.~,~.."~.,.,......,........".,.,~.."~..~. .._.___~..__-.,....~.,-."~",."-,_........_ _,~"...,.~,...,.......".~..~.......,.......,._.~..._ . ~, ...._ 7 2I2s1~~
expressed on specific pulmonary cell yp~, ~ also be covalently conjugated to the lipid carrier surface so that nucleic acid can be delivered to specific cell types.
Lipid carriers also allow for the delivery of relatively large amounts of nucleic acid, without a touc effect, such that therapeutically effectiw~e amounts of the desired protein can be expressed in vivo. For a review of the use of liposomes as carriers for delivery of nucleic acids, ~, Hug and Sleight, Biochim. Biophys.
Acre. (1991) '1:1-17; Straubinger et al., in Methods of E~rymology (1983), Vol. 101, pp. 512-527.
Lipid carriers, particularly lipossomes, have been used effectively, particularly to introduce drugs, radiotherapeutic agents, enzymes, viruses, transcription factors and other cellular effectors into a variety of cultured cell lines and animals. In addition, successful clinical trials exa~~nining the effectiveness of liposome-mediated delivery of small drug molecules and peptides which act extracellularly have been reported. however, while the ba~c methodology for using liposome-mediated vectors is well developed a~ has been shown to be safe, the technique previously has not been developed for aerosolized delivery of nucleic acid to pulmonary tis~e for in viv~o gene therapy. By in viuo gene therapy is meant transcription anrd/or translation of eaog~ously supplired nucleic acid sequences to prevent, palliate andlor cure an~mal or human disease.
In addition to the discovery that transformation of lung cells can be obtained using aerosolized lipid carrier-nucleic acid complaces, several factors have been identified that can affect the relative ability of patticular lipid carrier-nucleic acid complexes to provide transformation of lung cells following aerosolized delivery of a soluti~ containing the lipid curler-nucleic acid constructs and to achieve a high level of cxp~ession where ti~at is the desired endpoint. These factors include (1) preparation of a solution that prior to or during nebulization will not form maYcroaggr~gates and wherein the nucleic acid is not sheared into fragments and (Z) pmeparatian both of lipid carriers and of expression constructs that provide for predictable transformation of host lung cells following a~osolization of the lipid carrier-nucleic acid oo~plea and administration to the host animal. Other faa~ors include the lipid carrier:nucleic WO 93/ 12756 '~ '12, ~ ~ ~ ~ PCT/US92/ 1100 acid ratio in the solution f~ nebulizetion and the diluent used to prepare the solution. These facta~ts are discussed in die~Gl below.
Aerosol delivery of nu~ic acid-lipid c~a~rrier vonrnple~ces provides a number of advantages over other nodes of administration. For e~cample, aerosol administration can sere to reduce host to~cir. Such an ei°~ect has been observed with 11~ deuverlr of sorbs such as pcnts~idine aand cyt~, which can be highly ~cic wlu~r deliivared systematically, Oat are well toleaated when aerosolized. Additionally, the results in rodarrts with sized pentamidine a~ur~ly predicted rem~Its in human ~ ,~th AIZJS treated with aerosolized pent. ~, fog arampk, Dabs tt al. , Aa~tinicmb. .igsents ~n~Ot7rer.
(1987) x,:37-41; Debt et al., Mar. Rtv Rein. Dis. (19x:731-737; Debs et al., J. Inunurrol. (188) ~Q:348?-3488; A~ntgornc~y a 41., Lc~cet (1987) x,:480-483; Montgocnuo;y et al., Cheat (19~89!~ x:74? 751; ~,.oo~ung et al., N. Eng.
J. Meld. (1990) :719-775. Additionally, d clca~ance ~of circulating liposomes by tl~e livre~ snd Teen rr~ulod~c~lxlial ttystem ~s avoided, thereby allowits8 the suatain~, ps~noe of the adminOd s~lstanae at t~ site of inadrest;the lung. ra induced inactiof tlse t~ap~uti~c agent is also reduced: This method of transfecting lung calls also avoids- of the host mammal's gonads, thus avoiding tran~fectioo off' ge~sn line carps.
Other ~vanof the su6~eat invention include ease of administration i.e., the host mammal amply ~ the aeollzad lipid earrier-nucleic acid solution i~nta tt~e inta~od tistoe,tie long. Pure, by varying the size of the nebuliztd ~rtic~ some oontrvl y also be aWcised over where in the lug the aerosol is c~livre~d. )aalivesy nplsy be eatieneled oirer a long time period. Thus, there i~t a si~ificant inct~oe I~n the time pe~Od that target cells are eoposed~ to the expnsat~n aonatructs. Dis~Gri~ of the aerosol is ewe throughout areas of the lung ~ to the day. Se .advantages are significant, partieularl~r when oanpd to otter rays of istration such as intratraeheal dGli~y rwhich is i~nvmive, tlx ~m oars are delivered in a bole which may did dre nu~us and additly nay result in pooling of the introduced Eland in :coos of tha lung at borer elanation.
Further, _...-,~...~.,,a.,~,.-~,....~. """"-,-~",....~.,.._...,.~..,...~..:..-.,_..._. -."-,."~.:...,--....,a,..~...~.n,...,..~..,.-,....,.,._..r..... ... . -~'~"'"~ 93/12756 ~ ~ ~ ~ ~ ~ ~ PCT/US92/11008 damage from insertion of the int~atracheal tie may alter the ability of cells coming into contact with the expression constructs to be transfected.
The type of vector used in the subject application may also be an advantage. For example, most gene therapy strategies have relied on transgene insertion into retroviral or DNA virus vectors. Potential disadvantages of retrovirus vectors, as compared to the use of lipid carriers, include the limited ability of retroviruses to mediate in vivo (as opposed to ex vivo) transgene expression; the inability of retrovirus vectors to transfixt non-dividing cells;
possible recombination evaits in replication-defective retrovirus vectors, resulting in infectious retroviruses; possible activation of on~genes a~ inhibition of tumor suppressor genes due to the random insertion of the transge~e into host cell genomic DNA; size limitati~s (less than 15 kb of DNA can be packaged in a retrovirus vector, whereas lipid carriers can be used to deliver sequences of DNA
of z 250 kb to mammalian cells) and potential immunoge~city of the viral vectors leading to a host immune response against the vector. In addition, all ex vivo approaches require that the cells removed from the body be maintained in culture for a period of time. While in culture, cells may ua~dergo deleterious or potentially dangerous phalotypic and/or genotypic changes. Ada~ovirus and other DNA viral vectors share several of the above potential limitations.
Particularly for human use, but also for repeated veLexinary use, biodegradable lipid carriers may be used which are metabolized by the host mammal to naturally occurring compounds that are non-toxic to the host andJor are readily excreted.
The nucleic acid constructs generally will be provided as expression cassettes which will include as operably linked components in the direction of transcription, a transcriptional initiation regia~, a nucleic add sequ~ce of interest and a transcriptional termination region wherein the transcriptional regulatory regions are functional in the mammalian host lung cell. An intron optionally may be included in ttar construct, preferably z 100 by and placed 5' to the coding sequence. Generally it is preferred that the cxmstruct not be~oome integrated into the host cell geaome and it is introduced into tlu hod as part of a non-integrating expression cassette. A coding sequence is "aperably linked to" or "under the control of" ttanscriptional and/or translational regulatory regions in a cell when DNA polymerise will bind the promoter sequence and transcribe the coding sequence into mRNA, either a sense strand or an antisense strand. Thus, the nucleic acid sequence includes DNA sequences which encode polypeptides which are directly or indirectly responsible for a therapeutic effect, as well as genes 5 coding for active nucleotide sequences such as antisense sequences and riborymes.
The constructs for use in the invention include several forms, depending upon the intended use of the construct. Thus, the constructs include vectors, transcriptional cassettes, ezpression cassettes and plasmids. The transcriptional and translational initiation region (also sometimes referred to as a "promoter, "), 10 preferably comprises a transcriptional initiation regulatory region and a translational initiation regulatory region of untranslated 5' sequences, "ribosome binding sites," responsible for binding mRNA to ribosomes and translational initiation. It is preferred that all of the transcriptional and translational functional elements of the initiation control region are derived from or obtainable from the same gene. In some embodiments, the promoter will be modified by the addition of sequences, such as enhancers, or deletions of nonessential and/or undesired sequences. By "obtainable" is intended a promoter having a DNA sequence sufficiently similar to that of a native promoter to provide for the desired specificity of transcription of a DNA sequence of interest. It includes natural and synthetic sequences as well as sequences which may be a combination of synthetic and natural sequences.
For the transcriptional initiation region, or promoter element, any region may be used with the proviso that it provides the desired level of transcription of the DNA sequence of interest: , The transcriptional initiation region may be native .,..
to or homologous tb!the host cell, and/or to the DNA sequence to be transcribed, or foreign or heterologous to the host cell and/or the DNA sequence to be transcribed. By foreign to the host cell is intended that the transcriptional initiation region is not found in the host into which the construct comprising the transcriptional initiation region is to be inserted. By foreign to the DNA
sequence is intended a lranscriptional initiation region that is not normally associated with the DNA sequence of interest. Efficient promoter elements for transcription initiation include the SV40 (simian virus 40) early promoter, the RSV (Rous ''-"~'"- 93/12756 PCT/US92/11008 11 ~.~ ~~1 sarcoma virus) promoter, the Adenovirus major late promoter, and the human CMV (cytomegalovirus) immediate early 1 promoter.
Inducible promoters also find use with the subject invention where it is desired to control the timing of transcription. Examples of promoters include those obtained from a ~-interferon gene, a heat shock gene, a metallothionein gene or those obtained from steroid hormone-sive genes, including insect genes such as that encoding the ecdysone receptor. Such inducible promoters can be used to regulate transcription of the transgene by the use of external stimuli such as interferon or glucocorticoids. Since the arrangement of eukaryotic promoter elements is highly flexible, combinations of constitutive and inducible elements also can be used. Tandem arrays of two or more inducible promoter elements may increase the level of induction above baseline levels of transcription which can be achieved whey compared to the level of induction above baseline which can be achieved with a single inducible element.
Generally, the regulatory sequence comprises DNA up to about 1.5 Kb 5' of the transcriptional start of a gee, but can be significantly smaller. This regulatory sequence may be modified at the position corresponding to the first colon of the desired protein by site-directed mutagenesis (Kunkel TA, 1985, Proc.
Natl. Acad. Sci. (USA), $x:488-492) or by introduction of a conveni~t linker oligonucleotide by ligation, if a suitable restriction site is found near the N-terminal colon. In the ideal embodim~t, a coding sequence with a compatible restriction site may be ligated at the position corresponding to colon ~ 1 of the gene. This substitution may be inserted in such a way that it completely replaces the native coding sequ~ce and thus the substituted sequence is flanked at its 3' end by the gene terminator and polyadenylaticm signal.
Transcriptional enhancer elements optionally may be included in the expression cassette. By transcriptional enhanoer elements is intended DNA
sequences which are primary regulators of transcriptional activity and which can act to increase transcription from a promoter clement, and generally do not have to be in the 5' orientation with respect to the promoter in order to enhance transciiptional activity. The combination of promoter and a~hancer elements) used in a particular expression cassette can be selected by one skilled in the art to 6 ~ ~ ~ ~ ~ ~ PCT/US92/11 maximize specific effodts. Diffa~tt elemtnts can I~e used to produce a desired level of transgaae e~c~ssicei in a v~idl~ of tis~e and cell types.
For example, the hun CMV immediate early prosn~aiter-en~anoer element can be used to producx high le~rrel transgene ~pressic~ in many dift tissues in vivo.
Haamples of caber ad~noa e~at'SS wh~h confer a high 1e~~~el of transcription on linked ,genes in a of difhsrait cell types from many species include anhanaers frost SiV40 acrd ~tSV-L1"R. 1fie 540 and RSW-L'TTt are essentially constitutive. They may be aombin~d with over qnhancers which have specific effects, or the 'tc mhaa~ nay ~ u;~i alone. Thus, where specific control of transcription is dcs~od, t a~ta~r e~menp~ that are active only in a , dcve~opr~ntal-, or celhinclude fi~nrnunoglobulan, interleubn-2 (B.-2) and &glabin eahanoers ark of inta~st. 'Tis~e-, develop-rncatat , or call-spedfic caa be to obte~ t~ra~uga~ e~pr~ion in particular cell types, such as B-lymEp~ aid T-lymph, as well as myeloid, or erythroi~l ~rOgalitQr cells. ~ltively, a tissue-specific promoter such as that dezivr~d from the human cystic fibs errant conductance regulator (CFTR) gam can be fused to a very active, he~olpgous enhancer element, such as the S'~J40 a~ha~', in order to c~onfasr both a high level of transcription and tissoa-sic transge~ iption. In edition, the use of tissue-spocific peom~rs, such as L,CK, may o~llow g~ ~of trance transcription to T lymphocytes. Tissue traasorxption;of the transgene may be impo~uit, pa~iaula~rly in cases where tipue its of transc~n of the transgmit in tissues ot4er tl~t the target tiuue would be devious.
T~an~m red of two or mvae its o~c ao~mbinati~ons of enl~noer elements may sagni~ic~rrtly incr~e pose expmion when compared to the use of a single copy of an Wit; haa~oe aelements find use in the ion ~. The u~te of tw~mWaarit enh~n~r tt from the ward or ~t fianklag air win a single Prod can in some casts ~ ion in each ti~uwe in which etch individual enhanc~er acting a~lon~c vva~ld have u'~ effect, tby iu~r~asi~Ig the number of ie which ~ransa~iptio~t is o~,ined. In vt~' cs~, thr~ pre~cx of two dif~eerrslt enhance ~ ~sults in ~ of the elects. Evaluation of particular combinations of enhancer elements for a particular desired effect or tissue of expression is within the level of skill in the art; Although generally it is not necessary to include an intros in the expression cassette, an mtron comprising a 5' splice site (donor site) and a 3' splice site (acceptor site) separated by a sufficient intervening sequence to produce high level, extended in vivo expression of a tzansgene administered iv or ip can optionally be included. Generally, an intervening sequence of about 100bp produces the desired expression pattern and/or level, but the size of the sequence can be varied as needed to achieve a desired result. The optional intros placed 5' to the coding sequence results in high level extended in vivo expression of a transgene administered iv or ip but generally is not necessary to obtain expression. Optimally, the 5' intros specifically lacks cryptic splice sites which result in aberrantly spliced mRNA sequences. If used, the intros splice donor and splice acceptor sites, arranged from 5' to 3' respectively, are placed between the transcription initiation site and the translational start colon as diagrammed in (1), below.
Consensus seauences for the 5' and ~'~lice si . used in RNA splicing, 5' ezon iatron 3' ezon' C A UUUUUUUUUUU C G (1) 5'--or A G t_~'I;~ or A G U or or or or or or or or or or or N or Aor---3' A ~G~~ C C C C C C C C C C C U ~ ~~~ A
consensus sequence for consensus sequence for 3' splice site 5' splice site ("donor site") ("acceptor site') The sequence given is that for the RNA chain; the nearly invariant GU and AG
dinucleotides at either end of the intros are shaded.
Alternatively, the intervening sequence may be placed 3' to the translational stop colon and the transcriptional terminator or inside the coding region. The intros can be a hybrid intros with an intervening sequence or an intros taken from a genomic coding sequence. An intros 3' to the coding region, a 5' intros which is of less than 100 bp, or an intros which contains cryptic splice sites may under certain condition substantially reduce the level of transgene expression produced in vivo. However, unexpectedly, a high level of in vivo expression of a transgene can be achieved using a vector that lacks an intron.
Such vectors therefore are of particular interest for in vivo transfection.
In some cases, it may be desirable to use constructs that produce long term transgene expression in vivo, either by integration into host cell genomic DNA
at S high levels or by persistence of the transgene in the nucleus of cells in vivo in stable, episomak form. Integration of the transgene into genomic DNA of host cells in vivo may be facilitated by administering the transgene in a linearized form (either the coding region alone, or the coding region together with 5' and 3' regulatory sequences, but without any pkasmid sequences present).
Additionally, in some instances, it may be desirable to delete or inactivate a mutant gene and replace it with a desired transgene. This may be achieved by using an expression cassette suitable for homologous recombination in vivo. Thus, for example, a linearized plasmid comprising an expression cassette may be used such as is described in European patent applications 88/201743.7 and'EP89/202106.4. These applications disclose a pkasmid for targeting of a specific gene. For the present application, a linear plasmid can be constructed wherein the replacement gene is flanked by 5' and 3' sequences which are sufficiently homologous with the 5' and 3' sequences of the defective gene to provide for homologous recombination.
Where it desired to insert the replacement gene in the mutant gene (thereby inactivating it) a means for selection is included within the 5' and 3' flanking sequences of the pkasmid.
The incidence of transgene integration into genomic DNA may be increased by incorporating a purified retroviral enzyme, such as the HIV-1 integrase enzyme, into the lipid carrier-DNA complex. Appropriate flanlang sequences are ,...', placed at the 5' ands' ends of the transgene DNA. These flanking sequences have been shown to mediate integration of the HIV-1 DNA into host cell genomic DNA in the presence of HIV-1 integrase. Alternatively, the duration of the transgene expression in vivo can be prolonged by the use of constructs that contain non-transforming sequences of a virus such as Epstein-Barn virus, sequences such as oriP and EBNA-1 which appear to be sufficient to allow heterologous DNA to be replicated as an episome in mammalian cells (Buhans et al. , Cell ( 1986) x:955).

