United States Patent [19]
`Ansell et al.
`
`[54] COMPOSITIONS FOR THE INTRODUCTION
`OF POLYANIONIC MATERIALS INTO
`CELLS
`
`[75]
`
`Inventors: Steven Michial Ansell; Barbara Mui;
`Michael Hope, all of Vancouver,
`Canada
`
`[73] Assignee: Inex Pharmaceuticals Corporation,
`Vancouver, Canada
`
`[21] Appl. No.: 442,267
`
`[22] Filed:
`
`May 16,1995
`
`Related U.S. Application Data
`
`[62] Division of Ser. No. 316,399, Sep. 30, 1994, abandoned.
`Int. CL 6
`[51]
`.......................... A61K 37/22; C07H 21/02;
`C07H 21/04
`[52] U.S. CI .................................... 514/2; 514/21; 514/43;
`514/44; 424/450
`[58] Field of Search .................................... 514/2, 21, 43,
`514/44; 424/450
`
`[56]
`
`References Cited
`
`U.S. PATENr DOCUMENTS
`7/1983 Taylor ....................................... 424/85
`4,394,372
`4,897,355
`111990 Eppstein et al. .
`5,171,678 1211992 Behr et al ..
`5;234,767
`8/1993 Wallach ............................... 428/402.2
`5;279,833
`1/1994 Rose .
`5;286,634
`211994 Stadler et al ..
`
`FOREIGN PATENf DOCUMENTS
`
`2 552 679
`4/1985 France .
`2147263
`5/1985 United Kingdom.
`WO 91116024 10/1991 WIPO .
`WO 93/05162
`3/1993 WIPO.
`
`OTHER PUBLICXTIONS
`Ito et al., ''Synthetic Cationic Ampliphiles for Lipsome-Me(cid:173)
`diated DNA Transfection", Biochemistry International,
`22:No. 2, p. 235 (1990).
`Pinnaduwage et al .. "Use of a quaternary ammonium deter(cid:173)
`gent in liposome mediated DNA transfection of mouse
`L-cells", Biophysica Acta 985 pp. 33-37 (1989).
`Philip, R., et al., "Cationic Liposome-Mediated Transfec(cid:173)
`tion of Immune Effector Cells," J. of Liposome
`Resean:h3(1):71-84 (1993).
`Hawley-Nelson, et al., Focus 15(3):73 (1993).
`Stomatatos, et al., Biochemistry 27:3917-3925 (1988).
`Zhu, N., et al., Science 261:209-211 (1993).
`Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990).
`Chang, A.C.Y., et al., Focus, 10:68 (1988).
`Hope, M.J., et al., Biochimica et Biophysica Acta,
`812:55-65 (1985).
`
`1111
`
`Ill~~ 111111~11 1111~11111111~1111111111111 1111
`US005753613A
`5,753,613
`[11] Patent Number:
`[45] Date of Patent:
`May 19, 1998
`
`al., Molecular Pharmacology,
`
`Struck, D.K., et al., Biochemistry, 20:4093 (1981).
`Woodle, M.C., et al, Biochimica et Biophysica Acta,
`1105:193-200 (1992).
`Bennett, C.F.,
`et
`41:1023-1033 (1992).
`Wood, P.G., Methods in Enzymology, 149:271-280 (1987).
`Hyde, Stephen, C., et al., Nature, 362:25(}..255 (1993).
`Brigham, Kenneth L., et al., American Journal of the Medi(cid:173)
`cal Sciences, 298( 4 ):278-281 (1989).
`Behr, et al., Proc. Natl. Acad. Sci. USA, 86:6982-6986 (Sep.
`1989).
`Stewart, MJ., et al., Human Gene Therapy, 3:267-275
`(1992).
`Nicolau, C., et al., Methods in Enzymology, 149:157-184
`(1987).
`Feigner, P.L., et al., Natl. Acad. Sci., USA, 84:7413-7417
`(Nov. 1987).
`Gao, X., et al., Biochemical and Biophysical Resean:h
`Communications, 200(3):1201-1206 (May 16, 1994).
`Pickering J.G., et al., Circulation, 89(1):13-21 (Jan. 1994).
`Kaneda, Y., et al., Methods in Enzymowgy, 221:317-327
`(1993).
`Thierry, A.R.,
`et
`20(21):5691-5698.
`Keren-Zur, M., et al., Biochimica et Biophysica Acta,
`983:253-258 (1989).
`Nicolau, C., et al., Methods in Enzymology, 149:157-176
`(1987).
`Debs, R., et al., Am. J. Respir. Cell Mol. Biol., 7:406-413
`(1992).
`Brigham, K.L., et al., Am. J. Respir. Cell Mol. Bioi.,
`8:209-213 (1993).
`Canonico, A.E., et al., Am. J. Respir. Cell Mol. Bioi.,
`10:24-29 (1994).
`Mack, K.D., etal.,Am. J. Medical Sciences,307(2):138-143
`(Feb. 1994).
`Farhood, H., et al., Annals New York Academy of Sciences,
`716:23-35 (1994).
`Primary Examiner-Nathan M. Nutter
`Attome)l. Agent, or Firm--Townsend and Townsend and
`Crew
`[57]
`
`al., Nucleic Acids Research,
`
`ABSTRACT
`
`The present invention provides compositions and methods
`which are useful for the introduction of polyanionic mate(cid:173)
`rials into cells. The compositions are mixtures of cationic
`compounds and neutral lipids which are typically formulated
`as liposomes. The cationic compounds are quaternary
`ammonium compounds wherein the nitrogen has two
`attached long chain alkyl groups, at least one of which is
`unsaturated. The methods for transfecting cells involve (a)
`contacting the polyanionic materials with the compositions
`above to form a polyanionic material-liposome complex,
`and (b) contacting the complex with the cells to be trans(cid:173)
`fected.
`
`4 Claims, 4 Drawing Sheets
`
`Moderna Ex 1012-p. 1
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`

