CA2214029A1 - Single-vial formulations of dna/lipid complexes - Google Patents

Abstract

The invention relates to single-vial formulations of plasmid DNA/cationic lipid complexes for human clinical use prepared by various different processes, for example, by the process comprising the steps of: (a) autoclave sterilizing a cationic lipid solution at a concentration sufficiently high to substantially prevent lipid degradation during said autoclave sterilization;
(b) diluting the sterilized cationic lipid solution of step (a) to a degree sufficient to substantially prevent lipid aggregation during step (d) below;
(c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than isotonicity to form DNA/lipid complexes; and (e) adjusting the DNA/lipid complexes of step (d) to near isotonicity.

Description

WO 96/34109 PCTrUS96/05035 ~1 .
SINGLE-VIAL FORMULATIONS OF DNAILIPID COMPLEXES
FIELD OF THE INVENTION
The invention relates to - _' .;.,l fG,I ' i of plasmid DNAlcationic lipid - . ' - for human r clinical use, and related I .

The use of plasmid DNAlcationic lipid - .' 9 to transfer genes in vivo for the Ll~al..._..l of human diseases, including ' :y and ca, . .a;,cular disorders, is; ' r~ d1 in active human clinical trials.
The R ' I DNA Advisory Committee (RACJ of the National Institutes of Health (NIH) approved Nabel et al. to conduct a human clinical protocol using lipid mediated transfer of an _ encoding gene into tumors for i ;' .1 of re r~ ~ Nabel et al., Proc. Natl. Acad. Sci. USA 90:11307 (1993); see Nabel et al., Human Gene TheraPv 3:399 (1992); see also Nabel et al., Human Gene TheranY 5:57 (1994);
PCT Patent Al.' -: - WO# 94129469. The gene encoding a foreign major ' - . l' '~y complex protein, HLA-B7, was ;..l..~ ~ into HLA-B7-negative patients with advanced ' by injection of DN~lli,- - - .' using a DC-ChollDOPE cationic lipid mixture. Nabel et al., Proc. Natl. Acad. Sci.
USA, supra. Six - ' ;Idi- were ~ . ' ' without ~ . ' - in five HLA-B7-negative patients with stage IV ' (~, one patient received a second -' ;,~ ) Plasmid DNA was detected by p~ a~e chain reaction within biopsies of treated tumor nodules 3-7 days after injection but was not found in the serum at any time. F. - ' ~ I HLA-B7 protein was ~l ~.dll,d by -' ~ y in tumor biopsy tissue in all five patients, and immune, , to HLA-B7 and àul Rg tumors could be detected.
No ? ' ~ ' to DNA were detected in any patient. One patient d ~,dl~d ,~!J,. of injected nodules on two ', ' 1l~ : . which was e . ' by ll_ . at distant sites. These studies ;' alud the r. ' ' ~y, safety, and Ol~l.."_..li~, potential of 1i, ' ' : ' gene transfer in humans. See also Stewart et al., Human Gene TheraDy, 3:267 (1992) (safety and : y of gene transferin vivo with DNAllipid ~ '; Nabel et al., Human Gene Therapv, 3:649 (1992) (lack of L i ~y and gonadal 1 - ~ by gene transfer in vivo with DNAllipid c~ .' s!; San et al., Human Gene TheraPv, 4:781 (1993) (safety and nl lu~dl.ity of new cationic lipid mixture, DMRIEIDOPE, for human gene therapy).
As a result of the Nabel et al. study, Vical ' ~ alud proposed a ' L~..lel clinical trial using an improved cationic lipid mixture, DMRIEIDOPE. Vog ' _ et al., Human Gene Therapv 5:1357 (1994);
Hersh et al., Human Gene TherapY 5:1371 (1994); and Rubin et al., Human Gene TheraPY 5:1385 (1994).
The Food and Drug A~' Id6uil (FDA) has aui- i~d these clinical p.ul The FDA also recently allowed Vical 'c r aL d to conduct a clinical protocol using lipid mediated transfer of a cytokine encoding gene into tumors for ll~allll~lll of lil y. See Example 8. In ~,._ ' studies, the i..l, injection of a plasmid DNA ~AIJI ' vector ~ ~ ~ _ the human ;..1~l; ' 2 (IL-2) gene reduced the incidence of tumor f. Illai and slowed tumor growth. By local e~ of ~,y~l ' s at the site of the tumor, it is _.. ; ~ that lower levels of cytokines will be required for efficacy as L . ud to systemic - ' , and that these levels will be ~ur~ low to avoid r ~ ~ ~ _ toxicity W O96/34109 PCT~US96/05035 in the patient. The findings suggest that i..ll--' liO.. of IL-2 into a tumor, by il",all...,or injection of plasmid DNA t:A~llU ' vectors, can stimulate an, : response. In the proposed trial, this approach will be applied to human patients with solid ~ I tumors, using a plasmid DNA that encodes the human IL-2 protein and a DMRIEIDOPE cationic lipid mixture.
The w; ' , ~ad utility of a pul~llULIt:ui ' '!i~ ~ ' approach has been eai ' ' ' ~ from studies showing cationic lipid de, ' - delivery of DNA (Felgner et al., Proc. Natl Acad. Sci. U.S.A., 84:7413 (1987); Felgner, P.L., Adv. Druq Delivery Rev., 5:167 (1990); Felgner et al., Nature, 337:387 (1989); Brigham et al., Am. J.
Respir. Cell. Mol. Biol., 1:95 (1989); Muller et al., DNA Cell Biol., 9:221 (1990); Burger et al., Proc. Natl.
Acad. Sci. U.S.A., 89:2145 (1992)), mRNA (Weiss et al" J. Virol., 63:5310 (1989); Malone et al., Proc. Natl.
Acad. Sci. U.S.A., 86:6077 (1989)), and antisense oligomers (Chiang et al., J. Biol. Chem., 266:18162 (1991); Bennett et al., Mol. Phdll~dLoL~ 41:1023 (1992)) into living cells. Since the initial published di , i- in 1987, several reagents have become u , 'l~ available (Behr et al., Proc. Natl. Acad. Sci.
U.S.A., 86:6982 (1989); Rose et al., e i ' ~ 10:520 (1991); Leventis et al., Biochim BioPhys. Acta., 1023:124 (1990)), and additional cationic lipid reagents have been described reporting ad~ relative to the u;dl products (Farhood et al., Biochim BioPhYs. Acta., 1111:239 (1992); Gao et al., Biochem BioPhYs. Res. Commum., 179:280 (1991); Legendre et al., FhallllacL..IiLal Res., 9:1235 (1992); Zhou et al., Biochim. BioPhvs. Acta., 1065:8 (1991); r; - ~ . e et al., Biochim. Biophvs. Acta., 986:33 (1989)). The broad 3~ of this approach has been further e: '' ' ' in ,~I~ ' ~ ' in vivo studies showing cationic gene delivery to talh~L~HL~d blood vessels (Nabel et al., Proc. Natl. Acad. Sci. U.S.A., 89:5157 (1992); Lim et al., Circulation, 83:2007 (1991); Yao et al., Proc. Natl. Acad. Sci. U.S.A., 88:8101 (1991); Nabel et al., Science, 249:1285 (1990)), lung epithelial cells (Stribling et al., Proc. Natl. Acad. Sci.
U.S.A., 89:11277 (1992); Brigham et al., Am. J. Med. Sci., 298:278 (1989); Yoshimura et al., Nucleic Acids ~" 20:3283 (1992)), brain tissue (Jiao et al., Exp. Neurol., 115:400 (1992); Ono et al., Neurosci. Lett., 117:259 (1990)), Xenopus embryos (U ~ et al., Int. J. Dev. Biol., 35:481 (1991); Holt et al., Neuron, 4:203 (1990)), and the syâtemic ~ ' (Zhu et al., Science, 261:209 (1993); Philip et al., J. Biol. Chem., 268:16087 (1993)).
Yet, l~" ' ~ of plasmid DNA for i' , purposes requires that a "ha, ~c 11~ aL-.opi ' ' vehicle be found in which the DNA can be taken from the ~-.llJHIl9 site to the clinical site with a viable interim shelf-life. Although buffers c i ~ ~ " tris-(h~d~uA~ ' (Tris), usually ~ - ~ ~, chelating agents, are r N, used to handle plaâmid DNA in the research ' ' dllJIy, these buffers are ' i,~d for P~IILIIl~l~l use in the clinic. In their absence, a vehicle must be identified that provides for efficient handling of the plasmid DNA in the, 'a.,lL,i ~, setting, preserves the chemical and biological integrity of the plasmid DNA during shipping and storing, and allows efficient delivery of the DNA to the desired tissue target by the preferred route of ~ .dliun.
The inclusion of cationic lipids in a r., ' liu.. with plasmid DNA illll.d~ ad ' ' ~ y into the choice of a suitable vehicle for phall ~~ ' use. In this case, the solubility and stability of the W O96/34109 PCTrUS96/05035 individual o~pc- ly charged c r ' and the - , ' they form must be ~vvv -~ ' in a single medium. If ~lulai and ,ult~ dliun of the plasmid DNAlcationic lipid r ,' - is s~ ' Ld to be essential for the rl : v of the product, it is , dli~r, that the vehicle chosen for 1 ' lllaceuliL~dl use not interfere with this dLi-The stability of the product can be enhanced by frozen storage, which imposes ~ lli.. i s on the choice of vehicle for mixtures of plasmid DNA and cationic lipid. In this instance, the stability of the r ,' ~ formed between plasmid DNA and lipid must be rJ~ ' Ld in addition to that of the individual S r- IS. Sp61,ir- -~ly~ the fG, ' - must be designed so as to preserve the solubility and integrity of the plasmid DNAllipid -- ,' -- over the course of storage at the frozen ~ , dlL.~.
The Nabel et al. initial work in human patients P ~ :Idl~d a 1l, ~ ~",enl for administration of a ~t ' of plasmid DNA with cationic lipids for ~ce 'ul Lld"~reLliu" of tumor cells. SuPra. Indeed, many in vivo gene therapy c,,' ~ - have shown a 1~, 1,...~..l for _~ Idli~a of a mixture of DNA
and lipid for efficacy. In these ,u,ui S7~. the gene therapy dose has been provided to the clinical site in a multiple vial r 'ig, - because the plasmid DNAlcationic lipid ,' s are not stable. In this : '~ di- n, the plasmid DNA in vehicle is present in one vial. The lipid mixture is present in a another, separate vial either as a dried film or in solution. When the lipid mixture is provided in dried form, still another vial c - v the diluent for ~ J~di . of the lipid is provided to the clinic.
With this s 'iv of product, the clinic staff must prepare the patient dose at the site by r ~Ull ~ ~ several , : ' dilution and addition steps. First, the clinic staff must obtain the DNA in vehicle. Second, they must obtain the lipid. Third, if it is a dried film, which event is likely because of stability issues, they must rehydrate the lipid with a ~u, ': buffer by vortexing until h-Fourth, they must transfer the r. : ~ lipid into the DNA solution and mix. Finally, fifth, they must the amount to the patient. Optionally, the clinic staff may prepare dilutions of DNAllipid C~for the r~ : . of eec~ doses.
Although this type of s ';., dliOII is accl:,ui '' for Phase I clinical trials, it is - ' . - to 'dl,lUI~, difficult to manage in s '~ Illily with 1. ' ~ Y v ' ' s, costly to ship and store, and -,' d for clinic staff and ,JhJa;";d", to use. ' pr lallll~ there is no control or _ a""e of c: y in, ~, dliUn of the final product, which is the dose 1~ ~ I,d to the patient. These ~- liO-Is clearly Pl ~dle the need for a plasmid DNAlcationic lipid ~ Jl r, ~ lr for human clinical use.
Ar- . ~'~, it is an object of the invention to provide a . "' .;~1 rur ' - in which DNA and lipid are combined in a ,' ",ac~ aLG~"; '' vehicle.
It is another object to provide a . ~ "' viol f".l ' - that retains the potency of the DNA and lipid.
It is a further object to provide a single vial lu,l ': that enjoys stability over a time course of storage.
It is still another object to provide a single vial ~. I ' that is universal in 3~,' ~-W O96/34109 PCTrUS96/05035 lt is yet another object to provide a single-vial ft" ' that is safe as shown by suitable safety studies.
It is an additional object to provide a single-vial fo" ' i 1 that is easy for a clinic staff to prepare and a physician to - ' These and other objects of the invention will be apparent to the ordinary artisan upon 't dliu of the a,uel,;ri~ai as a whole.
SUMMARY OF THE INVENTION
The invention provides a process for making a - v .;al fu,l ' i of poly,' i'~l1i, ' in a 1' 11~ a, p~ '' vehicle for human clinical use in vivo or ex vivo ~.UIII~UUa;llu the steps of: (a) sterilizing a lipid solution; (b) sterilizing a r~H~ ~ liJe solution; (c) ' _ the sterilized p~l~,luoluul;de solution of step (b) with the sterilized lipid solution of step (a), in dilute form, at an ionic strength that is lower than isotonicity, to form p~1~, ' i ' '!i, ' c ,' and (d) adjusting the p~'~ ' li'e.'li, ' c .' - of step (c) to near ;aui ' '~y.
The invention further provides a process for making a _' .;JI rG~ ' t;"" of plasmid DNAlcationic lipid c . ' for human clinical use . i the steps of: (a) autoclave sterilizing a cationic lipid solution at high c di' , (b) diluting the sterilized cationic lipid solution of step (a); (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than ;aui ' '~ to form DNAllipid .t .' ;, and (e) adjusting the DNAllipid c~ .' ~ of step (d) to near ;aui ' 'ty.
The invsntion also provides a process for making a single-vial ru" j'~r of plasmid DNAlcationic lipid -- . ' ~ for human clinical use c . i g the steps of: (a) autoclave sterilizing a cationic lipid solution ata Cll r l~dliunsurri~ highto ' ~ preventlipid~dddl;u~duringtheautoclaveùl~," liu~, (b) diluting the sterilized cationic lipid solution of step (a) to a degree sufficient to ~ b~ prevent lipid e_ ._ during step (d) below; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c) to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than ;aui ' 'ty to form DNAllipid c ,' and (e) adjusting the DNAllipid r ,' of step (d) to near iaui ' '~y.
The invention moreover provides a process for making a _' . ' ~r I ': of plasmidDNAlcationic lipid - , ' for human clinical use - , i _ the steps of: (a) autoclave sterilizing a cationic lipid solution having a toll~ " . in the range of from about 0.5 to about 5.0 M; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a CUIII,I,..Ildi' in the range of from about 0.01 to about 1.0 M; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a ~ lldLiun in the range of from about 0.05 to about 10 mglmL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than iaul ' '~y to form DNAllipid - ,' and (e) adjusting the DNAllipid c ,' of step (d) to near ;au~ ' "y.