Downstream from and under control of the transcriptional initiation regulatory regions is a multiple cloning site for,insertion of a nucleic acid sequence of interest which will provide for one or more alterations of host genotype and modulation of host phenotype. Conveniently, the multiple cloning 5 site may be employed for a variety of nucleic acid sequences in an efficient manner. The nucleic acid sequence inserted in the cloning site may have any open reading frame encoding a polypeptide of interest, for example, an enzyme, with the proviso that where the coding sequence encodes a polypeptide of interest, it should lack cryptic splice sites which can block production of appropriate mRNA
10 molecules and/or produce aberrantly spliced or abnormal mRNA molecules. The nucleic acid sequence may be DNA; it also may be a sequence complementary to a genomic sequence, where the genomic sequence may be one or more of an open reading frame, an intron, a non-coding leader sequence, or any other sequence where the complementary sequence will inhibit transcription, messenger RNA
15 processing, for example splicing, or translation.
A number of nucleic acid sequences are of interest for use in vivo gene therapy of lung diseases or diseases of other tissues. When ~ it is desired to have an extra-pulmonary effect, nucleic acid providing for secretory leader sequence is included in the expression cassette. Where the nucleic acid codes for a polypeptide, the polype~ptide may be one which is active intracellularly, a transmembrane protein, or it may be a secreted protein. It may also code for a mutant protein for example are which is normally secreted but which has been altered to act intcacellularly. The nucleic acid may also be a DNA sequences coding for mRNA (antisense or ribozyme sequences such as those to HIV-REV or BCR-ABL sequences) or, for proteins such as transdominant negative mutants which specifically S~revent the integration of HIV genes into the host cell genomic DNA, replication of HIV sequences, translation of HIV proteins, processing of HIV mRNA or virus packaging in human cells; the LDL (low density lipoprotein) receptor, which specifically lowers serum cholesterol, and which can reduce the risk of heart attack in individuals with elevated serum cholesterol levels, and proteins such as granulocyte macrophage colony stimulating factor (GM-CSF) which can stimulate the production of white blood cells from the bone marrow of immunocompromised patients and produce significant anti-tumor activity or cystic fibrosis transmembrane conductance regulator (CFTR) for treatment cystic fibrosis. These, or other beneficial (therapeutic) nucleic acid sequences can be expressed in appropriate cells in vivo using this invention.
Examples of beneficial therapeutic nucleic acid sequences are those encoding molecules have superozide dismutase activity or catalase activity to protect the lung from oxidant injury; endothelial prostaglandin synthase to produce prostacyclin and prostaglandin E2; and antiprotease alpha-1 antitrypsin. Thus, this approach could dramatically impmve the treatment of acquired immune deficiency syndrome (AmS), cystic fibrosis, cancer, heart disease, autoimmune diseases and a variety of life threatening infections. For the treatment AIDS, anti-TAT, REV
TAR or other critical anti-HIV sequences may be usod, particularly for expression of the appropriate coding sequences in T lymphocytes, macrophages and monocytes which can be achieved following iv administration of the appropriate coding sequences; expression of wild-type CFTR gene in the lungs of cystic fibrosis patients (see Collies, Science (1992) 256:774-783) CFTR cDNA can be obtained from Dr. Collies at University of Michigan or Dr. Tsui at Toronto Sick Children's Hospital; expression of wild-type p53 in tumors of cancer patients with absent or aberrant expression of this gene, p53 is obtainable from Dr.
Vogelstein at John Hopkins Univ; antisense sequences to over-expressed, transforming oncogenes, such as myc or ras in tumors; genes which block activity of activated T cell clones which attack myelin in multiple sclerosis or other targets in autoimmune diseases. A T-cell lymphocyte clone activated to recognize and attack myelin can be targeted by using an anti-sense sequence, ribozyme sequence or transgene coding for a transdeminant negative mutant which specifically blocks surface expression on the T-cell of T-cell receptor components which mediate recognition and/or attack of myelin-sheathed cells.
The termination region which is employed primarily will be one of convenience, since termination regions appear to be relatively interchangeable.
The termination region may be native to the intended nucleic acid sequence of interest, or may be derived from another source. Convenient termination regions are available and include the 3' end ~f a gene terminator and polyadenylation ,~.."~ 93/12756 PCT/US92/11008 172.~~~~Q
signal from the same gene from which the 5' regulatory region is obtained.
Adenylation residues, preferably more than 32 and up to 200 or more as necessary may be included in order to stabilize the mRNA. Alternatively, a terminator and polyadenylation signal from different g~elgaies may be employed with similar results. Specific sequences which regulate post-transcriptional mRNA stability may optionally be included. For example, vertain polyA sequences (Volloch ,et al.
~ (1981) 23:509) and B-globin mRNA elements can increase mRNA stability, whereas certain AU-rich sequences in mRNA can decrease mRNA stability (Shyu ., Genes and Devel. (1989) 3:60). In addition, AU regions in 3' non-coding regions may be used to destabilize mRNA if a short half life mRNA is desirable for the gene of interest.
The construct may additionally include sequences for selection, such as a neomycin resistance gene or a dihydrofolate reductase gee and/or signal sequences to regenerate recombinant proteins that are targeted to different cellular compartments or secreted when the wild type sequ~e is not. Any of a variety of signal Sequences may be used which are well-known to those skilled in the art.
These signal sequences may allow generation of new vaccine strategies or produce soluble antagonists directed against specific cell surface receptors such as transformed oncogenes. The sequences for selection may be on a separate plasmid ~0. and cotransfected with the plasmid carrying the therapeutic nucleic acid.
Where a carrier is used, the selection plasmid may be complexod to a different carrier or to the same carrier as the therapeutic plasmid.
The recombinant coding-sequence flanked at its 5' ~d by the promoter and regulatory sequences and at its 3' ~d by a terminator and regulatory sequences may be introduced into a suitable cloning plasmid (e.g., pUClB, pSP72) for use in direct DNA uptake in host cells following introduction into the host T~~latiarn of C~~.es and Construction of Linid carriers Nucl~c acid sequences for use in the present invention, can be derived from Imown sources, for example by isolating the nucleic acid from cells containing the desired gene, using standard techniques. Similarly, the gene sequence can be generated synthetically, using standard modes of polynucleotide synthesis, well known in the art. See, e.g. Edge, M.D., Nature (1981) ?:756;