`

`U.S. Patent
`
`May 19, 1998
`
`Sheet 1 of 4
`
`5,753,613
`
`CHARGE RATIO ( +/-}
`
`FIG.
`
`I.
`
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`

`

`U.S. Patent
`
`May 19, 1998
`
`Sheet 2 of 4
`
`5,753,613
`
`L&..J
`
`L&..J
`
`0 z:
`0 "'
`
`120
`
`JOO
`
`80
`
`60
`
`I.LJ
`a::
`~
`0
`....J
`~ 4
`"#-
`
`DDAB
`
`DODAC
`
`DOTNA
`OSDAC
`CHARGE RATIO ( +/-)
`tSSSI I
`
`FIG. 2.
`
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`

`

`U.S. Patent
`
`May 19, 1998
`
`Sheet 3 of 4
`
`5,753,613
`
`>- 600
`0 :z:
`u.J 500
`0 .....
`.....
`L&.l 400
`:z:
`0
`...,
`6 300
`"'-Cl')
`:z: c
`cc::
`.......
`L&.l >
`:c
`....J
`L&J cc::
`
`200
`
`100
`
`0
`
`DDAB
`
`c:::::J 0.25
`
`DOTNA
`OSDAC
`CHARGE RATIO ( +/-)
`EZZ2 0.5 ~ 3
`
`DOOAC
`
`mnmn 4
`
`FIG. 3.
`
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`

`

`U.S. Patent
`
`May 19, 1998
`
`Sheet 4 of 4
`
`5,753,613
`
`- 40
`-
`
`~
`>-
`c.:> z
`Y.J 30
`c.:>
`"'-
`"'-
`Y.J
`z
`0
`t=
`c.:>
`Y.J
`"'-U')
`:z
`c
`0:::
`~
`
`20
`
`10
`
`.... OOTNA=DOPE
`
`+ DODAG: DOPE
`
`I~
`\
`\
`\
`\
`\
`\
`\
`
`/
`
`I
`I
`I
`I
`I
`I
`I
`
`0
`0.0
`
`0.5
`
`1.0
`
`2.0
`2.5
`1.5
`CHARGE RATIO (+I- )
`
`3.0
`
`3.5
`
`FIG. 4.
`
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`