= =

The invention ?''~ y provides a process for making a single-vial f~", ' - of plasmid DNAlcationic lipid cl , ' ~ for human clinical use c , v the steps of: (a) ~ ~ sterilizing a cationic lipid solution of DMRIEIDOPE, having a molar ratio in the range of from about 90:10 to about 10:90, and having a rs lldlion in the range of from about 2 to about 10 mg DMRlElmL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a CUllbclllldi - of < about 2 mg DMRlElmL; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a concb.,l, of < about 10 mg plasmid DNAlmL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than ;aût ~ 'y to form DNAllipid - ' s at a mass ratio of from about 50:1 to about 1:10 DNA to DMRIE; and (e) adjusting the DNAllipid c , ' s of step (d) to near ;so with sodium chloride.
The invention Pb,ll,c" c provides a process for making a _'~ .;al fl Illu6liun of plasmid DNAlcationic lipid - ' for human clinical use l ,;.;.., the steps of: (a) . ~obla~,c sterilizing a cationic lipid solution of DMRIEIDOPE, having a molar ratio of about 50:50, and having a con~b..l,di of about 8 mg DMRlElmL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a cs 'IdtiU~ of < about 1 mg DMRlElmL; (c) filter sterilizing a plasmid DNA solution; (d) adding the sterilized plasmid DNA solution of step (c), having a cûnc~..lldliùn of < about 5 mg plasmid DNAlmL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than ;aù~ ~ 'y to form DNAllipid cl , ' at a mass ratio of about 5:1 DNA to DMRIE; and (e) adjusting the DNAllipid complexes of step (d) to near ph~ ' ' salinity.
The: ~.,.. i . as well, provides a process for making a '~ d flJllllbdai of plasmid DNAlcationic lipid ' in about 0.9% sodium chloride with about 1% glycerol and about 0.01 YO Vitamin E for human clinical use r ia;..g the steps of: (a) autoclave sterilizing a cationic lipid solution of DMRIEIDOPE, having a molar ratio of about 50:50 molar ratio, and having a r ', " of about 8 mg DMRlElmL; (b) diluting the sterilized cationic lipid solution of step (a) with a diluent to achieve a c~ ~,dliun of < about 1 mg D'' ,'EI L, and with glycerol and Vitamin E; (c) filter sterilizing a plasmid DNA solution;
(d) adding the sterilized plasmid DNA solution of step (c), having a r - di' of < about 5 mg plasmid DNAlmL, to the diluted sterilized cationic lipid solution of step (b) at an ionic strength that is lower than iaui ' ~y to form DNAllipid ~ at a mass ratio of about 5:1 DNA to DMRIE; and (e) adjusting the DNAllipid cl ,' of step (d) to about 0.9% sodium chloride, wherein the final CU~bL..II_ of glycerol is about 1% and of Vitamin E is about 0.01%.
According to other e ' ' . the invention provides - 'l viol fo~ r of plasmid DNAlcationic lipid ' for human clinical use prepared by any of the above, ucesses.
In another ' - " 1, the invention provides a _' .;al rb~ of plasmid DNAlcationic lipid L ,' for human clinical use - i _ a cationic lipid s and a plasmid DNA c~ 1, wherein the plasmid DNA ~r L and the cationic lipid r , ' are combined at an ionic strength that is lower than ;aui ' '~y to form the plasmid DNAlcationic lipid c , ' s, and further c , _ a nearly W O96/34109 PCT~US96/05035 isotonic aqueous medium, optionally LIJ~ no buffering agent except the plasmid DNA itself. The single-vial fl liuil may be stable in frozen, IGrliy_~dlGd~ room IGIII,UGI G, or body IG---uG-dlL.G form. The single-vial D . It, may be stable in frozen, IGrliyGldl~d, or room tGIll~Jeldi G form for at least about 8 weeks. The single-vial rl Illl~lai ~ may further comprise about 1% glycerol and about 0.01% Vitamin E in nearly ,JI-, ~ ' v ' saline.
In still another: l 1, the invention provides a ~ ' .;.,l rul ' for human clinical use c" i;.;..J plasmid DNAlcationic lipid ~ ' having storage stability in frozen, IGDi~GldlGd, or room di G form for at least about 8 weeks.
in yet another ' ' l, the invention provides a . "' .;"l ru, j-~r for human clinical use 10 , ia;l,g plasmid DNAlcationic lipid ~ ~, ' retaining in vitro ~1 ~GI.liU~ efficacy of freshly prepared plasmid DNAlcationic lipid - ,' s for at least about 8 weeks.
DETAILED DESCRIPTION OF THE INVENTION
The objects of the invention are achieved by col.ll~" ' ' ~ of a plasmid DNA solution with a cationic lipid solution, ~J~GrGI ' 1~ in dilute form, at an ionic strength that is lower than ;au ~ t~" to obtain 15 plasmid DNAlcationic lipid ~ ' s, which, when admixed with solutes to generate iaui ~ ~" provide _' .;al fG. ' :- suitable for human clinical use.
We have d;~-,..._.Gd that the order in which individual plasmid DNA and cationic lipid are mixed to produce plasmid DNAlcationic lipid c ,' - is ~, i I to the final results, as is the ionic strength at the time of initial complex ru.l :- Optimal in vitro 11_ CLLi- GrR~iGll~y and ,UI~.G of 20 lipid ~ dlion have been obtained by slow addition of a plasmid DNA solution to a cationic lipid solution at an ionic strength that is lower than i~u; ~ ty. In this scheme, we have optimized in vitro 11 C~.,liu.~
Gfri ~ y and r.~.G..liui) of lipid ag~ - ~ even further by using the cationic lipid ~ , in a dilute form upon its - ' ~ : - with the plasmid DNA c pc : to form the DNAllipid ' ~r We have also found that alG.i' ~- of lipid mixtures by filtration c ~ ~ s their integrity. In 25 contrast, we have ': ~ ' that our lipid mixtures can survive alG-' i by autoclave IrGdl.. ~.. l.
Maximum in vitro lld~aOG~i Grri ~ y and ~.u.. of lipid d~.allai have been produced by autoclave alG.i' i In this scenario,in vitro ll~..;.r. efficiency is further ~ ~ ' and lipid ' _ adai- further .~..L~d by ~ - - - ~ the lipid mixtures at higher c~ during autoclave alG. ' ~-Preferred single-vial r. . ' of the invention have dCCuld "~', been obtained by preparing 30 cationic lipid mixtures c ~ ~ _ a cationic lipid cs~ and a neutral lipid ~ t, having a molar ratio in the range of from about 90:10 to about 10:90, ~ulGOGl.lbly about 50:50. A cationic lipid solution is prepared from the cationic lipid mixture by hydrating a dried lipid film with a suitable diluent",.GOGI.Jbl~ using water, to obtain a solution in highly cûnc~.llldluJ form. The cationic lipid solution is cc..i~idGIGd to be highly ~ : dlcd by having a ~r ll ~- in the range of from about 0.5 to about 5.0 M. This highly 35 c dlGd cationic lipid solution is s-~l-s~ subjected to al~li' ~- - by standard autoclave IIGdi 1, e.~., 30 minutes at 121~C, in the usual way.