W093/12756 ~~~~~,~ PCT/US92/1100~-~~

Nambair, et al., Science (1984) x:1299; Jay, Ernest, J Bias Chem (1984) ~,~Q:6311. Golly, ~c oligcxr~leo~i~a are pared by either the pho~tphotriester nu~hcbd as deecribcd by Bye ~t itl. , Na~ur~e (auupra) and Duckworth et al., N~rc~eic Adds ~Rets (1981)Q:1691, or the phosplromethod as described by cage, S.L., and Cuvthers, M.H., Tet: Let~c. (1981) x:1859, and Matt~xtcci, M.D., astd CaruthGrs, M.H., J. A~m. Chi. Sic. (1981) x:3185, and can be prepared us~g oommescially available aubornated oligonucleotide synthesisers. The gam saqu~x can be died widr die a~~e colons for the particular amino acid x~uenoe. In garl~al, one will sele4t grefarad colons for exsion in the indcd host. The come sequeruae is asetmbk~d from overlapping oligonucleotides prepared by s~nc~nd nusdsOda acrd assembled into a complete coding sequ~~ , ~,~" l3dge (1181) Notr~n x:7515; Nambair et al., (1984) Sc~~e~e t1; Jay ~ al., (1980 J. Biai G7. ;x:6311.
A particularly oonvmaient med~awd for obts~ning nucleic acid for use in the lipid cazrier-c adid prq~~tions, i~ by r~ooa~b~t ma~tns. Thus, the desit~ed gee can be eaaaisad l3nom a plasmid dying tha desi~od gene, using standard ~neatrictio~n mzand praceduns. 'ate ific D~iA cleavage is perf~rnsed by with the suitable rc~i enzyme (or ~) under ~nditiona which are gly undersZnod ~ the art, ant the :ors of which are spedfied by the ma~~ of these oa~~ly availu~ble restriction ex~aymes. See, e.g., N4w Ps~laad , Fiat . If desired, size separation of the cleaved fragtn~ts array be paefrocaaed bry poa~Crylamide gel or agarrose gel electrophor~,sis using standard dues. A gel description of size sepe~ation~s is faa~d in l~e~Ods fn ~tyogy (190) r499-560.
»arcxr rved fnrge~aats mr~y be bloat eodbd by treating with the large fragment of ~ colt DIrTA poi I ~orv) la, the gce of the four deoxynucleotide tr(p~spbates (dN'fPs) u~ip~,g stand td~hn. The Kienow tkagmeslt fills in at 5' sin~o-sttand~pd bot chews back prod 3' single stn~nds, aveu tho~h the drat d~'Iare Wit. If desired, selective xepair can be craned by sepply!~g golly one of tl~e, or selected, dNTP: within the limitns dic~od by the nee of the overhang. After tr~eatrnent with Kl, the mixture can be with e.g: phenollchlomform, "4.~Ai 93/12756 and ethanol precipitated. Treatment under appropriate conditions with S1 nuclease or BAL-31 results in hydrolysis of any single-stranded portion.
Once coding sequences for the desired proteins have been prepared or isolated, they can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art; the selection of an appropriate cloning vector is known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligation to other sequences is performed using standard procedures, known in the art. For example, ligations can be accomplished in 30 mM Tris-C 1 pH 7.8, 10 mM MgC 12, lOmM DTT, 50 ~eglml BSA, and either 1 mM ATP, 0.01-1.0 (Weirs) units T4 DNA ligase at 16°C (for "sticky end" ligation) or 1mM ATP, 0.5-1.0 (Weirs) units T4 DNA
ligase at 20°C (for "blunt exrd" ligation). Intermolecular "sticky end"
ligations are usually performed at 30-100 ~cg/ml total DNA concentration (5-100 nM total end concentration).
The nucleic sequ~ce is placed under the control of a promoter, ribosome binding site and, optionally, an operator (collectively refereed to herein as "control" elements), so that the coding sequence is transcribed into RNA in the host tissue transformed by the lipid carrier-nucleic acid. The coding sequence may or may not contain a signal peptide or leader sequence. A "promoter sequence"
is a DNA regulatory region capable of binding RNA polymerise in a cell and initiating transcription of a downstream (3' direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bound at the 3' terminus by the transcription start colon (ATG) of a coding sequence and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
Within the promoter sequence is a transcription initiation site (conveniently defined by mapping with nuclease S1), as well as profi~ein binding domains (consensus sequences) responsible for the binding of RNA polymerise. Nucleic acid "control sequences" or "regulatory regions" refer collectively to promoter sequences, ribosome binding sites, polyadenylation signals, transcription termination sequences, upstream regulatory domains, a~hancers, and the like, which WO 93/12756 , ~~~~~ ;~, PCT/US92/110~"'~
collectively provide fog the transcriptioa and translation of a coding sequence in a host cell.
The choice of regulatory its will depend on the host cell which is to be fed and the type of nc acid pration used. Thus, if 5 the host cells' endogenous ion and t~a~latiott ma~cb~n~y will be used to express a polype~i~ mf interest, control demd~ts fiu~ctior~al in the particular host which provide for boa are uamd. Seve~l promoters for use in mammalian cells are lmown in the art and include, but anp ~t limwi~od tc~~ a SV40 (Simian Virus 4~ early prom, a RSV (Roux Samna Virus) propiotcr, an Addnovirus 10 ma,~or lade promoter, at~d a human CI~IV (Cyll~megal~rua) n~ early one promote. O~r promatars which may be uslAd include tho~ derived from mouse mammary tumor virus (I~MTV, T7, T3, aund the lilac). Parf~culatly useful in the present i~nveution are tie RSV promoax and the C1~V ~, particularly the immediate early promoter from the AD169 strain of CMV.
15 In addition to the above soq, it auly be desirable to add to the nucleic ~I~cad co~rstruat xegulatory seqwl~iCh al1aw for ion of the e~cpressi~ of ttur polype~ide of inert aaqwpnoes. Ragul~ry sequences are known to thaw of skill in the art, and include tta~ which cause the eap~esai~ of a genre to bd honed an or off iir rosponae to a ~ ar physical 20 stimulus, including the pncx of a r~g~ compound. Such promoters can be used to regulate a~easian of the test by tlm use o$ stimuli such as interfitaon or gluco~rtiaoids.
Other tykes of regulatory clefts may also bye t in tire plasmid,. for eacample, seqmawes. Sluuh regulatory its include those obfiainable from ~3-inte~fiaon, heat thiamin or s~tetoid hormone responsive genes, including insect gc~s such a= the ecc~y~ona rexptor gene.
Since thd arrangement .of eukaryotic ptots is highly f~cible, combinations of ~oonstitutive end its can be aged. Ta~em arrays of two cc mope inducible day ink ttra levGi of induction above basalinc 1tvela a~f trantion which a~n be ac~i~eved vim a single inducible Wit. $y option ~~5 are i~ndmd DNA
sequencxs which are primary regulators of optional a~ivity which can act to ' ~~~n"~ 93/12756 increase transcription from a promoter element, and generally do not have to be in the 5' orientation with respect to the promoter in order to enhance transcriptional activity.
The combination of promoter and enhancer elements used in a particular nucleic acid construct can be selected by one skilled in the art to maximize specific effects; different enhancer elem~ts can be used to produce a desired level of transgene expression. For example, a tissue specific promoter such as that derived from the human cystic fibrosis transmembrane conductance regulator (CFTR) gene can be used flanking a very active, heterologous enhancer element, such as the SV40 enhancer, in order to obtain both a high level of expression and expression of the transgene primarily in airway epithelial cells in the lung. Tandem repeats of two or more enhancer elements or combinations of enhancer elements may significantly increase transgene eaprGSSion when compared to the use of a single copy of an enhancer element. The use of two different enhancer elements from the same or diffe~t sources, flanking or within a single promoter may be used. Evaluation of particular combinations of enhancer elements for a particular desired effect or expression level is within the knowledge of one skilled in the art. Promoter-enhancer elements which are least partially derived from CMV Townes and/or AD169 strains are of particular interest for providing a high level of expression of a transgene.
The termination region which is employed primarily will be one of convenience, since termination regions appear to be relatively interchangeable.
The termination region may be native to the int~ded nucleic acid sequence of interest, or may be derived from another source. Convenient termination regions are available and include the 3' end of a gene terminator and polyadenylation signal from the same gene from which the 5' regulatory region is obtained.
Adenylation residues, preferably more than 32 and up to 200 or more if necessary may be included in order to stabilize the mRNA. Alternatively, terminator and polydenylation signals from a different genelgenes may be employed with similar results. Specific sequences which regulate post-transcriptional mRNA stability may optionally be included. For example, certain polyA sequ~ces (Volloch et al., Cell (1981) x.:509) and ~-globin mRNA elements can increase mRNA

WO 93/ 12756 ~ ~ ~ PCT/US92/ 11 OQ~

Viability, whereas cer~in AU-rich sequences ie~ mRNA can decrease mRNA
stability (Shyu et al., C3enes and Deveiopmtat (1959) x:60). In addition, AU
regions in 3' non-oaring regions may be used to destabilize mRNA if a short half life rnRIrIA is desirable. A 3'-intrcm ahou~ld bie avoidedy particularly a SV40 3'-intron. If used, the 3'~intron str~ld be greatly than about 7p bp.
The nucleic acrid construct may: include soquames for selection, such as a nmomycin resistance gene, dihydr~olabe ~ geae~ , and/or signal sequa~s to regreomnbinan~ pins that are targcte~d to different cellular compartment or seed ~rhca the wild type ~oqua~oe is note Any of a variety of signal se~qu~s may be used which are w~rll known to thos~a skilled in the art.
The signal 9aqmay allow generation of new vaccine ~ pmduce soluble antagonists dfrecc;tly against specific odll rta~rs such as transfncm~ad onooge~~ The sequfor may be on a separate plasmid and cotnuasfectmd with the p~mid carrying tli~e nucleic acid boding for the therapeutic polypepride. The sp>auniid may be a~mp~.auod to a different carrier or to the same terrier as the ~dc plasmid.
An ion vector is oon~ruad so thdat the part~ular coding sequence is looted in tt~ valor with the ap~9~riate ndgulatory sequences, the positioning and orientation of the ending segue with r~p~Ct to the control ZO _ sequencms being such that the coding saequen~ is traaacxibod :under tht "control"
of the control scaqual~. modification of eequen~ ena6ding the particular pmtein of interest may be ~rabla to achieve this end. For eke, in some cases it may be neo~uy to modify the aeqso that it sway be attached to the 1 aequenoes with' the appropriate orimat; or to maigtai~a the reading frame. 'The control aaqand other regulxtoiy aequa~ may be to the coding sequence pr~o~r to inSartion into a vector. Alternatively, the coding sequnu~ can be dire~iy into an acpvector wlwi~ch ady contains the ooatsol seqand an appro~~ria~e r~eadio~ site whip is in reading frame with and udder regulatory ca~ra~l of the oonta~l. soquanc;es.
It may be derivable to pTOdutx au~nta or ana~bga of the proteins of interest. Mutants or aa,alogs may be plod by the dedetio~, of a portion of the sequmcx~ a~coding the pr~aa, by insaof a sequnryoe, a~rdJor by substitution _,._~,..._,~.,~.~,~..........._.....~..~__...-~,........,.",~...,...-,."..,..,~..~__._..___~..-_.,~"~,...""..,~a-".....,~..~. ....,...~.,...~
,~.....~. ..

~~""'""~ 93/12756 of one or more nucleotides within the sequence. The mutation can be one that affects secretion of a normally secreted protein, so as to eliminate or decrease systemic side effects of the protein, for example, tumor necrosis factor.
Techniques for modifying nucleotide sequences, such as site-directed mutagenesis, are well known to those skilled in the art. ~, gtg,,, Sambrook et al. , infra;
DNA
Cloning, Vols. I and II, supra; Nucleic Acid Hybridization, infra.
If the gene sequence of the desired protein is not known, it can be obtained using the following general techniques. The desired protein can be isolated from, for example, tissue samples containing the same. This is generally accomplished by first preparing a crude extract which lacks tissue components and several extraneous proteins. The desired proteins can then be further purified i.e.
by column chromatography, HPLC, immunoabsorbent techniques or other conventional methods well known in the art. Purification of the protein permits the sequencing of the same by any of the various methods known to those skilled in the art. For example, the amino acid sequences of the subject proteins can be determined from the purified proteins by rep~itive cycles of Edman degradation, followed by amino acid analysis by HPLC. Other methods of amino acid sequencing are also known in the art.
Once the amino acid sequences are determined, oligonucleotide probes which contain the colons for a portion of the determined amino acid sequences can be prepared and used to screen DNA libraries for genes encoding the subject proteins. The basic strategies for preparing oligonucleotide probes and DNA libraries, as well as their screening by nucleic acid hybridization, are well known to those of ordinary skill in the art. ~, g,g" DNA Cloning: Vol. I, supra; Nucleic Acid Hybridisation, supra; Oligonucleotlde Syethesis, supra;
Sambrook, et al. , supra. First, a DNA library is prepared. The library can consist of a genomic DNA library from the species of choice. Once the library is constructeti,~ oligonucleotides to probe the library are preparod and used to isolate the gene encoding the desired protein. The oligonucleotides are synthesized by any appropriate method. The particular nucleotide sequences selected are chosen so as to correspond to the colons encoding a known amino acid sequence from the desired protein. Since the genetic code is degenerate, it will often be necessary to ''~~,~~~~ 24 synttaxcize several oligonucleotides to cover sill, or a reasle number, of the possible nucleotide soquams which en<aode a paarticulat reg~pn of the protein.
Thus, it is gay p~efe~od in selecting a ncgiOn upon wb~Ch to base the probes, that the rqgion not aosttain aaasino acids whose are highly dente.
In ce~in ciroums, one a~ skill in tt~ alt pray find it desirable to Prepare probes th$t Sara fairly leap; andAor !a~comP~aLs roans of the amino acid sequence which would,, .a high dagnee of noctundancy in corresponding nucleic acid saqt~nces, pusrtic~arly if this k~thy aniWa~ redundant re~oo is highly chara~ct~ristic of the protean of interest. It tna~ also be dess~ble to use two probes (or sets of probes), ea~Ch to ~t regions ~ du; gone, in : a single hybtion experiment. Aut~amatt9id ~lig~onecleoti~dc syntbaxs has made ~e preparation of large of prab~ relatively straig,6t-rfo. mils ld~e act length of the probe e4iployod is not critical, gea~lly it is iced hn ~e art that pzobes from about 14 to about 20 base pairs are usuahy effective. ~~gc~r probes of about 25 to about 6i0 vase pours arc also used.
The d ude pnpbes are labeled with a naarlrer, such as a radyionucleotide o~ biptin using standard pssocedunes. Tll~e L~bedecl s~
of probes is then used in the awning step, whis~h sot of ~g the aingle-strandod probe to hyb~diac to isolatmd ssl7hNA fmtn thcC library, a~ocording to standard techniques. $ither stringent car dive hybrLdi~tiaw conditions could be ap~apriate, depending upon several , ash as the l~gth of the probe and wthe probe is derived finmn the aa~e >pecid as tie liubrary, or an evolutia~ary close or t . The atiOn of the aropriate cssnditions is v~ithi~nw the skill of the art. ~, , c~aic ,Acid F~br&~at~i~un, supra.
The basic roquirornent is that hybridisation oG~d~itua~ be of cent stringency so that seductive hybridization occurs; i.e., hyl~rydizati~on is due bo a scifficient degree of nucleic add horna~r (e.g., at 1~t ab~oqut ~~ %), as vppos~d to nonspecWc binding. Chnce a clone frown die ~d l~rary, has bean identified by pos~re hybnidiz~on, it can be , by ~s~ analysis and DNA soqu~aing that ire p~ ~a ~e~ for the desired protein. The desirOd »NA aequdsce ts~n be e.la~ed intp~ a cloning vector and further usmd, as d~cr~od berlow.

64157=467"
Pr~a~,~on ~ Lip,~d carriers Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations. However, cationic lipid carriers are particularly preferred because a tight charge complex can be formed between the cationic lipid carrier and the polyanionic nucleic acid, resulting in a lipid carrier-nucleic acid complex which will withstand both the forces of nebulization and the environment within the lung airways and be capable of transfecting lung cells after the aerosolized DNA:lipid carrier complex has been deposited in the lung. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Felgner, et al. , Proc.
Narl. Acad. Sci. USA ( 198" $4:7413-7416); mRNA (Malone, et al. , Proc. Natl.
Acad. Sci. USA ( 1989) $x:607?-6081); and purified transcription factors (Debs, er al., .l. Biol. Chem. (1990) x:10189-10192), in functional form.
Lipid carriers can be prepared from a variety of cationic lipids, including DOTAP, DOTIViA, DDAB, L-PE, and the like. Lipid carriers containing a cationic lipid, such as {N(1-2-3-dioleyloxy) propyl}-N,N,N-triethylammonium} (DOTMA), dimethyl dioctadecyl ammonium bromide (DRAB), or 1, 2-dioleoyloxy-3-(trimethylammonio) propane (DOTAP) or lysinylphosphatidylethanolamine (L-PE) and a second lipid, such as dioleoylphosphatidylethanolamine (DOPE) or cholesterol (Chop , are of particular interest. DOTMA synthesis is described in Felgner, et al., Proc. Nat. Acad. Sciences, (USA) (1987) 84:7413-7417. DOTAP synthesis is described in Stamatatos, et al., Biochemistry (1988) 27:3917. DOZ'MA:DOPE lipid carriers in the form of liposomes can b~ purchased from, for example, BItL. DOTAP:DOPE
liposomes can be purchased from Boehringer Mannheim. Cholesterol and DDAB
are commercially available from Sigma Corporation. DOPE is commercially available from Avanti Polar Lipids. DDAB:DOPE can be purchased from Promega. Biodegradable lipid carriers are of particular interest. Cationic lipids wherein the positive change is positioned very close to the lipid bilayer e.g.
DDAB, rather than projecting out from the bilayer and therefore more exposed e.g. ethylphosphatidylethanolamine (E-PC), are also of interest.

WO 93/12756 ~ PGT/US92/l ltd,, Cationic lipid canier:DNA complexes are intexnalized by cells by a classical receptor-mediated endocytosis using cell surface rectors which contain specific binding sites for, and are able to intetize, cetioni~ molecules.
Using agents such as cy~nlacras, growth , other sQlub~e pro6elhs and ceartain drugs, it is thus possible to seloctively up at down regu>ate these coon-binding receptors. The rate of up or down regulatan of these rDrs by the appropriate agent will allow selection of specific cells for mrharsrad or raduoed levels of trapsfection in view. Thus, t~ ux of spedfic conic hpirls can confer specific advantages far in vivw delivery. For eaamp~le, iv injection of DOTAP-captaining or ethylphosphatidyhcholine (&PC) lipid csu~s can target tltansgene e~cpression primarily to the lung std may offer irrareaaod y~lwantagas for lived delivery.
Furthermore, E-PC and DOr~AP, as well as I~-FE send ~EBt~, are fully met by calls, wDOT~lA gnat be ful~l.~ metabolized by cells.
Thesefare, DOTAF and L-PE, but pot DO'Tl, are su~ttable~ for repeated administration to mamtmajian hosts. Additionglly, c~ont~ the cationic lipid with a sa~copd lipid, ~y either cholesterol ~-30 mole Wit, preferably 33-50 mole pm~nt) or DOPE am maximise ~e ~n in viva. For e~rampk, miring choieste~l instead of DOFE with D4TAP, DOT~tA,, or DDAB
may subtly in~cr~due transgene expmcar in viva.
Particuhr cells within the lung ~r be la~tgby modifying the lipid carriers to direct Ihem to particular typea~of cells ~lsiAg .~t~g mad. Thus aptibodiea e~ ligands for pa~icvlar ~ may be employed, to target a cell associated wide a particular pram. A particular ligand or antibody may be oonju~ated to the lipiid canter in ac~~or~danco with conventional ways, asthar by cx~njugatin~g the sito-dira~irg beadle tp a laid for i~acorporation into the lipid bilayer or by pe~ovi~ling for a lung gtr on g lied pent in the bilayer fig linlung to a fu~ipoality of the aitawdieoctioapmd. Such toctnaiquma are well known tn those alcflled ip dze art. For ea~mple, ligand-directed DNA-polyc~iop t~omple~ce~ have been shown t4 >pfect hcpatocytes in the lives after iv injection. MoK peoc~e ~ iutrs~u1mo~ry tstrag may be achi~ev~d by a) alteci~g ~aa~o~d path size to~ preafly dirxt the aerosol to alveoli or proximal distal airways or (b~ to cowal~tly couple mionoclonal ,'""~"~' 93/12756 2 ~,~? ~ ~ ~ ~ PCT/US92/11008 antibodies to the lipid carrier surface, thereby targeting lung cells expressing the corresponding cell surface antigen or using specific cationic lipids that have specific affinities for specific lung cell types.
Non-cationic liposomes, particularly pH sensitive liposomes, offer another potentially attractive approach to ire vivw gee therapy. However, as compared to cationic liposomes, pH sensitive liposomes are less efficient in capturing DNA and delivering DNA intracellularly. Anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidylethanolamine, diolooyl~osphatidylcholine (DOPC), dioleoylphoshatidylethanolamine (DOPE), among others. These materials can also be mined with the DC7TMA and DOTAP
starting materials in appropriate ratios. Methods for malting liposomes using these materials are well lrnown in the art.
Unexpectedly, the lipid composition of the lipid carriers used for nebulization can dramatically affect the level of transgene expression produced in vivo. Thus, the liposomal lipid compositions generally have a composition of SOW molar ratio of cationic lipid to non-cationic lipid, but may range from 5 ~ to 100W. The diameter of the lipid carriers should generally be within the range of 2D _ 100 nm to 10 microns, preferably 100 nm to 500 nm. Other DNA sequences, such as adenovirus VA genes can be included in the lipid carrier-DNA complex formulation or be co-transfect~ed with the gene of interest. The presence of genes coding for the adenovirus VA gee product may significantly enhance the translation of mRNA transcribed from the plasmid.
The lipid carriers can comprise multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), or large w~ilamellar vesicles (LUVs). MLV
and LUV are preparod by vortexing rather than sonicating after addition of the aqueous material to the dry lipid film. If desired, the resulting lipid carriers can be extruded under high pressure through sized polycarbonate membranes to achieve more uniform size distributions. ' SUVs are prepared by extended sonication of MLVs to produce a homogeneous population of unilamellar lipid carvers. Commonly used methods for mating lipid carriers include Ca2+-EDTA

' 64157-467 chelation (Papahadjopoulos, et al., Biochim. Biophys. Acta (1975) X4:483;
Wilson, et al. , Cell (1979) X7:77); ether injection (Deamer, D. and Bangham, A. , Biochim. Biophys. Acta (1976) x:629; Ostro, et al., Biochem. Biophys. Res.
Commun. ( 1977) 7~: 836; Fraley, et al. , Proc. Natl. Acad. Sci. USA ( 1979) 7:3348); detergent dialysis ('F.noch, H. and Strittmatter, P., Proc. Nail.
Acad.
Sci. USA (1979) x:145) and reverse-phase evaporation (REV) (Fraley, et al., J.
Biol. Chem. (1980) 2,5:10431; Szoka, F. and Papahadjopoulos, D., Proc. Narl.
Acad. Sci. USA (1978) x:145; Schaefer-Bidder, et al., Science (1982) 15:166).
The various lipid carrier-nucleic acid completes wherein the lipid carrier is a liposome are prepared using methods well known in the art. ~, ~, Straubinger et al. , in Methods of Immt,~nology (1983), Vol. 101, pp. 512-527.
By "lipid carrier-nucleic acid complex" is meant a nucleic acid sequence as described above, generally bound to the surface of a lipid carrier preparation. The lipid carrier preparation can also include other substances, such as enzymes necessary for integration, transcription and translation, cofactors, etc. Furthermore, the lipid carrier-nucleic acid complex can include targeting agents to deliver the complex to particular cell or tissue types.
To prepare the complexes, generally, the nucleic acid material is added to a suspension of preformed MLVs or SUVs only after the lipid carriers have been prepared and then vortezed. When using lipid carriers containing cationic lipids, the dried lipid film is resuspeaded in an appropriate mixing solution such as sterile water or an isotonic buffer solution such as l OmM Tris/NaC I , or S S6 dextrose in sterile water, sonicated, and then the preformed lipid carriers are mixed directly with the DNA. The lipid ,carrier and DNA form a very stable complex due to binding of the negadlkely charged DNA to the cationic lipid carriers. SUVs find use with small nucleic acid fragments as well as large regions of DNA ( z 250 In preparing the lipid carrier-nucleic acid complexes, care should be taken to exclude any compounds from the mixing solution which may promote the formation of aggregates of the lipid carrier-nucleic acid completes. Large particles generally will not be aerosolized by the nebulizer and even if aerosolized would be too large to penetrate beyond the large airways. Aggregation of the lipid carrier-nucleic acid complex is prevented by controlling the ratio of DNA to lipid "''"w 93/12756 PCT/US92/11008 2921~~.A3 carrier, minimizing the overall ooaoaitratLOn of DNA:lipid ~mgleac in solution, usually leas than 5 mg DNAJ8 ml sswlution, xnd the avoiding cheiating agents as EDTA, and iigai~t am~o~mts of salt which tend ~o p~a~ote macroag~egatia~. The pr~tad earGipi~t is water, elwater or another solution having low or no ionic strazgth. Further, the volute must be adjusted to the miaimum for deposition in the lungs of the host mammal, but taking care not to maloe the solution too conaeatratod so that age ford.
Tlzc chaitx~ of lipid carti~xs anl~ the tta~lon of lipid carrier-nucleic acid completes thus involvas a two slid p~oc~t. That firs is to identify lipid carniers and coooeatradon of lipyd c~rnuclaic acid complexes that do not when the cx~or~ats arc ~nbined art dur~pg tire s~nifiwrant agitation of the mut<u~e first occurs during tha nd~uliastion sly. The second step is to identify among first ate ideatified as of uat~rtha first step (i.e. do not agg~ata) those oomplr~ that pride 8~dr a high lev~eh°of transfection and can of a gate of iat~est in tai cerlix in the lung. '~'he ievel of ea:pression and the cdl typo in rich siou of tire ~recrombi~t g~lne is owed may be determined at the mRNA level amilor at the levtl of p~lypede err . Gene product array be quantitatad by measuring its bbl activity in tissues. For exaanple~ enzymatic activity can be mdahy biola~al ~rsa~y or by identifying the gate product in trsns~eabed odla by imm~ro~tain~' taehpiqrrms such at ping with an antibody which ally mooogrriu~ the gam pro~rrCt or a sported gene praduat grout in the expression .
As an examplie, a reporter germ CAT (which chlorampheaicol acetyl trans~Case) can be inked in the e~r~pt~sion cute and used to evaluate caGh lipid curia aoalr of iata~t. 'dire DNA:lipid cagier compla~es must be misted in which ~ob,not tir~mael~res induce aggregation of the DNA:lipid carries comb such as she water. Tie ~neasicxr cassette (DNA) is minced together with the lipid carry to be tied In m~ip~e different ratios, ranging as an e~campia fiom 4:1 to 1: 1 (mic~o~ama~~NA to nrm~moles cationic lipitn. The remrtts provide inf~mat~dn worr~rning ~Ivhich ratios iesult in aggrega~ian of the DhlA:lipd c~ ~ ~ ~ ~,ful for use in vivo, and which remain in a faxm euitable~for aaoa~oliution.
__..~_..~,......,.".._....,.~...................~...,~."~:..~..........~...."~.
.........___ .~..~"..., ,~,.~,.~,.~.,~~,."~.......~...",~...."~,..""..~..._.-..._ __. _ ._ WO 93/12756 ~~~'~~~ PCT/US92/110(~,,, The ratios which do not result in aggregation are teaded in animal models tn d~mine which of tt~e DNA:lipid carrier rata confer the h~gheat level of tranagane eapresaimon in viv~n. For ale, the o~imal DN~II~:lipid carrier ratios for SUY for D(~T'M~/DOPB, E-PC:c~ho, tthyldhnyarl~tidyl-choline and DDAB: Chol are 1:1 or 1:2.
A~mini,~iQn The matnn~lia~n boat may be any ma~ma~al having symptoms of a geneti~ly-based disorder. Thus, the aub9aCt ~li~tio~ fnuws use in domestic animals, feai stocac, such as bovine, ovine, arid pamciae; as well as primavtes, particularly harnans. I~ the a~cthad of the inv~nti~, a~naattion in viveo is ob~inad by ink a non-ink tbehtpaati~c p~~smic~; into the mammalian host cc~m~leaed to a lipud , pn~tiiavlatly ~ ionic lipid c~ia more particularly, for hum~a use ac for roped ap~rliaationa a biairrrdable lipid carrier. For introduction into ~e ~nalian ~b~t acry phyaidlogirxlly acxeptable madiura may be ea~ployad fcx adminithe DNA or lipid car~cie~~ such as deiani~od water, 5 % deatao~a in water, acrd tl~ iilme. (Xta~ arts ray be included in tl~e fornwLuti~ such as atabiiiae~s~ biocsda,: etc, ~ that they area the criteria outlined above, i.e. do n~ c~tsa aggaegation of the onrrrpiexes.
The variaua components lied above find eartaasi~re eaelnpliion in the literature and need not be dcaaribed in per here.
The lipid curia nucleic act camplea is aao.~iizod by any appropriate rnedrad. Fog use wide humans or o~CC per, the aerosol is gdnerated by a aredical ~ ayatan wit delivan the a~oaol through a m~, f~emask, era. fiom which the n~mmalixs boat ttan draw the aerosol into the lungs. Variaoa neb;rlizas are lmcnrn inn the art and aan be used in the method a~ the prr~at inventim. ,~, for Vie, ia~ tt al., U.S. Patcnt No. 4,26$,~6I); Lebmbeclc, tt off, U.S. Pa~m~ No. 4,,4" U.S. Patent No.
4,Odb,l4b; IIavatad, tt ~, U.S. Pa~tt No. 3w826,255; Koip~t, a o1., U.S.
Paxent No. 4,649,911; Hoadoni, a al., U:S. t No. ~4,51~,829. The selection of a syst~ d~a~a on w;r alvaor or a~i~tway ,detivc~ry (i.e., trachea, primary, :ec:oor~ry c~ tertiary bronco,, arc.), is das well as a"'"""~193/12756 2 ~ ~ ~ ~ ~ PGT/US92/11008 verifying that the specific nebulizer system deer not dasadue or inactivate the nucleic acid construct.
A convaaidnt way to insure effipctive delivery of the nucleic acid to the alveoli is to select a nebolizer which paiodaces mtl~ small particles for example, particles with a mean particle diam~r of less than 5.0 microns (Ecm).
More preferably the psticles have a auaa p~uiticle diametrr ~f about 0.2 to about 4.0 ~cm, and most preferably tlm particle have mean diametdr of abet 0.2 to about 2 ~, ainoe larger ~ ( ~ 5 Vim) ~e g~e~hy d~osite~d in the proximal airways or na~opharynx. As an a to small mean particle diameters to a~iave substantial distalairway arid alveolar tion, a very high dosage of teat lipid carrirsnucleic said prepluratian ;can be administered, with a larger mean pard~ dial. A proviso to such am ~p~oaCh is that the particular lipid camia~~nuQieic acid cx~mp1lex is t~ is knot too irritating at the required dosage and that there be a su~cdrnt number of ~artici~es in the total particle population having a diamt~ in the 0,~ to about 5 ~p range to albw for deposition in the alvoo~.. Far airwax delivery, the mean particle size will be ls~es. Far e~am~e, suitable mmnt,particle dratme'~ts will gay be less than about 15 dun, more preferably fmm about 4 ~ctn, sad most p~refiembly from abaut S ~cm to about 10 ~cm.
~ of irebu~s useful ~pr alveolar delivery include the Acorn 1 nebulizer, aml tht R~espi~rgard Iii NSy~s~n,' bath available commly from Marquest Medical Pmdnc~t, Inc., ~nglcywpod, CO. Other comnally available nebuli~ for use with tire insl~t intioe include the UltraVen ~ nebulizer available from I~atliacl~dt, Inc. (Maryland Htd~ghtg, MO);
the Writg6t nobulizer (Wright, B.M., Laa~ (~9~8) x:24-25~; and the DeVilbiss nebolizer (Mercer a al., Win. Ind. Iiyg. Assoc. ~ (191$) ZQ,~6-7$; T.T.
Mercer, Chest (1981) ~:6 (Sup) $'13-81'n. Nebuliza~ useful for a~ir~vay delivery include those typ~ally used in the tteatrn~t of asthma. Such atbuli~rrs are also comme~ally available. tyne of sicdt in the apt can deta~rmin~ the usefulnss of a particular nebulizer by measuring the mss p~Cticle s3~e gau,~tatad thereby with for example, a 7 gage r ctor (fox Prr'ducts, Albuquerque, NM).
Concxntrations of the lipid carrier nucleic aa~ oomplrac from ~ ~p~~