WO96/34109 PCTrUS96/05035 Al~ aL._I~, a cationic lipid solution can be prepared and sterilized by filtration or i"_ ~D or other d,u,ulI, ial~ means at any desired cc c ~ :lai the higher c l~aiiar, of cationic lipid solution being ad~, i g for protecting the material during sterilization by aulûL.ia~,~, llealllll:lll.
- Upon obtaining a highly c llal~d cationic lipid solution, one p.~r~l.. hly dilutes the solution, ,u~r~,_"y using sterilized water I~L, sterile WFI). Other suitable diluents, such as salines, can be lud for water, and are selected on the basis of having low ionic strength, that is, lower than i~u; ~y. The cationic lipid solution is diluted to achieve a c : a ~ in the range of from about 0.01 to about 1.0 M. Ori 'I~, at this point, suitable r. Iu.y agents may be illllu~ d, as di~ d below, pa, li.,~da;l~ to facilitate the , of DNA and lipid during the cc ' liun step (~).
Plasmid DNA, which is p'~ ~ ' 9. ' in quality, is I . ' ' obtained. The plasmid DNA, in aqueous solution, such as water (~k, sterile WFI) or other a,, u,ulial~ diluent, such as saline (~Ek, , by ' -' saline), is sterilized. Sterilization is ~,~r~ by filtration, for instance, through a 0.2 ~m filter. It is preferred that the diluent have an ionic strength that is no higher than ;au ~ y.
The sterilized plasmid DNA solution and diluted sterilized cationic lipid solution are usually brought to room I di ~ prior to - ' ' The DNA solution may be used at crz l,_ - within a range that extends from about 0.05 to about 10 mglmL. The solutions are then ' d, ~ rel "~, by c , " d addition of the plasmid DNA solution to the cationic lipid solution at low ionic strength with mixing to form DNAllipid ~ . ' - By low ionic strength is meant lower than ;~ui ~y. C7 mixing of the lipid solution and plasmid DNA solution as they are combined is preferred, with a further short period of mixing after the c ' i is complete. The mixing may be achieved by l,.,.i _ manual agitation (shaking), __L ~ ~ mixing or stirring, or other suitable means.
DNA and lipid are combined to produce - ' - at a mass ratio that is optimal for Ll fc:
~rr- ~y, as evaluated, for example, by in vitro 11_ fu: assays.
The L , ' are b~ adjusted with a tonicifier to ; ,ui ~y for, ' y ' _ e' .~ For example, adequate sterile sodium chloride stock may be added to give a final Jrc 1_ of about 0.9% NaCI (j~, ,' y ' g ' saline). The final ru. '; ~. can next be a-qpt- 'Iy filled into sterile ', ,-~_ lud vials, and can then be stored frozen at about 10~C to -70~C,, ~r~
about -20~C.
At the clinic site, the DNAllipid vials are c( .~ thawed and typically mixed (e.a., by vortexing or manual agitation, i_, shaking). They can be ~ ~ at room i alL.~, and are -' ~d 2d~, ~_ '~ within 24 hours of thawing. Each vial may deliver DNA in unit dosage form, or, all~luali.
may cu.. ,lilule a multidose container.
For use by the, ' ~ ~ . the ' .;..l ru~ ' i - are provided in cc l,_: up to about 10 mg DNAlmL, ,u,~ru. "~ up to about 0.5 mg DNAlmL. As will be r~ c _ ~ ' by those in the field, an effective amount of DNA will vary with many factors, including the condition being treated, the chala~ liL.s of the CA 022l4029 l997-08-27 W O96/34109 PCT~US96/05035 patient, and other factors. Typical doses will contain from about 5 mg to about 10 ~L~g DNA, although wide va.idliu,,a from this range are possible while yet achieving useful results.
Other preferred single-vial iOI ' liùns of the invention include pll~ v 'l~ aGc~"i '' ~ . ~
as called for by accepted ,' ~~ac~li"dlpractice. These may ~ buffers, a: ' ~ amino acids, - ' 'i~la, starches, sugars, ' ' " a, rdCi la, , ~' _ agents, t( ';cla, wetting agents, etc.
It has been found that the single-vial rO, provided herein maintain pl~y ' l ' pH without the use of buffering agents except the plasmid DNA itself While additional buffering agents may be omitted, they may dll~.-.dli.~ly be added as occasion provides Preferred r. . F - are suitable for ~ that is pa,~"ld~dl, that is, by any means other than oral. Pa.~ -dl - .dliul~ includes ; : s, such as i l-u~.-uv~ llddll~,idl~ i--ll . ' - ' _, ;~1- ' I l i--ll . ilUIledl~ iull_' ~~, and i-~ lalilial '; ' 5, infusions, and by inhalation.
Injections include -' : dOUn through " '~y,i _ and catheters.
In pdli' ' 1'~ preferred - b~" Is, the invention provides v~ l r-,. ~ having final c~ dlions of 0.9% sodium chloride l~"Jh~ ' ' saline) for iau~ y, 1% glycerol as an e,-,lairi~, (and a ".yl, ot~ , and 0.01% Vitamin E as a IJ~:a~vdlN~ (and an ' ~).
CATIONIC LIPIDS
Cationic lipid reagents that are in use today for DNA 1. '~6UII are r~ d as lipid vesicles or li, - - cu,.i _ cationic or positively charged lipids in r ' ' " with other lipids. The rl ':
may be prepared from a mixture of positively charged lipids, _ ';..,l~ charged lipids, neutral lipids, and 20 ' ' ul or a similar sterol.
The positively charged lipid can be one of the cationic lipids, such as DMRIE, described in U.S.
Patent No. 5,Z64,618, or one of the cationic lipids DOTMA, DOTAP, or ~ ' - thereof, or a -of these. DMRIE is 1,2d 11ialylUAy,Ulu,uyl-3-dimethylh~dlU~y~lh11- ' bromide, and is preferred See Felgner et al., J. Biol. Chem. ~f;9-7!;50(19941. DMRIE can be Sylli' - ' according to Example 1.
Neutral and r _ ';.al~ charged lipids can be any of the natural or synthetic I ' s, b 'i, ~ ' or mono-, di-, or llial,ylylyi,~i.ula. The natural ~h- ,'-1i, '- may be derived from animal and plant sources, such as 'y ~ V ~ 'yl~: ~ e, i, _ ~ ~ P , i ~la~li or r ~ ~
Synthetic l'-~,' 1i, ' may be those having identical fatty acid groups, including, but not limited to, dimyristoylphospha~tidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, " uy\p~rS,' il~;'-' - and the r l~_r ~'V synthetic rh- .' i 1~ ! and ,' ,' lidylgl~ .v!s. The neutral lipid can be ,' ,' ~' '~' e, Cdll" ~i~', rhc~ di' ' ' e, mono-, di- or IOd~.yl~ ll ' or ~-- ' v thereof, such as " ' ~1, ' , ' i 1~1~i' -' (DOPE), which is preferred. DOPE can be ~ ul-dsed from Avanti Polar Lipids (Alabaster, Ala). The ll~ydli.~l~ charged lipid can be .' ,' 1y!91~elul, ,'- .' " acid or a similar rh~ .' li~'' analog. Other additives such as 35 .' ' : ul,yl~ ' fatty acids,, ' v li, 's, p-- v' " 5, 9 v' ' neobee, - orany other W O96/34109 = PCTrUS96/0503 9.
natural or synthetic , ' er' ' ~ can also be used in liposome ru",.u6liun~, as is c~r,. ..i 'Iy known for the ".,, . - of li, s In a L pC "' for preparing cationic 'i, -~ s, the cationic lipid can be present at a I Jr lldi - of between about 0.1 mole % and 100 mole Y~, Lr~rb~l3r'1~ 5 to 100 mole %, and most, ~rb~ bl~ between 20 and 100 mole % The neutral lipid can be present in a CUnC~:Illldliua of between about 0 and 99.9 mole - %, ~.~rb~ , O to 95 mole %, and most ~JIbrbl_~y 0 to 80 mole %. In order to produce lipid vesicles or 'i, having a net positive charge, the quantity of the positively charged component must exceed that of the r,bsdl;.~ly charged ~ , Jr 1. The "?li._l~ charged lipid can be present at between about O to 49 mole % and, brbl~.bly O to 40 mole %. Ch ' ul or a similar sterol can be present at 0 to 80 mole %, and, brb~ O to 50 mole %.
Lipid ~ r having at least one amphipathic lipid can ;, i3. ~ assemble to form 'i,-s Lipid reagents having a cationic lipid species can be prepared as cationic 'i,-- s The - pr I lipids can be dissolved in a solvent such as r' ' Ulu~lll. The mixture can be _., dlbd to dryness as a film on the inner surface of a glass vial. On ~ in an aqueous solvent, the --n,' pc ' Iipid -' ' will assemble ih M,s into 'i, . See Example 2.
The 'i, - - can be analyzed for potency by in vitro lla--;~lL - assays. In these assays, plasmid DNA and cationic lipid - ,' - are formed by mixing of the two sb,ualdlul~ diluted r r ' The mixture is then added to cells in culture and 1- Ct:b assessed according to the I .re' b of Example 3.
PLASMID DNAs The plasmid DNA required for 'i, ' mc~ tPd gene transfer has been widely and routinely prepared in the ' ' dlulr~ for many years. S ' ~.ok, Fritsch, and Maniatis, Ml' ' Clonin~: a LaLûldl Y Manual, 2nd ed., Cold Spring Harbor LaLu,dluly Press, New York, 1989.
These plasmids aLcul J -~ can be selected from among p-.' ~UIib and: ' yUIib vectors, pBR322-and pUC based vectors, and their deli.dli._~, etc. They can utilize any of various origins of ,., " for instance, prokaryotic origins of ~I,' i . such as pMB1 and ColE1, and ' ~rul;b origins of ~I,' .
such as those r " ~".. " in yeast, fungi, insect, and ' cells (~, SW0 ori). They can GIdle any of . genetic elements to facilitate cloning and bA~UI ' n, such as ir' r '' genes, ~G~ Lbl~ leaderpeptide , er,,introns,l~ ' i Pl " ~,Kozaksey- -- ~s.P~'lraJ_.. ~Iai signals, 1~ i,ui l~ , 5' UTRs, 3' UTRs, etc. The selection of vectors, origins, and genetic elements will vary based on ", and is well within the skill of workers in this art.
Genes encoding any of diverse ~lll al proteins (or peptides, p~ s. "1~cn, ui .' r Ui ' S, amidated proteins, etc.) can be inserted into the plasmids for delivery into cells. These genes may rs : genomic DNA, cDNA, synthetic DNA, Fe'~ ~ _liJb~ r~ -liJd, etc.
Transfer of mRNA, antisense oligomers, and triple helix agents are also expressly r .' ~ as falling within the scope of the present invention. These ., -e~ may be obtained using chemical synthesis or W O96t34109 PCTrUS96/05035 gene . ' i t-~ , They can be inserted into plasmids, and the plasmids ' , ll~ lJJùbLd into host cells for p~pal, :iun.
Host cells can be selected from among prokaryotes and t~ dl yUII:S, including bacterial, yeast, fungi, insect and ' cells. Preferred hosts are bacteria, such as E. coli. Any suitable strain of E. coli is -or j : ~
Plùpagdi - of plasmid DNA~ i ~, hosts can be carried out using known IJIu~,eaa~:5. Such p .~ ~ may utilize b~lola, ' ~ ,lu~a, lu.l..~ ,.a, etc., according to batch fe~....ldl fed batch f~ll IdOul1, c..,.; ~ culture, Type 1, Il, and lll ~" Ldliull, aseptic ~l Idliun, uu~laOI i f~" ~ n, protected ~., : . etc. Fitting the c ' (~, medium, i dllJI~:, pH, hours, agitation, aeration, etc.) for ~.(n~ _ to the Lb._ : :CS is empirical and well within the skill of those in the art.
r".i~i ~r of plasmid DNA to ~ ,acL..li-,al grade quality may proceed using well 6ai '' ' p,. Allel"dl;..,ly, ~Jlucesads for ,~ iun of I ' ,,,aceuli~.dlgrade plasmid DNA disclosed in Example 8 are preferred. A~ for reducing RNA ~ ~ di- in cell Iysates using didi _~
earth materials also according to Example 8 are also preferred in purifying plasmid DNA to phz.",ac"~.li"al grade aldndd.da.
REGULATORY PRQCFSSEC
Gene therapy requires approvals from several different " ': y agencies in the United States, including the Food and Drug A~' ~.dliu.l (FDA). The FDA oversees and regulates inter alia that medical drugs are safe and L~R"d";uua and how they are '~Li ud (~L., GMP). Similar approvals are required by most foreign countries. The . _'l .; ' fo" ' i of the invention comply with these r,_ ' y r. .
APPLICATION
In one i,, ' : DMRIEIDOPE cationic lipid mixtures are prepared at from about 90:10 to about 10:90 molar ratio,, e~ at about 50:50 molar ratio. A cationic lipid solution is prepared from the cationic lipid mixture by hydrating a dried lipid film with a suitable diluent, I e~el hl~ with water, at ~
about 2 mg DMRlElmL, I ~, hl~ at about 2 to about 10 mg DMRlElmL, most ~ at about 8 mg DMRlElmL. This highly L lalud cationic lipid solution is ~ subjected to sl~,i' i - by standard autoclave ll~,i 1, e.q., 30 minutes at 121 ~C, in the usual way.
Upon obtaining an ' ~d highly ~ lldlLd DMRIEIDOPE lipid solution, one then dilutes the solution, I Lre~, ' 1y with sterilized water (e.~.. sterile WFI), to < about 2 mg DMRlElmL, p,~ to <
about 1 mg DMRlElmL, most I ~ to < 0.2 mg G'' I I ' Other suitable diluents, such as salines, can be ': ' for water, and are selected on the basis of having low ionic strength, that is, lower than iaui ~ ~y. Optionally, at this point, suitable 1~, ' y agents may be h,l,.' d, zd~dlll _ 'y by addition into the diluent, for , l d into the cationic lipid solution. For example,; ' fiLla may be bllllld~ -d,, ~ glycerol, in amounts to provide a final r~ - ~.dliUil in the ' .;il WO96134109 PCTrUS96/05035 ~u" ': - of from about 0.5 to about 5% glycerol,, ~re~ ~ 'y 1% glycerol. A ' ' ~ ,, t~e,va~ s may also be added at this time,, ~ Vitamin E, in amounts to give a final c~ - ai' in the ' '~ .; ' f....~da ~ of from about 0.005 to about 0.05% Vitamin E,, ~r~lably about 0.01% Vitamin E.
- Plasmid DNA that has been purified to ,' UlaLL.. ~ ur~l quality is ~ ' ' obtained. A
process for the ~ iun of ,uI,a,,,,àLL~LiLdl yrade plasmid DNA is provided in Example 4. The plasmid DNA, - in aqueous solution, such as water (~L, sterile WFI) or other ., r~r iale diluent, such as saline (~, ,' ~ ' -' saline), is sterilized. Sl~, ' li_a is pl~,ably by filtration, for instance, through a 0.2 ~m ' _ filter. It is preferred that the diluent have an ionic strength that is no higher than ;sui~ ~ ~y.
The sterilized plasmid DNA solution and diluted sterilized DMRIEIDOPE lipid solution are usually broughttoroomI~ IdlUI~priorto c ' - TheDNAmaybeusedat c ll nrwithinarange that extends from c dliui,sthat are quite high, for example, 10 mglmL, to r - llai - that are quite low, for example, 0.02 mglml. The solutions are then s ' I, I ~f~, ''y, by LOIllll'' ' addition of the plasmid DNA solution to the DMRIEIDOPE solution with mixing to form DNAllipid ~ , ' at low ionic strength. Low ionic strength means lower than ;au' ~ ~y.
DNA and lipid are combined to produce -( , ' s at a mass ratio of from about 50:1 to about 1:10 DNA to DMRIE,, Lr~.dbly at a mass ratio of from about 10:1 to about 1:5 DNA to DMRIE, most, ~, ' 't at a mass ratio of about 5:1 DNA to DMRIE.
S ' , 'y, the; , ' are adjusted with solutes to iaui ~ y for, ' ~. ' ' )y' ' ~ , d lr In pal i' ' Iy preferred bJ~ the invention provides _' .;al f~ ,uuldi having final con~,~.. lld;' of 0.9% sodium chloride, 1% glycerol, and 0.01% Vitamin E.
UNIVERSALITY
S _' . ' fo, ' - of the invention have been ', 'l~ tested with several different plasmid DNAs, for exampie, plasmids c -i _ reporter genes, for instance, luL;r~laae and 1~ gà6Llua;llase, and plasmids Op~ldli..,ly encoding rJr't~,_,.lid~s suitable for human gene therapy, ~, HLA-B7 and human IL-2.
See Examples 5-10. A~'' 'I~, we have applied the present invention to numerous diverse cationic lipid species and cationic lipid mixtures, for example, DMRIE, DMRIEIDOPE, DOSPA, DOSPAIDOPE, HP-DORIE, HP-DORIEIDOPE, T-MU-DMRIE, T-MU-DMRIEIDOPE, T-MC-DMRIE, T-MC-DMRIEIDOPE, ~-Ser-DMRlE, ~-Ser-DMRlElDOPE"~AE-DMRlE"t~AE-DMRlElDOPE, Arabinose-TU-T-DMRlE,Arabinose-TU-rDMRlElDOPE, C~ t~se-TU-T-DMRIE, ~ rtose-TU-T-DMRlElDOPE, Glucose-TU-T-DMRlE, and Glucose-TU-T-DMRlElDOPE. These results ' :~dle the ._.~ My of the present invention.
SAFETY
s- _' .; ' R ll~ulai of the invention - " DNAIDMRIE-DOPE at a mass ratio of 5:1 DNA
to cationic lipid in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E ' ,aled no signs of toxicity in acute .J._ - toxicity studies in mice, repeat dose safety studies in mice, and repeat dose safety studies in cj~ 1~ monkeys.
STABILITY