WO 93/12756 ~~ ~ PCT/US92/1 t00, can be d~e~ennined by eluting the complex dmr~om sad assessing the optical density at an appropriate wavdength and comparing the staadrurd cloves.
Results are generally expressed at mass ~dian aerod~~~aaaic diar ~, geometric standard deviation (Raabe, J. Aeraso~i Sci. (19T1) x:289-303).
The amount of lipid carriers .used will be an arhouat sufficient to provide for adequate transfection of calls after miry of the DhTA or complexes into the lung and to lmovLde foi a therapanic lcvd of transorlpt~n and/or translation in transfectad cells. A critic lewd of tram and/or ion is a sufflciatt amount to treat ar pavlliate a of tlrre hit following adminia~ati~un of the lipid nuelCic aced to the lsoat mammal's lung, particularly the alveoli ctr airway. Thus, an "ova ate" of the aerosolized lipid nucleic acid ps~q~ration, is a dose' atde~at to effect treatment, that is, to cause alleviawtion or neductiaa~ of aympbonte, to inh~it the worsening of symptoms, to prcvr~t tlt~e onset of sympp~o~, ~i due liloe. a dosages of the present rpasiti~ans wiich institute an ~e amow~tt cad be determined in view of this disc~sure my once of ordinary mall in the art by donning routine trials with app~priate controls. Comparison of the rule tc~d~maat groups to the controls will indicate a partic,Yular dce~e is eff~tive ~ preventing or reducing particular symptoms. Appropriate doses are discussed further below.
26 _ While thoFe is no dit~o~ct nm~Od of m~e~uuing the a~i amount of lipid caaier-nucleic acrid complex delivered to the alveoli, ~toQcho~riv~eo~lat' lavage ($AL) can be used to indirxtly ~nalveolar oonc~ts~vns of any e~pressad and secreted protein, usually 1~-24 hrs after i~ to allow cru~oe of the pmtein deposited in the la~ar airways and bro~hi.
The total anwunt of nucaac acid ddivexed to a mammalian host will depend upon many ~ct~s, including the total amount a~amsol~Zed, the type of nebulizer, the particle size, bathing pa~rns of the nutnnun host, severity of lung disease, conceal and nuan of the lipfid nucleic acid complex in the ae~li~d sdution, and largtM of inhalaltion ~e~py. Thus, the amount of Gxpmased pr~tdv mea~u~od in the a~wCOli may be twubstantially less than what wauid.be acpectad to to a~prasad fmm the amot~t of nucleic acid present in the aerosol, since a large portion of t~ oomplea may be exhaled by the subject ""'"""~ 93/12756 PGT/US92/11008 33 ~~~~~~3 or trapped on the interior sof tt~ rbbulizer appauatus. For example, appcoxin~ately one third ~ the lipad carrier-nue~C acid dose that is placed into the nebulizeer remains in the nebuli~ and associalled tubing ainhalation is compl~d. This is true endless of the doses size, dutati~on of inhalation; and type of n~ebulizGr used. hdoreovar, rsion of the residue and readminiatration does not ntiy incnGase the doss dali~od to the subject;
about care third rcn~n~ in the nebulize.r. Fu~th, ever ~ action of airway diepositia~, a portion of the dose is still deposited in ~e airways.
Additionally, efficia~cy of exprn of the arty p~otein,~will vary widely with the exon system used.
Despite the intmscr~g faaOrs deacril'od above one of ordinary skill in the art will be able ~adily to design effective protaools, rticvl~arly if the particle size of the ae~o~ is o~ptim~ed. Hasdcl ~ e:ti~ates of n~ebul~er efficiency, an effective dose detivered usually lies in the range of about 1 mg/ent to about 501 mg/ent, although mare or less may be found to be effective dep~ding on the subject and deed result» ~t i~ genaxally desirable to adminrtster highs daaat why tn~ng merle severe ~oonditi~s. Generally, the nucleic acid is not integralted into the host cell genome, thus df necessary, the treatment can be repeated ~ ~ ~ hoc ba$is deeding upod the results achieved.
If the traabnent is repeated, xhe mama~aliaa hdst is ~itoc~e~ll to assure that there is no adverse immune rrspoose to the tttatt. The icy of treatments upon a number of factors, mch as that amamt of lipid caaiec nurdeic acid eompla~c administered per dose, as well as the-health sad hiuory of the subject.
As used 'hey, with oCfaCmce to , "lipid csiniiet-nucc acid aeaoaol"
refers to the amount of lipid c~rriez-nucleic said oomglex that is plalced in the nebulizer and subjected to aerosolization. The "an'oont nebt~iized" of "amount aemsotiaod" of the co~np~ mesas the amouMt that actually 'leaves the apparatus as an aerosol, i.e., the antou~t placed into the aatna less tl~ amount ret~ned in the reservoir and on the itmar of the ~ at thwe conclusion of a trea~nent session.
In partiaulaar treatment ray for ale, in the treatment of cancer, it may be nooe~sa~ry to admini~r tial doses at. intervals ranging WO 93/12756 ~ ''~~,~~~~~ PGT/US92/110.~

from every 8 to 12 hours to once a month. Tire frequency of treatzneat can be monitored by evatuatirrg whether there has barn until significant amelioration or complete disapgaaa~raanoa of tire c~nca with a pparticuia~r treat~t .
Treatmeart may have to be aradified if c~o-~g host tooti~ity develops.
Similar a~dministratia~r protocols also may be dievrelop~d in fad example, patients where all macmsoopic evid~ee of tumor lws bears raaav~ad, in order to prevent tumor recurrent due to peraistmoc of undeta~lbed miacrc~i To trees pulmonary infec~ooa std a' brcuichis anal pneumonia, it will usually be na~saty bo atlminiatsr at laid oae dense per day aver a period of about 4 to about 21 consecutive days or lmrger. The t is visually carried out on consecutive days because sow assess of lungs open up to paretration and depositiar of the nucleic xid with itrcxeasiug luti~an: of thwe iefec~ion. The success of the treatment c~~n be monitored and ~e ad~aamnstra~ion ~lte~red by ass~in~g convartional clinical crit~eri~a; o:g, chuicrg of r~rdiogtrc infiltrate, improvod arterial PO~ (e.g., >?0 m, ~ in ~dysp, re~i~ratory rate aadlor foyer. For the treatEn~ent of gaic di~rd~, au~ch a~~ cystic fibrosis, the lipid cannier-mreleic acrid ao~rlea will be ad~n~i:at flat ial~als, from once a week to once ewer one to sevc~l , in ardex t~ pnavide wild-type GFl'R p~ot~ein in critical host airway caeus, aide these cells cloatinue to turn over.
It may also be paasib~le to stsrbly the f~;PTR gate ino~l appiate .lung stem cells, which would tbeer prawide a cons of ~~nal airway cells with~t ~oquiring lifirdong tc~et. P~ntia~ tic of the gene pmduct can be rnea~rud, by ding the acts of gare;,~c~easion on survival of tcansge~uc host mautamala its which ~tlre tre~ue iult expirss~ed.
Production of significant amounts of a t~nsgdse peoduct wild:, ubstaat~lly prolong the survival of the afi'>ld host.
Where aeon of the polyp~i~ielprottin o>' eve the mItNA
itsetf confers a fed biocissaioai ptyp~p upon tb~ hosts the pre~oe of a new plres~type or of an old pb~wtyp~ ~y ~ ; ~r ple, as a result of transformation of the host cells, there may bit hoed production of pr~cistisrg des~abl~e pr~odtx~ far~ly prv~duced in inert quantities or there may be lion or ~evron auppnan ~ an ~r~a~irab~e 8ena pmduct using '"~"""~ 93/1275b ~ ~ ~ ~ ~ ~ PCT/US92/11008 antisense, ribozyme or co-suppression technologies; in the case of reduction or suppression, a reduction or elimination of the: gene product may be determined.
The potential toucity of the tra~ent may be evaluated by behavioral manifs, and where approprliate, by aaalysit of biopsy specimens.
5 Thus, behavioral a~ctivi~ty which dis~as, such as changes in activity level, changes in eating and drinlong pollens and the like, can be monitored, as well as esvi~noe of ne~is, edam or inflarn~matian in biopsy specimens.
The subject oornpositions can be pmaovided for use in o~ or more procedures. Kits will usually include the DNA either as nahOd DNA or 10 compleaed to lipid catyrias. Additionally, lipid auri~ars ma~~ be pmovided in a separate ccmtainer for oompleaing with the provided DNA. The DNA or the lipid carrier/DNA oompleaas may be present as oonoa~trates whip may be further diluted prior to use or they may be provided # the conoon of use, where the vials may include ~e or more dosages. Contly, singe dosages may be 15 provided in sterilized oontaicxus suitable for use with a nebur, so that the physician or veterinanibn may employ the diners directly with a nebulizer, where the containers will have the desired amount and c~o~ntraticm of agents.
Thus, the kit may have a plurality of contains c~taining tie DNA or the DNA/lipid crier oos~tpl~ in appr~oprriave psdpcrrtiohal ar~unts, and optionally, 20 appropriate diluent and arid solutions. When die cont&in~rs contain the formulation for direct use, usually drone will be no need farr othea re~ge~nts for use with the method.
25 Uses of the subject invention i~elmde but are riot limited to the following. The pre~t invention is particula~y useful for the delivery of substances directly into the lung for the prevetrtion aadlor tit of pulmonary disorders such as lung cue, emphysema, asthma, lung ia~Ctions such as chronic bronchitis and prreumaniA, degmaative dish of the lung, as well as gaietic 30 disorders such as cystic flbrdns sad a-1 ~amitrypsin de~ci~y.
For the treatarent of lung t<mnaics, gores eacoding bm~ic peptides (for example; thaapc~tic agents ash as ricin, dip~tlurria t~ouin acrd cobra venom ~_~.~_._..,...T.,._ .,m..~_........~.~....~..
........".~_~......".......~....~.___ ....~..........."."....~:,..-~......",..e...~,....,.,....,..a_.........._....~.___.__ _.._. _ _ WO 93/ 12756 ~~ ~ ~ ~ ~ PCT/US92/ 1100,,,, factor), wild-type tumor suppressor genes (such as p53, genGS decoding for mRNA
sequences which are antis~e to transforming aoc~ge~es, or aver antineoplastic peptides, such as tumor meiosis factor ~ sod other aytokines, or ttansdominant negative mutants of transforming onooganals), can be insecrted into the nucleic acid constnyct and using the above gibed ~net~c~ds, complexed to a lipid carrier and delivered for acpuo~n at or oear the tumor ~tite. Some tamers such as coon tumors can be spocifr~ly mrg~ed by mcorporald~g targeting ag~ts, such as antibodies directmd against tumor cell sua~face antigens, onto the lipid carrier. ~, e.g., Laserm~n, et al., Nature (19~U) x$$:402-6~4; HuanB, et al., Biochemistry (1981) ~:4~299-4238, f~ m~hOds of inccrtporati~g antibodies onto liposomal surfaces. Sinrilatly, Bares coding fox g~tide: to display antiviral amdJor antiba~ct~rial activity, or s4imulalae the host' im~nuae system, can also be administered to the lung in order to ~It pulm~r ind~ecti~s. Thus, the genes awading many of the various cym(~ functi~al fits thereof), such as the intcrlaaldna, in~r~Ons, aml c~olQaY stunulatipg fal~tors, will find use with the instant invention. The gene sequences ~ for a number of these substances are known. Interferon-~ prods szpt aeii-pneum~pcyst~e carinii pua~~nonial (PCp) aeti~vity in immvmodt mice with PQ" following altrosol delivery of the pe~ide. Heck et al., 1. surd Irrrmtrn. (1991 59:3859~38a6a.
Oe~ ~di~ng antiots will ~o find use fir the treatnu~t or prevention of lung daac~ge due to dati!~~e g disoaders paused by sm~king and other environmental agents. For example, gates arrandi~ng superoxide dismutase (SOD) or catalase, as well as a-1 antitrypsin, will be particularly useful for this purpose. 'These gene sequ~ces are known. See, e.g., Long et al., Biochem, (1984) x:4828-4837 for the a-1 anti~ypsin gene sequence. For the treatment of genetic dieotdors, such as cystic fi~aeis a~ad ernp~Ysema, functional genes, corresponding to g~ lanawn to be ant in the pat~kicular disorder, can be administered to t~ subject. For ~, i~ is laaown; that ~dividuals lacking suffiaent xwels of a-1 antitcypain are prone to ysanta and other pulmonary disorders. Thus, this gene tin be admi~ti:l~ed~prvphyly, as well as in response to clinical maniac of the disdt~, for both the prcventian and/or tteaonent of this di:orda. Similarly, the gene ~ttvolvad inn cynic fibrosis has been ...."-,....,..,..,."~.....~~..~.,...-.a,~......,~~...~...,.m...,..,..~~.,~....~..~..~..~...:. ..,.. _ .-identified. Goodfellow, P.,111otvre (1989) ~:lOEl-103; Romtnens, et al., Science ( 1989) x:1039-1054; H~dsley, et al. , Sci. Win. ( 1989) x:28-30. Thus, this gene, or fragments which e~u~de a biologically carve ion product, can be delivered to a mammalian host suffering from ll~i~s disa~der.
"The inva~tion also finds use for the ddivexy of substances into the systematic circulation via the lung. For example, as e~lained above, a number of substances, such as cytokines, are toxic when administered using conventional methods of delivery. Sae, e.g., Debs et el., J.'hr~urrw~ol (1980 ~Q:3482-3488.
The invention allows tlret d~livay of these Suba~nees fag able, ~ treat cancer, as well as bacterial and viral infections, syst~tlly. This approach already has shown promise for tlur trot of extra-pnim~nary c~~r in' humanss.
The insta~it 4~h~Ods aho find use in antiaense y, for the delivery of olig~uclable to hybridise to ~ ~mp~ry scqu~ca, thereby inhibiting the transai~On atxllor train of these sequ. Thus, DNA or RNA coding for prittans ney for the progress of a particul9ur disease, can be targeted, tha~eby dpting the di8ease process.
For a review of antisense and aliga~ucleotid~a useful in the llama, ~, TJhlmann, E. and Peyman, A., (~tdrt: lttv. (1990) ~Q:54~-584.
The following eaainples are provi~dad far illustrative purposes only and are riot inte~ad to limit the scope of the ~t imeation.
The practice of the prra~nt invedtion employs unless otherwise indicated, conventional yechniqu~es of cell cnltw~e, moleatlar biology, microbiology, reao~mbi~nt DNA, and immuno~gy, wbidh arl~ within the skill of the art. Such txh~qudv ate e~plaiead fully in, the literature. ,$~, ~,g"
Sambrook, et al. , Moledular Cloning: A Lai~a~l~oocy Manual, $eao~d Bdit (1989) Vats. 1-3; DNA Toning {1985) Vois. 1 and II, D.N. t~rlovnt (al.);
Nucleic Acid Hybridization (1984), B.D. Hames, et el. , (~ds. ); Perbal~, B. , A
Practical Guide to Molecular Clod (1984); Methods it Hazymwdogy .(the aeries), Academic Puss, Inc.; '~a~s: A Survey of lldblea~lar Clog Vectors and Their _,_._.._~..~...........,....,..............-..~.,."....~..-..,..~.~..~"..-.:.~"..~~,~.,.,......-...~..___ .~..",~"",w,~~"" ~~",.~",",~,~".~~, _._... .

WO 93/ 12756 ~ '~~, ~ ~ ~ PGT/US92/ 110 Uses (198'x, R.L. Rodriguez, et o1., (eds.), Hutterwotths; and Miller, J.H., et al., Expwiments in Molacular Cieoetics (1972) Call Spring ~r Laboratory.
Up to 48 mica may be plod ie the ehan~bar ~t one time. The amount of the total amaumt of DNA-lied oompl~c pled in the nebulizer that is dalive~nod to tlx lungs of each n~a~e is app~imatedy 0.02 % .
Thd lipid c~i~s usod were plmd pRSV-CST, as d~eecribed by Gorman, et o1., FrvG. 1V4~~1. Aid. Sci. USA (9$~2) ~:6'7?7-6781; and Juang, and Corman, Marl. Gdl. R'ipl. (1990) ,IQ:1805-181~i~ pallid containing the CAT
gene dri~ea by the RSV leog tamiuat report; ~i pl~lid P~SV-~'ga~, as described by Hazinah ~ sl., Me. J. R~espir. G~"tl~' 11,~v~T $i~o~l. :(1991) 4:206-209.
The p&SV-CAT pls~aa~ud wan odd to lid sad iatered to 25 grad a BALB/c ask as faliaws. Two mg of pRSV-CAT
was miu~~cl with 4 ~cmoies of (CiI8C~1 BRL, C~a~rd Tsluui, NS~Icholesteml (2:1) sma~tl wt~ lipoeos is p~hospbe bind saline and then nabttlized in an Acorn i (l~arq~st dal P~ducts, Inc., Inglewood, CO) to groups of rata ac mike in lnt~ ntrsa~ily ire chamber (Intox Products, Albuquerque, NM). The sadte procedure was followed with 0.5 mg pRSV-CAT mined with 1.0 1 DOTMA-cholesterol (2:1), as well as 2.0 mg pRSV-CAT abna. 1'wo to five days later, amimal~ !ur~aaacrificed and lungs collected Lungs were alto collected fi~ unod co~trols~. The luegs were hornogd and ells di~ptOd with three fio-ibaw cycle. CAT activity in aliquots from the lung Iuctracts was mauled ps~g a assay as described by Wolff, tt e1., Sceen~t (1990) ?r4Z:1465-141. As can be ~a in Figure 1, animals ~rdmiaistened 2.0 mg RSV-CAT w~h ~.0 ~cmol DOT~tA~lcholesterol (2:1) eacpres~od the CAT profit in the lusrgs while ~hc cabal arias wen as animals uoceiving aelipued RSV-CAT DNA alb, o~ lawvlsr doses of R8V-CAT:DO~1'MA:chol ao~pnpla~ut did not. A sir pcoo4dure was followed with respect to pRSV ~-gal, with the ~tion that 50 mg of pRSV-~B-gal was minced -~''"'~193/12756 with 50 ~cmoles of DOTMA/choles~ol (2:1). The pte:emx of ~B-gal activity was detamin~ed using a staadard 6istoci~emical staining praaodure. ~S-gal activity was present in the airway epitlu;lisl calls of exposed rats.
Also teslied was a plasmid containing the CAT .gene driven by the CMV promoter. This plasmid was made as gibed in Hutng, M.T.F. and German, C.M. Nuc. Acids Res. (1990) 1$:937-947, with the exception that a CMV promoter and a hybrid intron sequence were used rather than the SV40 promoter in the plasmid p~IL.I. CAT, desca~b~ld thm~n. Briefly, the CAT lipid carrier was constructed by first malci~ag a pML-b~tsad pl~nid containing the CMV
promoter immediately follbvved by a portion c~ the 5'-un~ leader from the adeawiras-major late (~Al~.) region. This region coatainad all but the first nucleotides of the first exon of the tripartite larder plows a potion of an intervening sequence (IVS) from the AML region. A c a~onu~a~eotide was inserted which merged with the adenovirus intro to peavidC a furaal splice acceptor sequence derived from an Iga variable region. Bothw~l, tt dl., Cell (1981) x:625-637. This plasmid was then c.ut at tvws r~ic~i~ sibs bordering the intmn (G'IoT and Psti) to reaarrre a 292 by fng~t. A matching s~nthatic oligonucleotide linker was inserted. The pied was tee pCIS-CAT.
To test for ex~s~ion of the CAT gene using ~CIS-CAT, 12 mg pCIS-CAT was minced with 24 of DO'~"~'MA/D~PE (1:1). Female ICR
mice were placed in three different aerosol rac~ving chambats. All mice received the same amount of the CAT expression d oanq~1~exed ~0 liposomes, as described above. Animals 1-3 were abed b the aesosoi i~ an intox designed aerosol camber. 'Aiu~mals 4-7 were apoaed b the aerosol ~ a modified rat cage containing dividers for individual mice. Awiasals 8-10 were plaood in a smaller, similarly modified mouse cage after being put fn the rr~ltr~n~rs used in the Intox chamber. 48 hours following aa~o~a~lization, !he aaiaa~s wave seerific:od and whole lungs assayed far CAT expmeasion using the chaomato~;raphi~c CAT assay.
As can be seen in Figure Z, a single aerosol dose of a CAT ~ayc-a~prcssion plasmid complexed to lipoammes can produce high-lyres transgene acpressian in the lunge of mice. S'rgmf' res<tt Ll~veis of trnnagwne expression are pre9ent in the lunge of all 7 mice (numbers 1-3 ~d 8-10) which were to ._.._.._._...."".,~.,_.-...~,-,.,~..-...--"-~"~., ..a.
".~.,..,...~.,_,....~...,..~_.. ,..,~"."",~,"".~"~, ~..~~~".,~,.,.,..,.~.._.~.._ _ _ _ _ .

WO 93/ 12756 '~~, ~ ~ ~ ~ PCT/US92/ 11 the aerosol mist in Intout noSe~anly expaeure tubes which were constivcted to maximize the amount of aeroaol that the mica . The amount of variation seen here is comparable to that soap is othex eapain~aits and may have several ~cp~lanations, including variatia~n~s is ~ ~ the aerosol mist, 5 indi~ridua~l vatiatio~ns in ecy of nasal frl, etc.
Two nronxh old, fdnale, ICR n~Ce ~e~e used in a'~ a~peri,ments.
,p"pj~s~ The p~iaol a~lt~utsferase (CAT) gene was used as a repo~r to measure ~d~sion reveals (Cancan et al., Pros. Nat'1 Aaad Sd (USAF (1982) 79: 67'7-6781). ThC p>asmid used omrtains the CAT greae fwed to the human cy~megalaninis ~ irate early pcamota~r-en~r~cac eie~tt (pCIS-CA'T~. T~ pla6mid eves purred using alkaline lySiS and amtneaium pta~on (Satabrocrla et al. (1989) ,, and the nucleic xid c~no~n~asm~nd by U'V abserptiat at 260 nm. The CAT gene is not presaot in d~iraryotic aells. ~s prod<r~ct is at1 a~zyme which cataiyus the transfer of acetyl groups frmn a~o~tylCoA to the aubsttate chla~pheaicol.
illl~Ls~~i~l. R'«e pea small unilaradlar v(ap~OUa~dy 1t14 nm in ) containing the cationic lipid DMA as D0~'~I:DiOPB (1:1 ara~e r~io). is (N[1 2,3-dioleyloc~y~rOpyl]-N,N,i~.triethylamamnium ~Bym~ Carpor~rptiom); and DOPE is the neutral. lipid dioLdaylplaspi~akld~rlathanda(Ava~tti Pear Lipids). Stock sol~ions of the lipids were diasalved in chbr~orm and st~orag under argon at -20°C. Lipids ware mid in a mund-botto~r~ed flask and eyapcnated to dryness on a rotary evaporator under zed~uoo~d per. Ip~oulble-distillac~ was added to produce ~anal lipid oooa~Ira~wons of 1eet~h, and tire ra~llting mix was sonicated for apptrraicn~r CIO minutes in a bad soniealor (I~~bo2atory Supplies, Hicksvyll~e, Nl~. The ~paawere Stored atalear argcam at ~t°C unttl use.