W O96/34109 PCTrUS9610503 The above single-vial ru~luuldliul1s were found to be stable. A,,c~ dlt:d testing e.;' ~d full stability over a time course of 8 weeks at 20~C"~Oiy~ldi , and room l~ e.dl.J.t:. This testing showed full stability over a time course of one month at body i , dlU~. These studies d~ Idl~d the utility of the ~ , L of the ru~llluldliull. Example 11.
HUMAN GENE THERAPY
The single-vial ru~ ~ lians of the invention are suitable for human clinical use in vivo ~Example 12-13) or ex vivo (~ U.S. Patent No. 5,399,346 to Anderson et al. for "Gene Therapy").
Particular aspects of the invention may be more readily u-,.R:,~luod by reference to the following ,' which are intended to exemplify the invention, without limiting its scope to the particular 10 ~ ,''i~d P-bc' PREPARATION OF 1.2-DIMYRISTYLOXYPROPYL-3-DIMETHYL-HYDROXYETHYL AMMONIUM BROMIDE (DMRIE) DMRIE was ;~y~LIl~ a;~ed using minor ~--GdiR-.di c of the, .r ' t: ~ ~r~ r d for the synthesis of 15 DOTMA (Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987)). Thus, 3 :" II-y' 1,2-~.. , " ' was r ' d with myristyl mesylate - ,' ~ ~, basic catalysis to generate the c~ r " ~, diether.
S- ' , to ~ . I..yl . ' , iR~ai of this lipophilic amine, q alli~di' was effected by ll~d~
with 2 ! ' :' ~' at elevated; , L:l. The ' . : " , ' ~'ly purified product exhibited IR, TLC, and elemental analyses ~ : with those predicted for the desired hyJIu~.y "~b M salt.