41 ~~~~~03 Twelve mg of plasmid aomplexed to 24 ~cmols of D01'ASA:IhOPE (1:1 mole ratio) liposomes was aerosolized and administered to mice over two diffearmt araosol pm7iods on the same day. In order to present aggregation anal preci~ of the oppositely charged components, the plasmid and tt~e lips were dilated separately in sterile water prior to mixing. Six mg of plastid DNA end 1~ ~cmols of D01'MA:DOPE (1:1 mole ratio) liposomes were each dfluted~ to 8 ml with water and mixaed. Four ml was then placed into each of two Aoo~' I nebulizers (Marquest, Englewood, CO), the animals phradd into an Intox small animal exposure chamber (Albuquerque, NI1~, and an air flow rate crf 4 L min'' used to ges~erate the ae~ol. Appro~cimately 90 minutes wme requdnld to aerosolize 4 ml.
The animals were remo~rod from the chamber for 1-2 hours arid then the above procedure was rated with a second 4 ml dose.
Organs were dissected from animals sacrificed in a C02 cbatnbier at periods from 1 to 21 days fol~Owing aaosdlization, washed in cold phosphate buffed saline (PBS, and homogaaized using a hand-held tissue homogaiizer in 230 mM Tris-HCl, pH. 7.5, casi~tining 5 mM EDTA
for lungs and spleen and 250 mM Tris-HCl, pH 7.5, containing 5 mM EDTA
plus the protease inhibitors aprotinin, E-64, arnd leupeptic (B~ehringer Maimheim) for liver, heart and kidneys. The inhibitors pucveat dagtadat~on of acetylaEed chloramphenicol species gauatatd during the assay, dyareby ~Irllowing optimal detecti~ of CAT expression.
Following homogaiization of the tissue, cells were lysed by three freeze/thaw cycles, the lyaat heated (65°C for 10 minutes), find centrifuga~ed (16,000 x g, 2 minutes). The protean ocmoentd~tions of the etltttaets were measured using a Coomasste bloc-baaod assay (Hio-Rad). Protein conolsnt<ations were normalized and a volume of extract added to 1~ ~cl of iA0 ml~t acetylCoA
(Sigma), 0.3 ~cCi of [14C]-labelled chlorampha~ic:ol (Anam), and ~'tstilled water to a final volume of 180 gel, and allowed to react ~! 39°C for 8-1~ hours (Gorman et al. (1982) . Following the reaction, the ac~ylaoGed and tinaettylated chlorampheniool spedes were attracted with algid ethyl , spotted on silica TLC plats, and developod with a ehlorofarm:(9S:~v/v) solv~t. The TLC plates were ezpbsed to photographic film (Kodak X-GMAT) * for one to three days, then evaluated by visual inspection.
Preraration of Genomic DNA and Southern Hybridization. Immediately following aerosolization, mice were sacrificed and their lungs removed.
Genomic DNA was isolated and analyzed by Southern hybridization (Sambrook et al. ( 1989) using a ~Iybor.~d N+membrane (Amersham). A CAT probe was prepared from a 1.6 kb fragmEmt of the CAT gene labelled with a-[32P]dATP by random priming, which yielded a probe with an approximate specific activity of 2 z lO9dpm/~cg. After hybridization, the membrane was washed three times in 2zSSC, 0.1 % SDS at 65 ° C for 20 minutes and exposed to film for 24 hours. In order to determine the approximate transfected CAT gene copy number, blots were also hybridized with a 1.1 kb BSU 36-1 single copy probe from a mouse factor VUI-A
genomic clone (L,evir.~son et al., Genomics (1992) 13: 862-$65). Relative amounts of the CAT plasmid deposited in individual mouse lungs were quantitated by phosphorimagining analysis using a Molecular dynamics 400A phosphorimaginer ~' (Johnson et al., Elecsmphorexis (1990) 11: 355-360). The amount of retained probe in each lane following hybridization with the CAT probe was normalized to the amount of DNA loaded per lane using the counts measured after hybridization with a Factor VIa-A single copy probe.
In Sitr~ bnmunochen~pj~~ for CAT enztr~e. At selected time points following aerosolization, mice were sacrificxd and their lungs immediately removed. The lungs were slowly inflated with phosphate buffered saline (PBS) containing 33 % by volume OCT (ll~les, Inc.), placed in a tissue cassette filled with OCT, and frozen in 2-m~hylbutane chilled in a dry icelethanol bath.
Cryosections were cil~ at 5 hem and collected onto salinized slides. CAT was detected after fixation of cryosections for 10 minutes in either 4 % acetone or 2 paraformaldehyde in IPBS containing 0.1 % Tweea 20 (PEST). All subsequent dilutions and washes were also done in PBST.
Following fuation, sections were washed three times (5 minutes each) then covered wiith 10'96 normal rabbit serum for 10 minutes at 20°C. The serum was replaced v~rith diluted (1:500) rabbit polyclonal antibody against CAT
(Drs. Parker Antin and David Standring, UCSF Medical Cater). The antibody *Trade-mark covered section was gently overlaid with a siliconized coverslip and incubated in a humid chamber at ~t ° C for 24 hours. Slides were then warmed to 20 ° C and washed three times.. The presence of bound rabbit antibody against CAT was detected by covering sections with biotinylated, affinity purified, goat anti-rabbit antibody (Vector Laboratories) diluted 1:300 for 1 hour, followed by washing (3 x minutes) and rep~lacemeat with streptavidin labelled with alkaline phosphatase (Zymed, South San Francisco) for 20 minutes. Immobilizxd alkaline phosphatase was detected using ~~P-red (Zymed) as the chromogea, with endogenous alkaline phosphatase being ilihibited with levamisole (Zymed). To control for potential 10 spurious adherence of the streptavidin conjugate to bronchiolar epithelium, some sections were treated with fi~ee avidin and biotin prior to application of the primary antibody. Other controls, run concurrently, included the use of normal rabbit serum in place of primary antibody and the use of lung tissue from untreated mice.
Photo-microscopy w.as performed using Kodak Elctachrome 64T film X50 (Fig. 6 A,D) and X250 (Fig. 6 B,C,E,F).
Initially, mice were exposed either to an aerosol generated from a solution containing 12 mg of a CMV-CAT expression plasmid alone or to an aerosol generated from a solution containing 12 mg of CMV-CAT completed to 24 ~cmoles of DOTMA:DOPE (1:1) liposomes. Aerosols were administered to animals after they were placed individually in nose-out cones and inserted into an Intoz small animal exposure chamber. The mica showed no apparent ill effects or respiratory distress either during or after aerosol exposure. Figure 7 shows the results of CAT assays from extracts of the lungs of mice sacrificed 72 hours following aerosol administration. Significant CAT gene expression was seen only in mice exposed to aerosolized DNA/liposome completes.
How long CAT protein was present in the lungs of mice and whether expression of the reporter gene was limited to the lung was also investigated. Despite inter-animal variation, high levels of CAT activity are present for at least 21 days following a single aerosol dose of DNA/liposome complexes (Fig. 8A). No CAT activity was detectable in extracts from the heart, *Trade-mark WO 93/12756 ~~~~~ PCT/US92/110~", spleen, ladneys or liver of animals that sbavvud high level eacprnn in tire lung (Fig. 8~), sugg~ng that transgeae exprc~ian followiutg eeeoaol delivery is restricted to the lung. This is a~iatent withNpzior absWons showing that penetratlcm of very high rno1ecuiar we~ht auk ~ the respiratory epithelium of normal aoima~ is very limited. DNA~'liposom~e complexes have molecular vwag6t~ gtmttr then x06 daltans.
Ald~owgh tyre small aniaoal ao~p~u~e chamber used in these eaperim~cnts is deeigne~ to qtly ~iva~ ~ aaol~c~1 dose to up to 48 aaimala, we have ob~tv~ed aignificaflt vas in the ~evel:of CAT activity in the lungs of mice within a ~gla eapaiment. O~ a lion for this variability is that the atnaunt of DNn~e boandqd~sited in the lungs of mice is rrot uniform. ~ oto tee this hypis, lrlitial dung deposition of liposomas was mid using fluoneecencx a~ysu and initial lung deposition of DNA was mea~od uSouthern blot anal.
AeraSOlizcd cationic liposonbs -or pI~AI~ip~o~ alone or DNA/liposome comgloocea containing 0.5 nwla petoaat of a ~uorescently labelled lipid, rhodaminc-phospbatidylethanolamine, v~re administered to mio~.
Lnmec~ately fdg aet~otiration, the awc~e Od and their lungs removed, homogenised and rho~damiae flu~o~e~a~ot nr~ua~ using a fluoarimeter.
The mcawerad fl~uo~~e per wet 0~6~6 t 0:02 (~$.D.) of the total amount eeeosoliaead. '1'~is suggests that less then 10 wg ~ t~ ~2 mg of DNA
aer~osoliaed per eaEt wits al~ally dapo~d~te~d in t!u 1. in edditicm, there was no scant diffiaeace ~ lipud depoe~id~a betw~aea< ar~ec~cving liposomas alone and those raraeiviag tYc DhToarae ooh. Since it is possible that a disruption of tire caa~l have ooa<nrd~ Burring ~bulization, the amount of CAT Bade deposed d~iag saa~oli~tiod (Fi~,.9) was also assessed.
l.mmodiatrdy follaw'sag aeroeal detivay of Dl~A~lipoeonle ~, mice were sacrifioa~ and total lwrg DNA psepat~od. Sin bvprobed with «[szP]_ labelled CAT gene. frlaballod baoda weave d and stcatdd less than a 4-fold diffia~ae in ~aa~d dbct~em~ ~u~s is the ~~ne invent (Fig. 9). These r~ltsi aft that tire mv~us* to m~aued vaW lion m SAT gene ltviels followi~og cklivary (fir do tar»fc~d) is not only a fim~on of the ~ 93/12756 ~ ~ ~ ~ ~ ~ ~ PCT/US92/l10~8 amount of complex initially depo$ted in the lung, but also may reflect differences in the site of uptake, rate of lung clearance, andlor va~r~ation in the ability of different lung cell types to express the transgme.
To d~cxmine the types and pe~ntage of lung cells which were 5 transfectad in viva, lungs of mice sacrificed 72 hours folla~r~g exposure to an aerosol containing DNA/liposome oomplexe$ were ciywoaecri~ned, probed with a polyclonat anti-CAT adtibody and oountasxain~ed to detect intzacellular CAT
protein (Fig. 6). Lung sections talmn from DNA/liposome treatod mice had a diffuse immunostaining pattern involving bron~iQlar and alveolar components.
10 The bronchiolar epithelial cytoplasm ata~ed with t inl~ity and uniformity.
CAT antigen was defected (ax demonstratod by red staining) in nearly all conducing airways with only rare individual ~ 2-3 ell clusters not staining (Fig. 6 A,B). The di.~e alvealar paten wad due to moderately intense staining of the majority of alveolar lining calls (Fig. 6C). Theoe area oeeasianally faded 15 into small, randomly shod regions where baing odl stainimg was faint.
Focal, intense staining (a~rmws) oocunnd in tlu; ~ytoplaszn of scatted, individual, alveolar lining cells (Fig. 6C). Controls included tip of the p~imaty antibody with normal rabbit serum (Fig. 6D) and use ~f lung suctions from un~at~od animals (Fig. 6 B,F). Lnmunostainit~g was not det~xable in either of 20 _ the control preparations. P~camination of m~ulie sections of lung from treated and control mice demonstrated no t ins which would indicate adverse effects of the aerosol t~aatrnent.
30 Two mopths old, female, rCR mice obntiunod &nm Simonsen, Cilroy, CA, were used.
~t~e.

WO 93/12756 ~~ PCT/US92/110(=
The p>a~nid liposome used, pZN'32, contains the human CFTR gene coding region fused to the human cytomeg~t~la~iras imp early pmmoter-enhancer element shown in Figures 3-5 atta~d hemto. A fall restriction map of the immediate early and prow of ~ICM~' (Towns) and IiCMV
(AD169) is provided in Figs. 11A and 11C. '~ two seqare c~ompare~d in Fig. 11B. p2N32 was purified using alyeis and ammonium aar~e precipitation, and the nn~CC acid aoaicent~ttid~ measot~ed by UV absorption at 260 nrn.
Lipid carriers were peered as small unidiameliar vesicles (approximately 100 ram in diaar~) ooathe c~~icmic lli~pid DDAB (dimethyl dioctadocyl ammonium bromide) as DDAH ch~stterol in a 1:1 molar ratio.
DDAB was pm~haaed fm~m Sigma, St. Loais,1~0, and choles~ol was purchased from C~IBioChem, Saran Diego, CA. Stock sa~u~s of the >iwere dissolved in chlosasform. Iipyda were mixod in a ro~d?~c~tta~ed flash and evaporated to dryness on a rotary evaporatar under reduced ~. ~~,~ water was added to produce fir~l lipid conom~o~rat~t of 10 m~M eai~h, and the resulting mix was aonicated far mately 20 minutes in a berth sorricator (laboratory Supplies, I~icksviLle, N~.
Twrdve eng of -pZN32 c~amphra~d to 24 dads of DDAB: cholesterol (1:1 mole ratio) liposomea was aerosolized over two diffcrmt aacosol periods on the same day. To prevent agg~ation and station of tire oppositely charged components, the liposotnes ahd DNA v~rere dih~ted separately in sterile water prior to mixing. Sin mg of DNA and lit enrols of I~DAH:clwl (1:1 mole ratio) liposomes were ales thd to 8 ml with and meal. Four ml of the DNA-lips ~ vwut then pl~roed into o Aca~n I neaulizers (luta~rquest, Englewood, CO), and the animals glarxd in as IOtOx ~»all animal exposure chamber (Albuquerque, ~. An sir flow rate of 4 L min'' was used to generate the aerosol. Nin~r miautea were roqu~ed to line this volume (4 ml) of DNA-liposome mixture. Tla; animals were ren~arved ~m the for 1-2 hours and then the above procedure was reed wid~s ~t and 4 ml dose.

'~;~,~,~ for the human CFTR yrntein in mouse lanes.
At selected time points following aerosolization, mice were sacrificed and their lungs immediately removed. The lungs were slowly inflated with phosphate buffexed saline (PBS) containing 3.3 % by volume OCT (Miles, Inc.), then placed in a tissue cassette filled with OCT, and frozen in 2-methylbutane chilled, in a dry icelethanol bath. Cryosections were cut at 5 ~m and collected onto sialinized slides. CFTR protein was detected after fixation of cryosections for 10 minutes in either 4 % acetone or 2 % paraformaldehyde in PBS
containing 0.1 % Tween 20*(PBST). All subsequent dilutions and washes were done in PBST. Following fixation, sections were washed three times (5 minutes each) with PBST then covered with 10 % normal rabbit serum for 10 minutes at ° C. Immunolocalization of CFfR was then performed using an affinity purified rabbit polyclonal anti-CFfR antibody, a-1468, provided by Dr. Jonathan Cohn, Duke University. T'he serum was replaced with a-1468, diluted (1:1000). The 15 antibody-covered sa:tion was gently overlaid with a siliconized coverslip and incubated in a humid chamber at 4 ° C for 24 hours. Slides were they warmed to 20 ° C and washed tb~ree times. The presence of bound rabbit antibody against CF'IR was detected by covering sections with biotinylated, affinity-purified, goat anti-rabbit antibody (Lipid cagier Laboratories), diluted 1:300 for 1 hour, 20 followed by washing; (3 z 10 minutes) and replacement with streptavidin labelled with alkaline phosphatase (Zymed, South San Francisco) for 20 minutes.
Immobilized alkaline phosphatas~e was detecLod using AP-red (Zymed) as the chromogen; endogamous alkaline phosphatase was inhibited with levamisole (Zymed). Other controls, run concurrently, included the use of normal rabbit serum in place of primary antibody and the use of lung tissue from unmice.
Photo-microscopy v~ras performed using Kodak Ek~chrome 64T film at X50 and X250.
Photomicrographs of frozai sections (viewed at different magnifications) of nnouse lung 48 hours following aerosol exposure to pZN32-DDAB: cholesterol 1:1 mole ratio liposome completes and lung from un *Trade-mark WO 93/12756 PGT/US92/110~
4s ~~~6 control art shows in Pigs. l0A-10E. As by thl~ intet~e staining with the polyclonal anti-CFA, antibody, a-14b8, the ave~rmajority of the airways were tracrsfeeted with the human Cgene. fee Pigs. 10A, lOC and 10E. By visual inspection, exseatially all the cells in transfea~l~ed airways stain positively, demonstrating drat the owscwma,~atlty of ~hway cells are transfeatod with the lawn CPTl~ art vfvn with a single 1 die of pZN32 cacrnplexed to CAB-choles~l 1:1 mile rat~or gpaea~. Re~aitative seetionx are shown in Ffgvit 10. T'b~e waa nw~ c evirdadae of lung damage, inflammation or edema praeot in and of the p~:N32~.DDAB:aholeonerol-1:1 tno~rratio lipos~re-animals. p2-DDA~B:chol-1:1 mole ratio liposoated and contnvl animat: could _ be diat~id histologic~lly.
Sigmfxsmt expn~ion of the hun~en CPTR gee is pre~nt in .:at least 50 % of all the airways and at least ~% of all csF the airvuay lining calls ~,bY visual inspection) in mouse lung$ for at least 60 days following a single aerosol ~dox of pZN32 oompleaed to DDAH-~chWol 1:1 mole rats lipoeo~mes. FxGtions of mouse lungs from oootrQl animals (Figs. 10B ~tl 101;1) do not show any detectable staining fear CFT~, that all time CF1'R boa pit in Fig. 10A, lOC sad 10E is due to trausfecxioa of lung oe~t with the hun~n CFTR gale.
Cell,C~: NC1-H69, NC1~H82, and NCI-I~320. H6p sad H320 cells were grc~m in RFMI-1640 with 1096 fetal bavrine spun (FHA) anc~, H82 cells were grown in Dulbocoo's minimum aseatial mediym (D~B~-H'~ 1 with 10 % FHiS .
Lips wnae pr~patred as fadlows: a total of 4 males of lipid dissolved in chl~oform, (or in cxhanol (DOrI'MA)) were e°vaporatod to dryness o~n a rotary evaporator. One ml of S0 mM Tris, 0.5 mM EDTA, ~0 mM
NaCI, 100 mM ZnCl2 burr per 20 mma~i~es c~f lipid ways added, and the mixture was sartioeted in a bath aonicna~ (Iabo~toty y Co., Hilvnlle, N5~ for 20 min. The resulting lip~o~mes have ~ approximate meati liar of 100 t 25 ~"'"""'"'~ 93/12756 ~ ~ ''~) ~ ~ ~ ~ PGT/US92/11008 nm. The following lipoaome prepa~ati~s wen used: pure D4~'MA, DOTMA:chol in a 2 to 1 mar ratio, pure L-~"E ar L-PE:ch~il-b-a1a in a 6 to 4 molar ratio.
Fen traasfection of crlls, 2 a 10'6 cells in 4 ml of serum-free medium were plated in 104 mm plastic petri d(Falcon, pxnard, CA). The plasmid DNA lip~o~ oomp~a~ was pr~urod.!by fir,~t adding, 1) DNA and then 2), liposomes and mixing gently. The complat was tlKrt susp~ed in 1 ml of serum free medium anti ~a~ddod to the cells. Faur hoofs later, 'the sells was washed twice, resuspended in 10 ml of ae~rum-ecrnmum, and subsequently harnsted, .eit~r 44 twura later. fust prior to h~vas~ing, the cells were washed 2 times, alud the plates vverre then; with a~, rubber policeman.
The cells were ~trifugatad at 1,000 x g for 5 min, and 0.1315 ml of 0.25 M
Tris buffer wet added to each pellet. The cells weae froe~a-rd~awad 3x, heated at 65 ° C
for 10 min, and spun at 12,540 a g for 10 min The su~nt was assayed for protein and 24 ~cg of aupa~ant pm~ia per s~nple was uxd~ to measure CAT
activity, as der~cribed above.
$g: The ability of cationic loon's to ~uate tr~sfee~ion of two different human small cell lung canar' lines (H-b9 at~d ~-82) and a squs cell lung cancxr line (H-520) was assessod. All three lines were very efficiently transfected by RSV-CAT when complexed to 3 different cationic liposome formulations.
These human cell lines were ether as or more efficiently trantcfected than rodent cell lines transfectod under comparable conditions.
._..._.-,.......~--_.....,......,.,~.~.,..~."~»,..",».,...~"...-.....,-~.~,....",~,.~.~.,.~"..,~~.~".,...,~. .~ -...~.,.~.,.. . ... -_..

As shown by the above results, a single aerosol dose of an expression liposome, containing a gene of interest, complexed to cationic liposomes transfects the majority of the cells lining both the conducting airways and the alveoli of the lung, the gene produ~:,t is present in the lung for at least 60 days, the expression 5 appears to be lung-sF~ecific, and there is no histological evidence of damage following exposure. Thus, the aerosolized cationic liposomes mediate efficient transfection of non-dividing as well as dividing cells. This is important because many airway epithelial cells are well differentiated and divide slowly or not at all.
The lipid carriers appear to be both well toleratcd and non-immunogenic.
10 Additionally, the effexts of repeated aemsol administration of the DNA/liposome complexes is effective and is non-tonic. The cationic liposome-mediated DNA
delivery by aerosol p~mvides high level, lung-specific transgene expression in vivo.
The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and 15 modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims (20)

CLAIMS:

1. A composition comprising:
a nebulized transfection agent, prepared by nebulizing a mixture comprising complexes between transcription cassettes or expression cassettes and cationic lipid carriers in a pharmaceutically acceptable carrier or diluent, wherein said mixture is substantially free of macroaggregates of said complexes and said lipid carriers have a diameter from 0.1 microns to 10 microns and the ratio of DNA to lipid carrier is in a ratio of 4:1 to 1:10 micrograms DNA to nanomoles cationic lipid and wherein said transcription cassettes or expression cassettes comprise a DNA sequence capable of producing a transcription product in a mammalian cell transfected by said nebulized transfection agent.

2. The composition according to claim 1, wherein said DNA sequence comprises an inducible promoter.

3. The composition according to claim 2, wherein said inducible promoter is a cell specific promoter, a tissue specific promoter or a hormone responsive promoter.

4. The composition according to claim 2, wherein said DNA sequence comprises an SV40 enhances element whereby transcription from said promoter is enhanced.

5. The composition according to any one of claims 1 to 4, wherein said cationic lipid carrier comprises a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA);
dimethyldioctadecylammoniumbromide (DDAB); 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); lysinylphosphatidyl-ethanolamine (L-PE); dioleoylphosphatidylethanolamine (DOPE); and cholesterol (Chol).

6. The composition according to any one of claims 1 to 4, wherein said cationic lipid carrier comprises cholesterol and a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA); dimethyldioctadecylammoniumbromide (DDAB);
1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and lysinylphosphatidyl-ethanolamine (L-PE).

7. The composition according to any one of claims 1 to 4, wherein said cationic lipid carrier comprises dioleoylphosphatidylethanolamine (DOPE) and a lipid selected from the group consisting of N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA);
dimethyldioctadecylammoniumbromide (DDAB); 1,2-dioleoyloxy-3-(trimethylammonio)propane (DOTAP); and lysinylphosphatidyl-ethanolamine (L-PE).

8. The composition according to any one of claims 1 to 7, for use in the treatment or therapy of a human or animal body, said human or animal body having an aberrant expression of said transcription product.

9. The composition for use according to claim 8, wherein said use is use for inhibiting growth of tumor cells in a mammal.

10. The composition for use according to claim 8, wherein said use is use for providing or enhancing production of a polypeptide in cells in lungs of a mammal.

11. The composition for use according to claim 10, wherein said cells are distal airway cells or proximal airway cells.

12. The composition for use according to claim 10, wherein said cells are tracheal cells.

13. The composition for use according to any one of claims 10 to 12, wherein said cells are normal cells.

14. The use of a composition as defined in any one of claims 1 to 7 for the manufacture of a medicament for the treatment of a human or animal body, said human or animal body having an aberrant expression of said transcription product.

15. The composition according to any one of claims 1 to 4, wherein said transcription cassettes or expression cassettes and said cationic lipid carriers are present in said mixture in a ratio in the range of from about 4:1 to about 1:10 micrograms DNA to nanomoles of cationic lipid.

16. The composition according to any one of claims 1 to 4, wherein said cationic lipid carriers are small unilamellar vesicles.

17. The composition according to claim 16, wherein said small unilamellar vesicles comprise (a) dioleoylphosphatidylethanolamine (DOPE) and N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) or (b) dimethyldioctadecyl-ammoniumbromide (DDAB) and cholesterol.

18. The composition according to any one of claims 1 to 4, wherein said transcription cassettes or expression cassettes and said cationic lipid carriers are present in said mixture in a ratio in the range of from about 1:1 to 1:2 micrograms DNA to nanomoles of cationic lipid.

19. A composition for use according to claim 8, wherein said use is use for altering the phenotype of cells of a lung of a mammal.

20. A kit for use in the treatment or therapy according to any one of claims 8 to 14, said kit comprising in combination:
a container containing said transcription cassettes or expression cassettes;
another container containing a specific amount of cationic lipid carriers; and instructions.