CATIONIC LIPOSOME PREPARATION
DMRIE was ~ylllh6a;~t:d accordiny to Example 1. DOPE was ,uululla;.~.l from Avanti Polar Lipids (Alabaster, Ala). Cationic ~i, ~ were prepared by mixing a ~' ' urullll solution of the lipids in a sterile glass flask. The solvent was removed by e. . , under reduced pressure to produce a dried lipid film.
25 Vials were placed under vacuum overnight to remove any solvent traces. The lipid mixture was hydrated by addition of sterile water for injection.

IAI VITF~O TRAN; ttL; l lrJN PROTOCOLS
Plasmid DNAlcationic lipid ,' were prepared by mixing an aliquot of an pth,. ' ;ida 30 solution with an aliquot of a liposome solution at room i , di 1:. Different ratios of positively charged - to F l~llull~ui ' ~ can be used to suit the need. The methods are a "fi~_: of those described in Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413 (1987), and Felgner and Holm, Focus 11(2) Spring, 1989.
Tl f~.; were carried out in 96-well plates, as follows:
35 (1) The wells of a 96-well ulil~. plate were seeded with 20,000 to 40,000 cells per well;

CA 022l4029 l997-08-27 W O96/34109 PCTrUS96/OSO35 (2) S; ' .;JI r., ~ - of plasmid DNAlcationic lipid ~ ' s were prepared in sterile vials or tubes;
(3) Dilutions of the ' . ' fl,., ' liuns prepared~as in (2) were carried out by serial d6UI~ into cell culture medium (with or without fetal calf serum) ~ :, ' l~d in 3~ ~ ~r ial~ volumes in 96-well plates;
- (4) The cell culture medium covering the cells that had been seeded as in (1) and grown to rll e was removed by ~, dliu.\, (5) The cells were washed with an -'' ' volume of cell culture medium without fetal calf serum, and the wash medium was removed by e, di- 1, (6) A volume of the diluted plasmid DNAlcationic lipid complexes was added to the washed cells in a well of the ulil~, plate; the volume 1, '~ d usually consisted of 80 to 100 ,uL;
(7) Adequate 50% fetal calf serum was added to each well to bring the cùnL~ dliull of serum in the wells to 10%;
(8) The plates were b. ' at 37~C (5% CO2). At 12 and 24 hours post llall.rL~.; . an aliquot of 10% serum in Opti-MEM~ reduced-serum media (GIBCO BRL Life T~ - ' ' y, MD) was added to each well;
(9) At the end of the ' (usually 48 or 72 hours), the medium covering the cells or a whole cell Iysate was assayed for ~AIJII ~ activity.
Where~-gd6,,i'- wasthereportergene,the-, I was ~d 1l i :,i 'Iy,using -' ~ r~11 red~-5dla~(CPRG) as a substrate, reading the plates with a olil~ reader at 405 nm.

PROCESS FOR THE PURIFICATION OF
PHARMACEUTICAL-GRADE PLASMID DNA
Cell Lysis. A cell paste was ll r ~ in 6 mL per gram wet bacterial weight of cold Solution 1(61 mM glucose + 25 mM Tris buffer pH 8.0 + 10 mM EDTA at 5~C) with stirring at room alL.~. To this solution 12 mL per gram wet bacterial weight Solution 11(0.2 N NaOH + 1% SDS) was added and mixed end-over-end until ~ ~9 This was ' ~ ' on wet ice for 3~ 10 minutes. To the Iysed cell solution, 9 mL per gram wet bacterial weight of cold Solution lll (3.0 M
30 p~rl acetate pH 5.0 at 5~C) was added, mixed end-over-end until a white r;
appeared, and ' - on wet ice for .".~"l ~ '~ 5 minutes.
Filtration. The cell debris was removed from the Iysate by filtration, LL~llliruy-: or s- ' The . llat was collected and clarified by adding 3" ~~ ILI~ 25 911 Celite~' :' iearth and filtering through a ~, ~rer "~ pltl,oalLd) filter ' ~ (Whatman # 1, 113 or ~ ' :' arranged in a table top Buchner funnel. All~lua~ , the cell debris was removed from the Iysate by direct Celite@3 aided filtration. In this case, 3~, 1 ~ ILI~ 90 911 Celite'~' diai L earth was added directly to the Iysis W O96/34109 PCTrUS96/05035 solution and mixed by swirling until ~1 - The Iysate was then filtered through a ~ r ,u,~cGal~d) filter ' a"a ~hài # 1, 113 or ~ ' t: arranged in a table top Buchner funnel.
DNA P~u, i rul~lh~l~"d glycol ~PEG, e.a., PEG 8000) was added to the filtrate to 5 ~5%
~wlv), plus NaCI to 0.3-1.5 M. The PEG s--~, was stirred I rr~aLly overnight at 2 8~C. The DNA
1 ~.,;,u;ldle was collected by adding ~ 25 gll of Celite~ did~ rDu ~ earth to the PEG S_-r - -and filtering through a ~,u.er~.~,51~ ,u~r~,Odl~d) filter ' arranged in a table top Buchner funnel. The DNA ,ulu~,;r; dl~ was captured in the Celite'~ cake and recovered by s--r ' ~, the cake in TE buffer ~0.01 M Tris base pH 8.0 + 0.001 M EDTA).
RNA, Protein and I i~ , v'~aaLLhdride Removal. ~ acetate was added to the TE buffer to 2.5 M and stirred for ~,, uAi~l~alal~ 30 minutes at 2-8~C. The , which still c ~~
die earth, was filtered through a ~JIrr~ ' Iy ~ oal~d) filter ' a..a arranged in a table top Buchner funnel. The DNA filtrate was then optionally clarified by alJh ~ I filtration.
Final DNA P", A final DNA ~.", Iiu-- was prlrul ' with 0.6 volumes of cold~ , ~p, ' for a minimum of 2 hours at -20~C. The ,u., , I~d DNA was ~,~..lriruudd in a Sorvall table top LL.. llirL_ for 30 minutes at Z000 x 9 or , .' I. The DNA pellets were Ir, ~ hd in column buffer prior to gel filtration ' I i _ ~,' ,.
6el Filtration Chl~ lu~. . ' r. A rhalllld~;a S 1000 tandem size exclusion column, DNA exclusion limit of 20,000 bp, ~Phdl ~ Pia-,dl~.. d~, NJ) was poured. The S 1000 matrix was an inert and highly stable matrix that was prepared by co. ' ll~ cross linking allyl dextran with N,N' Illt:lh~, ' - ~; ' The column was poured in two rhdlu._~;a XK261100 columns ~PI.a.. aL;d, ria~,al~ ay, NJ) with a final bed height of 80 85 cm ~7 ~y8orln) resulting in a total column volume of ,, uJd~dl~ly 900 mL and a total length of d~,plu,.i...dlul~ 160 cm. The columns were 'i~;' 'l~ pressure packed in one direction, reversed and c -: ' in series for:, '' di- and ~ , - The column was e, "' dl~d in column buffer and run at an 3p~ u~JI;aL~ flow rate. Cleared Iysate plasmid DNA was filtered through a 0.2 ~m filter and loaded onto the column. Column operation and rldbi ti were i l_d with a Pha.llld.dd FPLC ~Phd~l ria-,dlL. a1, NJ). Fractions ~a,r u)dllldlal~ 0.5 5% of column volume) were collected over the product elution zone and analyzed by 0.8% agarose gel ~IU~llUr~ ra;a. Arr.u,u.iale fractions were pooled and , ., tud with 2 volumes of cold ethanol. This column purified DNA was stored frozen at 20~C until needed for ~.L~Jdldliùn of bulk plasmid DNA.
Bulk Plasmid DNA r,l,,- dliUII. The ethanol r.r~ l, column purified DNA was spun at maximum speed in a Sorvall table top C~.~tli6 ~ for 30 minutes at 4-10~C or ~ ' : The pellets were air-dried and pooled. The pooled pellets were ,. , ' ' in vehicle, for example, sterile WFI or l.h~ ' ' saline. The DNA was then filtered through a 0.2 ,um filter into a pyrogen free container. Samples were optionally taken for quality control testing and the ,. ' stored frozen at -10~C to -70~C" r, at-20~C.

W O96/34109 PCTrUS96/05035 HLA-B7 plasmid DNAIDMRIE-DOPE lipid c ,' . intended for use in human gene therapy, - dl'~dlLdi77 vitro potency as - ~' ~ial rull ' l;uns of the invention. See Nabel et al., Human Gene Therapv 3:399 (1992); Nabel et al., Proc. Natl. Acad. Sci. USA 90:11307 (1993); PCT Patent A~," i WO# 94129469; Nabel et al., Human Gene TheraPy 5:57 (1994); Vogelzang et al., Human Gene TheraPy 5:1357 (1994); Hersh et al., Human Gene Therapv 5:1371 (1994); and Rubin et al., Human Gene Therapv 5:1385 (1994).
An HLA B7 encoding plasmid was c~ ~.u.,lcd of about 5000 bp in size. It derived from a pBR322-based plasmid c a bacterial origin of r~ It encoded the heavy (human HLA B7 cDNA) and light (~ r~-2 ,_' ' ' cDNA) chains of a Class 1 MHC antigen '~ ~, lLd HLA-B7. These two proteins were ,A~ ;,aed on a bi cistronic mRNA. Cukalyuli,, cell t:A~ ;Un of this mRNA was :'~, ' l on a Rous Sarcoma Virus (RSV) promoter sequence derived from the 3' Long Terminal Repeat (LTR).
'Y" was also J, ' on a llan~ ,liua ~ 'ya ~l ~lalion signal sequence derived from the bovine growth hormone gene. CAIJ.U of the heavy chain was regulated by the 5' c~p d, protein ll. ' liul. start site. EA~.~ of the light chain was regulated by a Cap ' d~ Tr Enhancer (CITE) sequence derived from the '- ,'-' ,.,caldili, Virus. The plasmid also encoded a k.. ,u;.. r~ .a~e gene derived from Tn903.

W O96/34109 PCTrUS96/05035 The HLA-B7 encoding plasmid was purified to pha~ r~ ' grade - ' da as del~ ' by the criteria given in Table 1 below.

TABLE 1: HLA-B7 PLASMID DNA QUALITY CONTROL CRITERIA

TEST SPEGlrlCATlON METHOD
Size Identity A~, uAilllal~a. Agarose Gel [I~"l",' l~a;a 4900 bp Realli.. liun Sites A r u~dlll~l~s ~ ' Agarose Gel [~ r~ a;a XhollXbal- 3500 & 1400 bp, BgllllXhol - 2100, 1700 8 1000 bp Circular Plasmid DNA > 95YO of visualized nucleic Agarose Gel [I~,llupllO,~a;a acid A2601A280 Ratio 1.75 to 2.00 UV Absu,'-E coli DNA < O.01 ,ugl~g plasmid DNA Southern Slot Blot RNA N .- ' ' on gel Agarose Gel Elc.,lll,,Jhult:a;a Protein L~ BCA C~' :liL Assay Pyl~ t~, Not pyrogenic at 5 ,uglKg Rabbit Pyrogen Assay rabbit body weight '- ' i < 0.1 EUI~g plasmid DNA Limulus A~ ' lcyle Lysate (LAL) Assay Sterility No growth through 14 days Fluid T' ~ Assay Potency 50 200% of reference In Vitro Tl~llal~l,liulll Fl ~SLC~.e General Safety Test Passes per 21 C.F.R. 610.11 Tr_ f~. err ; was dLt~, ~' by HLA-B7 gene LA~ aa;un in SW480 cells, a human colon P 'I -~ . cell line, ATCC # 228-CCL, or UM449 cells, a human ' ~ cell line, Alfred Chang, University of Michigan, following in vitro 1, 'u,,; using HLA-B7 plasmid DNAIDMRIE-DOPE lipid ~
formed by the :hc~ ' ~.1 of the invention in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E.

W O96/34109 PCTrUS96/05035 From 200,00 to 400,000 UM449 cells were seeded per well into a 6-well plate the day before Il. CL_ ' Cells were a >75% confluent -~ . prior to ll .~,:..liun. The cells were llàll~r~lad with 10,1lg plasmid DNA in the presence of 2 ~9 DMRIE (Sylllll~a;~t:d in house) and 2 ~9 DOPE '~, _ "has.,d - from Avanti Polar Lipids, Alabaster, Ala). The cells were ' -l~d at 37~C, 5% CO2 Ih.~ ,.' l. Reduced serum media, e.q., Opti-MEM~' reduced-serum media (GIBCO BRL Life T~' ~", s, G ' ~b~J~u~ MD), _ . r~ with fetal calf serum, was added to the cells 1-4 hours and 24 hours post-l,. 'C.,liull. Cells were l~a~r,~l~d 48 hours post-l~ ~L_ HLA-B7 ., on the cell surface was measured by labelling with anti-HLA-B7 mouse antibody, followed by a rluul ~aCu.\l se -- ' y antibody (anti mouse IgG - ' ' antibody R ph~..L.~. y 11ll bl ~ ~ "_ ) I r' daC~.. I staining of the cells was analyzed by flow cy~ lly. A two-fold increase in mean 1' ~ ? intensity was observed for ll_ h~l~d cells in contrast to negative controls (non-lla"al~LI~d cells or cells ll C~Ll~d with an irrelevant gene). Potency was e~u;. ' I to that of freshly prepared plasmid DNAlcationic lipid - ,' IL-2 plasmid DNAIDMRIE-DOPE lipid r ,' , intended for use in human gene therapy,~alLdin vitro potency as - ' ~;àO rul ' of the invention.
A plasmid encoding IL-2 was cû.l~llu~ d of about 5000 bp in size. It derived from a pUC-based plasmid cu"i ~, a bacterial origin of r ,' i It encoded an IL-2 fusion protein. The protein was - ~., d by cloning a portion encoding a short segment of the 5' :lallalal~d region and the first six amino acids of the leader peptide of the rat insulin 11 gene 5' of the human IL-2 coding sequence minus the first two amino acids of its leader peptide. This fusion protein was placed under the ~ ' ~ulil, Il i~, ' control of the l,yt _ ' .;-ua (CMV) " l~ early 1 r ~ This sequence D " l~d ~ ,., of a r , ~ ~ mRNA c i ~, a 5' ..llàualal~d sequence from the CMV
": early 1 gene, including the 800+ bp intron, the IL-2 fusion protein coding , and a 3' ....~6l~d sequence derived from the bovine growth hormone gene having ll iuUua .du..11~ -signal , r~ Theplasmidalsoencodeda' ~.,;"r~;;,i genederived from Tn903.

W O96/34109 PCT~US96/05035 The IL-2 encoding plasmid was purified to ~' ludG~u~ .adeslalldallla as d~ d by the criteria given in Table 2 below.

TABLE 2: IL-2 PLASMID DNA QUALITY CONTROL CRITERIA

TEST S~EOlrlCATlON METHOD
Size Identity Ap~.. uAilllal~s Agarose Gel [IL~ll.r' ~a;a 4900 bp R6alli Sites A~, UAi"lal~a ~." ": ' Agarose Gel EIL~II p~ ~a;a EcoRI - 3000 & 1900 bp, Ncol - 3700 & 1200 bp Circular Plasmid DNA > 95% of visualized nucleic Agarose Gel [IL~I-upl,u,t~,;a acid A2601A280 Ratio 1.75 to 2.00 UV Al3so,L
E coli DNA < 0.01 ~91~9 plasmid DNA Southern Slot Blot RNA r:~ . on gel Agarose Gel [IL~,III, ' ~a;a Protein < 0.016,ugllJg plasmid DNA Protein Slot Blot Residual Ethanol < 500 ppm Gas Chlul,.ai ,'1 P~ y Not pyrogenic at 5 llglKg Rabbit Pyrogen Assay rabbit body weight r- ' Iu,;,~ < 0.1 EUI~g plasmid DNA Limulus l,r- bc yl~ Lysate (LAL) Asâay Sterility No growth through 14 days USP Direct Transfer Potency 50-200% of reference In Vitro Trallru~,L;ù
ELISA
General Safety Test Passes USP General Safety Test //1/ VIT~O POTENCY OF IL-21DMRIE-DOPE COMPLEXES
Tlallalu: t~R ~ was 1~:, ' by IL-2 gene, .s ~r in B16F0 cells, a mouse cell line, ATCC # CRL 6322, following in vitro llàllar~_ using IL-2 plasmid DNAIDMRIE-DOPE lipid , ' formed by the ... '3d('~,y of the invention in 0.9% sodium chloride with 1% glycerol and 0.01%
Vitamin E.
From 200,00 to 400,000 B16F0 cells were seeded per well into a 6-well plate the day before l-_ CL_ ' - Cells were a >75% confluent ' 1~. prior to 1, Cu~.liun. The cells were llallar~cled with 2.5 ,ug plasmid DNA in the presence of 0.5 ,ug DMRIE (s~lllh6a;LLIl in house) and 0.5 ,ug DOPE

W O96/34109 PCTrUS96/05035 ("...,,hased from Avanti Polar Lipids, Alabaster, Ala). The cells were ;Il~.ubdlud at 37~C, 5% CO2 ' ~
A reduced serum medium, e.n., Opti-MEM'~ d~,bed scrum media lGlBCO BRL Life T~ ' ~, s, G -' ' y, MD), , ' l~d with fetal calf serum, was added to the cells at - : of l~a";.r~ r and 24 ~ hours post llall~r~"i Cell ~u~u~lllaldul was hal~c~l~d 48 to 80 hours post llan~r~
IL 2 e~,u~l in the cell 5l~ Illaldlll was measured by an enzyme amplified ~e"~;li.ity ~ IM ~5 ELlSA, Medgenix r ~ -: 5, Fleurus, Belgium). Potency was ~, . ' I to that of freshly prepared plasmid DNAlcationic lipid complexes.

The stability of IL 2 plasmid DNAIDMRIE DOPE lipid c , ' formed by the ' ~ of the invention and stored in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E was E.' lod at ~20~C, 2~C, 25~C, and 37~C. An ~ .a, of DNAIDMRIE-DOPE - ' in 0.9% sodium chloride with 1% glycerol and no Vitamin E was evaluated at the 37~C storage I , di ~. The stability of the materials in the study was analyzed by a number of methods, including 96 well l~ar,~r~ assay. The results of the study showed that IL-21DMRIE DOPE ~( , ' - and free DNA in 0.9% sodium chloride with 1% glycerol and 0.01% Vitamin E retained full stability over 57 days of storage at -20~C and 2~C. At 25~C
IL 21DMRIE-DOPE - ' ~ retained apparent full activity over 57 days of storage; free DNA showed a half Iife of 500 days for c .~.. from circular to linear form at this Ir"".~,dl...~. At 37~C, IL 21DMRIE-DOPE
c ' s showed a half-life of 34 days; free DNA was r .~ d from circular to linear form with a 290 day half life at this l~ lai ~. In - , in the absence of Vitamin E, IL 21DMRIE-DOPE c showed a half-life of 12 days at 37~C; and free DNA was - ..,. I~d from circular to linear form with a 108 day half-life at this ~ Idlu.~. Therefore, the presence of Vitamin E at the level of 0.01% in the subject bc " I provided 2.7 to 3 times greater stability to the - of the vial.

A. LIPID FORMULATION and AUTOCLAVE STERILIZATION.
1. PI~lJaldl of the DMRIEIDOPE Bulk Solution.
Using an _ ~yli~,dl balance, the batch amount of DMRIE Br was weighed out.
Working in a ~~..lilaled laminar flow hood, the DMRIE Br was placed in a clean 50 mL round bottom flask.
Using a 5 mL glass pipet, five (5) mL of ' ' uru~lll was added to the round bottom flask from above. The flask was swirled to dissolve the DMRIE Br.
Using a SMI pipet, the batch amount of DOPE was added.
The neck of the round bottom flask was rinsed with about five (5) mL of r'' uru~lll and the contents swirled gently for at least one minute to mix. Any solution adhering to the neck after swirling was rinsed into the flask with '" ' r'' uR..Ill.

W O96/34109 PCT~US96/05035 Using a rotary ~........................... dpUld~UI~ the ' ' U~u,l~ was removed from the solution from above. The flask was kept on the rotary e~, di ~ untii ce~ u~ was no longer visible in the ~
A dea;.,cd was IhGI~__"y wiped down with alcohol and placed in a ~..Li6led laminar flow hood.
The round bottom flask from above was placed in the d~ Ca~ . The desi,,cdlu, was cc : ' to a vacuum pump, the dl ~ e~d~ualed~ and the round bottom flask ~ d under vacuum for at least 12 hours.
After at least 12 hours exposure to vacuum, the deJ;I~cdi was isolated from the vacuum pump.
Working in the ~,..li6l~,d laminar flow hood, the ~4,: ~ al~ was c ' to a nitrogen gas source, the gas turned on, and the vacuum released. When the d - lù~ was filled with nitrogen yas, the gas source was removed.
The round bottom flask was removed from the ~le-~ lu,. Using a sterile ', - ' ' pipet, sterile water for injection was added to the flask. The flask was capped, the liquid swirled, and vortexed for at least 5 minutes or until luh~l' di ~r was achieved.
2. i'~"a,dliun of DMRIEIDOPE bulk liPid mixture vials for autoclave Irt:dlult:llL.
The solution from above was lldll~ d in --0.5 mL aliquots to clean 2-mL Type 1 glass vials.
Each vial was capped with a clean teflon-coated gray butyl stopper, and the cap secured with an aluminum crimp.
The filled vials from above were l,ui ' .~d using a standard liquid cycle on the ~ ul,l~ (no less than 121~C for 30 minutes). If i...ll~cl~d vials were not used ~ ' 1y, they were stored at 2 to 8~C.
The ' .~d vials were not held at room ~ , u for more than 6 hours.
B. PLASMID DNA FORMULATION.
1. Sterile filtration of IL-2 Plasmid DNA Standard Bulk Solution.
Working in a ~~..li6lGd laminar flow hood, the plasmid DNA standard bulk solution was filtered through a 0.2,um sterile filtration unit. The filtrate was collected in a sterile d, ~- '' tube. The tube was stored at 15 to 30~C.
C. PLASMID DNAIDMRIE-DOPE LIPID MIXTURE FORMULATION AND FILL.
1. P~UIJdldi of the DMRIEIDOPE lipid solution.
Working in a ~..li6l~d laminar flow hood with all materials at room l~ JLIdlul~ (15 to 30~C), sterile water for injection was Lld~ d into a sterile li, - ' 250-mL bottle.
An dU~,.I,iale amount of stock glycerin and an a,, ., idle amount of stock Vitamin E was added to the bottle from above and swirled to mix.
An au,u~, idle amount of autoclave sterilized DMRIEIDOPE bulk lipid mixture from above was added to the solution from above and swirled to mix.
An aPP~r iale amount of the filtered IL-2 plasmid DNA solution from above was added to the DMRIEIDOPE lipid solution from above. The bottle was capped and the mixture was swirled ~ by hand for at least one minute.

.

~ ~ =

WO 96/34109 PCTrUS96/05035 An ., up,idle amount of stock (5%) sodium chloride injection was added to the IL-2 plasmid DNAIDMRIE-DOPE lipid complex solution from above. The bottle was capped and the mixture was swirled ~,;grn ly by hand for at least 1 minute.
2. FilllFinish.
Working in a ~,.lilaltd laminar flow hood, 1.2 mL of the IL2 plasmid DNAIDMRIE-DOPE lipid complex from above was ar~p~ filled into prepared 2 mL type I glass vials using a Drummond Pipet-Aid and sterile ', Q~'' 2 mL pipets.
The vials were sealed with prepared 13 mm teflon-coated gray butyl stoppers and 13 mm tab-top aluminum caps. All vial caps were crimped with a hand crimper.

PHASE I HUMAN CLINICAL TRIALS
Direct ;..I~di injection of plasmid DNA ~:AIJII ~ vectors provides a method for the i"ll~d : Jr of IL 2 genes into the tumor. In the Phase I study proposed in this protocol, the sponsor tests for safety and dose a, i of the direct gene transfer approach for delivering the IL-2 gene directly into solid tumors and 1~, hr r'~ I ~ ~ of the IL-2 gene is - 'il ' The IL-2 produced should elicit an ~ ' _ antitumor response which in turn may lead to a systemic ' _ -' ~' of other tumor cells. Dose 1l r 1! are ~ 1~6lud with specific immune ", ~r. -S,r~ '; '1~, the phase I protocol is designed:
1) to minimize the risks to the patient;
2) to derive the maximum i"r. . regarding the , . of ,~ ~l ' I genes in vivo;
3) to maximize potential benefit to the patient; and 4) to gain new ' . 'L '9 regarding gene delivery to tissues in vivo and the immune response to tumors.
The . b; : .~s of the clinical plan include:
1) To confirm the in vivo eAIl~U ~ of the IL-2 gene in the tumor cells.
2) To r!: , the safety and toxicity of direct _' ' injections of b~ tda;~ld amounts of a DNAllipid mixture, IL-21DMRIE-DOPE, into patients with advanced 'ig y.
3) To d~l~l ~ the biological activity and, ' --' : of ll~dll_l with IL-21DMRIE-DOPE.
4) To chalaclLIi~: the clinical response to escdldi " doses of the study drug by assessing the size of the injected tumor and of other tumor masses that may be present.
The product is L d of plasmid DNA coding for IL-2, ~ d in an injection vehicle with the cationic lipid mixture DlAh~ OPt. When ~,-' d into the target tumor tissue, the lipid ~
ll_ h ~ of cells with the plasmid. On ll~l : of the plasmid into cells, the ru ' : gene is eA~.,eased. The lipid mixture is a ~ ' i of two ~r ' DOPE (CAS name: 1,2-dioleoyl-sn-glycero-W O96/34109 PCTrUS96/05035 3,'-;p~v ' ' ~ a) and DMRIE ~yllLh~ ud as DMRlE-Br (CAS name: I+l-)-N-(2-h~-huxytlhYI)-N,N-dimethyl-2,3-L;~ ' jlo~y)-1-~ r bromide) which are assumed to be rapidly : bc' The, '~ ~ ~'li, ~ ' mixture and injection vehicle are produced in aLGull' with the "It: ' - ' ' "y of the invention. The plasmid DNA is R..lllalal~d with DMRIEIDOPE lipid mixture in the injection vehicle which s~ : 1% glycerol and 0.01% Vitamin E in normal aqueous saline (O.9% sodium chloride in sterile water for injection). The dosage form is delivered by injection into solid tumor tissue.
The c~ - lld" of plasmid DNA and DMRIEIDOPE in each dose package is spec!fied in the following table:
TABLE OF DOSE PACKAGES

Dose Package Plasmid DNA Mass dried Volume sterile v llaliun (mglmL) DMRIEIDOPE (mglmL) Injection vehicle (mL) lO,ug 0.01 0.0041.2 30 ~ug 0.03 0.0121.2 100 ~9 0.10 0.041.2 30~,ug 0.30 0.121.2 This is a Phase I ~ ?1 study in which up to 25 patients are enrolled for injections directly into tumor nodulés with a lipid-~G~I ' Ird IL-2 plasmid complex. Solid tumors ( ' ' ~ bony tumors), and ,: of 1il ' renal cell Call a, and hepatic -l~ s of advanced cc~:~.ldl ca",;"ull,a, and l~,. . ' are the tumor types to be evaluated.
Eligible patients have a primary tumor nodule injected several times at specified intervals with a specified dose of the study drug (see below). There are four groups with 5 patients each treated at the ..,;bEd dose (10, 30, 100 or 300 ~g), with a group of 5 patients retreated at the maximum tolerated dose (MTD), or at 300 ~g if the MTD is not reached. The highest dose that does not yield Grade 3 or higher toxicities is ' ~d the MTD. All toxicities are graded according to the World Health Olya~ ai (WHO) Re ~ :- for Grading of Acute and Sub-Acute Toxic Effects.

CA 022l4029 1997-08-27 W O96/3~109 PCT~US96/0~03 TABLE OF SPECI~ltu DOSES

Dosage No. Of Dose PerTotal No. Of Days GroupPatientsTreatment Tl~ai Between Per Group Per Patient Tl~a~ s 1 5 10~9 6 7 Il 5 30~9 6 7 lll 5 100 ~9 6 7 IV 5 300 ~9 6 7 The study drug is P' : ~d and toxicities are ~d. Tumor lesions are selected for ll~d~ if they are acc~ '' to lldi -' . by direct needle injection. These Ill~la~lali., lesions are located at any ' ' site such as skin, nodes, lung, liver, soft tissues etc. Bony tumors are excluded. The amount of study drug material injected into each tumor is based on the algorithm outlined below. The p,t~..,ib~d dose (10, 30, 100 or 300~19)iS thawed and diluted with injection vehicle to the 3" ~, ial~ volume. If s y, the study drug is injected with the aid of ~l. .' or CAT scan ~; ' of the - Id~lda;~. Prior to injection, following ,' of the needle, gentle , d ' is applied to the syringe to ensure that no material is injected ;.,IIu. '~. After injection of the drug and with the needle still in place, the dead space is flushed with 0.25-0.50 mL of sterile normal saline (0.9%
sodium chloride in sterile water for injection).
Tumor Diameter (cm) Volume of Injection (cc) 1.0-1.5 1.0 1.6-2.0 2.0 Z.1-3.0 3.0 3.1X 4.0 Vital signs are measured every 15 minutes at the start of, during, and after the injection for at least 2 hours or until the patient is stable. If the systolic blood pressure drops below 80 mm Hg, the injection isl~ y and the patient is closely ad and treated a~ r ial~ until blood pressure is r s Patients are closely ~d for toxicity for 34 hours post injection, then 24 hours and 7 days after the first and second : For injections 3-6, patients are ~d for 3-4 hours post injection then 7 days post injection as long as they have i~""cd no toxicity during the 4 and 24 hour st ,~ v periods following injections 1 and 2.

CA 022l4029 l997-08-27 W O96/34109 PCT~US96/05035 TABLE OF SCHEDULE FOR POST-INJECTION MONITORING

Treatment # 34 Hrs 24 Hrs 7 Days 14 Days X X X ---X --- X ---Before each L , : injection, patients are evaluated for toxicities from the prior injection and injected with the next dose only if no Grade 3 or higher toxicity occurs. A tumor sizing is done at each dl..,..o(al injection of the nodule. If the tumor shrinks to a point where it can no longer be injected, c~ doses are ~ ' ~ ~ ~d into another tumor nodule if any are present.
After the 6th injection, patient follow-up includes e.~' i with tumor sizings at weeks 8 and 16. After the week 16 visit, patients are evaluated a minimum of every 4 months.If a patient r,A~JLli stable disease or a partial response (see below) at 4-8 weeks after the last injection of their initial course, helshe may receive an additional course of ll~alll-r,--l identical to the first course of 1~ ; or the next higher dose. The patient must, however, continue to meet the entry criteria.
Adverse events are ~- Gd, and patients are removed from the study if ullacL~J '' toxicity (Grade lll or IV) develops.
Classical, ' ~c~' ., ' studies of drug ' ~,iL . half time" : ' ' . and excretion are not entirely relevant to in vivo gene injection and , . ~ However, the fate of the plasmid and detection of the gene product (IL-2) are relevant to the ' ..', ~ of this agent. In addition, immune a-.li.ai is 25 i , lalll. Therefore, as part of the ~,.. a.,t of the efficacy of this study, - '.11 gene transfer and e"~,.t..~;u.. is evaluated by molecular and i ' ' analyses. The following pal_ : ~ are measured to evaluate the tumor Ir c~ and ~AIJII ~ of IL-2: 1) the presence of DNA from the IL-2 gene is assessed by PCR - , ' C;~ai of cells obtained by biopsy of the treated site after the injection of the study drug, 2)i '~~ ' ~ ' staining of tumor biopsy samples is used to assess ~ ", response and 30 soluble IL-2 eJ~Ir~;u~, 3) serum IL-2 levels are measured pre-llr,ai I and 2 times post the start of therapy, however, the detection of serum IL-2 levels is not liL;~Jal~d due to IL-2 ~ : ' ' ~" 4) PCR analysis uf pr,li~Jllr,,al blood samples is used to test for the presence of plasmid DNA after the start of llr,allll.:llL and c , _ ~,d to pre-therapy, but detection of the gene in r~-~;.' dl blood samples is not . i , d, 5) the cellular immune response is evaluated by l.._a~....i ~, baseline and pOst-llr,allll_..llL-2 induced a..li., of 35 PBMC by Ih,~ ' - uptake assay and NKI LAK response in ~ ,' dl blood pre-therapy and post-therapy, and 6) an attempt is made to excise tumor tissue from another site prior to ll, l for di;~ ~ .

WO 96/34109 PCT~US96105035 y, cryo; t;~lvai and to evaluate r i ~ al blood 1~, ' yl~ ' -' reactions to the tumor before and after 1,.
As an -'' ' part of the ~. ' i of the efficacy~of this study, the clinical response is J measured. Standard oncologic criteria are applied to d~l~l whether or not a patient responds to the study drug. All tumor - ~ la are recorded in ~ : a and cr ~ the longest diameter and the r ~ ~ ~ diameter at the widest portion of the tumor. The tumor response d~ri Iisted below are used to compare current total tumor size to pre-lu I total tumor size.
There is a complete tumor response upon ' ., a"Le of all clinical evidence of active tumor for a minimum of four weeks, and the patients is free of all 51~ . i of cancer.
There is a partial tumor response upon fifty percent (50%) or greater decrease in the sum of the products of all ' ~ a of ~ '' lesions. These " ' : - in tumor size must endure for a minimum of four weeks. No ' increase in the size of any lesion or ~, a"..e of new lesions may occur.
The a~ , iàle ' _ ~_ tests used to d~ ..,al,dl~ the response must be repeated four weeks after initial ~b~ v: - in order to d~ I this duration.
There is stable disease upon less than 50% decrease in the sum of the products of all ' : a of - ' ' lesions, or an increase in the tumor mass less than 25% in the absence of the du.. ' r of new lesions.
There is r ~_ I ~ disease upon tumor, ~v bi.S;U~ as defined if one or more of the following criteria are met: 1 ) 3~ ~ ~ . e of any new ~t 's).2) increase in tumor size of 225% in the sum of the products of all d;a",dlL,a of I ' ' lesions, 3) _ " I clinical 'l iUld'- that cannot be dllH' to ll~ai ~ or other medical c~ " and is assumed to be related to increased tumor burden, and 4) ., _ of tumor related a~ , deemed clinically: ' : by physician.
The principles of informed consent described in Food and Drug A ' ~,di' (FDA) R v ~ 21 C.F.R. Part 50 are followed.
The ~" ., idl~ approvals are obtained from the relevant h,~lilui ' Review Board (IRB), the l;~r ' ~ DNA Advisory Cc ~ (RAC) of the National Institutes of Health (NIH), and the Food and Drug A~ (FDA).
While particular bc ' : of the invention have been described in detail, it will be apparent to those skilled in the art that these - bc ' Ia are e , ' y rather than limiting, and the true scope of the invention is that defined within the attached claims.

Claims (14)

1. A process for preparing a single-vial formulation of plasmid DNA/cationic lipid complexes comprising the steps of:
(a) providing a sterilized cationic lipid solution.
(b) providing a sterilized plasmid DNA solution;
(c) in this order, aseptically adding the plasmid DNA solution of step (b) to the cationic lipid solution of step (a) to form plasmid DNA/cationic lipid complexes in a solution, said solution having an ionic strength that is lower than isotonicity;
(d) aseptically adjusting the solution of plasmid DNA/cationic lipid complexes of step (c) to near isotonicity; and (e) storing the solution of plasmid DNA/cationic lipid complexes of step (d).

2. The process of Claim 1, wherein step (c) comprises freezing the solution of plasmid DNA/cationic lipid complexes.

3. The process of Claim 1, wherein the cationic lipid solution of step (a) has a concentration in the range of from about 0.01 to about 1.0 M and wherein the plasmid DNA solution of step (b) has a concentration in the range of from about 0.05 to about 10 mg/mL.

4. The process of Claim 1, wherein the cationic lipid solution of step (a) is prepared by autoclave sterilizing a cationic lipid solution having a concentration in the range of from about 0.5 to about 5.0 M.

5. The process of Claim 1, wherein the cationic lipid solution of step (a) is a cationic lipid solution of DMRIE/DOPE having a molar ratio in the range of from about 90:10 to about 10:90.

6. The process of Claim 5, wherein the cationic lipid solution of DMRIE/DOPE of step (a) has a concentration of ~ about 2 mg DMRIE/mL, wherein the plasmid DNA solution of step (b) has a concentration of ~ about 10 mg plasmid DNA/mL, and wherein the plasmid DNA/cationic lipid complexes of step (c) are formed at a mass ratio of from about 50:1 to about 1:10 DNA to DMRIE.

7. The process of Claim 5, wherein the cationic lipid solution of DMRIE/DOPE of step (a) is prepared by autoclave sterilizing a cationic lipid solution of DMRIE/DOPE having a concentration in the range of from about 2 to about 10 mgDMRIE/mL.

8. The process of Claim 1, wherein the cationic lipid solution of step (a) is a cationic lipid solution of DMRIE/DOPE having a molar ratio of about 50:50.

9. The process of Claim 8, wherein the cationic lipid solution of DMRIE/DOPE of step (a) has a concentration of ~ about 1 mg DMRIE/mL, wherein the plasmid DNA solution of step (b) has a concentration of ~ about 5 mg plasmidDNA/mL, and wherein the plasmid DNA/cationic lipid complexes of step (c) are formed at a mass ratio of about 5:1 DNA to DMRIE.

10. The process of Claim 8, wherein the cationic lipid solution of DMRIE/DOPE of step (a) is prepared by autoclave sterilizing a cationic lipid solution of DMRIE/DOPE having a concentration of about 8 mg DMRIE/mL.

11. The process of any of Claims 1-10, wherein the cationic lipid solution of step (a) comprises an emulsifier.

12. The process of any of Claims 1-10, wherein the cationic lipid solution of step (a) comprises water as the solvent.

13. A single-vial formulation prepared by the process of any of Claims 1-12 that is stable in frozen form for at least about 8 weeks.

14. A single-vial formulation prepared by the process of any of Claims 1-12 that retains in vitro transfection efficiency of freshly prepared plasmid DNA/cationic lipid complexes for at least about 8 weeks.