`5,753,613
`
`1
`COMPOSITIONS FOR THE INTRODUCTION
`OF POLYANIONIC MATERIALS INTO
`CELLS
`
`2
`FIG. 2 illustrates the fusion of lipid/DNA complexes with
`RBC ghosts.
`FIG. 3 illustrates a preliminary survey of the transfection
`abilities of cationic lipid vesicles formulated with 50 mole %
`This is a Division of application Ser. No. 08/316,399 5 DOPE.
`filed Sep. 30, 1994 and now abandoned.
`FIG. 4 illustrates the charge ratio titration of DOTMA-
`:DOPE and DODAC:DOPE transfection efficiencies in
`BHK cells.
`
`BACKGROUND OF THE INVENTION
`Gene therapy is an area of current interest which involves
`DErAILED DESCRIPTION OF THE
`the introduction of genetic material into a cell to facilitate lO
`INVENTION
`expression of a deficient protein. There are currently five
`major methods by which this is accomplished, namely: (i)
`Abbreviations and Definitions
`calcium phosphate precipitation, (ii) DEAR-dextran
`The following abbreviations are used herein: BHK, baby
`complexes, (iii) electroporation, (iv) cationic lipid com-
`hamster kidney; RBC, red blood cells; DDAB, N,N-
`plexes and (v) reconstituted viruses or virosomes (see 15 distearyl-N,N-dimethylammonium bromide; DODAC, N,N-
`Chang, et al., Focus 10:88 (1988)). Cationic lipid complexes
`dioleyl-N,N-dimethylammonium chloride; DOPE, 1,2-sn-
`are presently the most effective generally used means of
`dioleoylphoshatidyethanolarnine; DOSPA, 2,3-dioleyloxy-
`effecting transfection.
`N -(2( sperminecarboxamido )ethyl)-N ,N-dimethyl-1-
`A number of different formulations incorporating cationic
`propanaminium trifluoroacetate; DOTAP, 1,2-dioleoyloxy-
`lipids are commercially available, namely (i) lipofectin 20 3(N,N,N-trimethylarnino)propane chloride; DOTMA, 1,2-
`(which uses 1,2-dioleyloxy-3-(N,N,N-trimethylarnino)
`dioleyloxy-3-(N,N ,N-trimethylarnino )propane chloride;
`OSDAC, N-oleyl-N-stearyl-N,N-dimethylammonium chlo-
`propane chloride, or DOTMA, see Eppstein, et al., U.S. Pat.
`'d Rf
`4 2 h dr
`h 1
`No. 4,897 ,355); lipofectarnine (uses DOSPA, see Hawley-
`n e;
`• room temperature; HEPES, -( - Y oxyet Y )-1-
`Nelson, et al., Focus 15(3):73 (1993)); and lipofectAce (uses
`piperazineethanesulfonic acid; FBS, fetal bovine serum;
`N,N-distearyl-N,N-dimethylammonium bromide, or 25 DMEM, Dulbecco's modified Eagle's medium; PBS,
`DDAB, see Rose, u.S. Pat. No. 5,279,833). Other research-
`phosphate-buffered saline; EGTA, ethylenebis
`ers have reported alternative cationic lipids that work in
`(oxyethylenenitrilo)-tetraacetic acid; SF-DMEM, serum-
`essentially the same manner but with different efficiencies,
`free DMEM; and NP40, nonylphenoxypolyethoxyethanol.
`for example 1,2-dioleoyloxy-3(N,N,N-trimethylarnino)
`As used herein, the term "alkyl" refers to a saturated
`propane chloride, or DOTAP, see Stomatatos, et al., Bio- 30 hydrocarbon radical which may be straight-chain or
`chemistry 27:3917-3925 (1988)); glycerol based lipids (see
`branched-chain (for example, methyl, ethyl, propyl,
`Leventis, et al., Biochem. Biophys. Acta 1023:124 (1990);
`isopropyl). Preferred alkyl groups for some substituents are
`lower alkyl groups containing 1 to 3 carbon atoms. For other
`lipopolyarnines (see, Behr, et al., U.S. Pat. No. 5,171,678)
`and cholesterol based lipids (see Epand, et al., WO
`alkyl group substituents, long chain alkyl groups containing
`93/05162).
`35 from 16 to 20 carbon atoms are preferred. All numerical
`ranges in this specification and claims are intended to be
`Others have noted that DOTMA and related compounds
`inclusive of their upper and lower limits.
`are significantly more active in transfection assays than their
`The term "acyl" refers to a radical produced from an
`saturated analogues (see, Feigner, et al., W091/16024).
`organic acid by removal of the hydroxyl group. Examples of
`However, both DOTMA and DOSPA based formulations,
`40 acyl radicals include acetyl, pentanoyl, palmitoyl, stearoyl,
`despite being the most efficient of the cationic lipids in
`myristoyl, caproyl and oleoyl.
`effecting transfection, are prohibitively expensive. DDAB
`As used herein, the term "pharmaceutically acceptable
`on the other hand is inexpensive and readily available from
`anion" refers to anions of organic and inorganic acids which
`chemical suppliers but is less effective than DOTMAin most
`provide non-toxic salts in pharmaceutical preparations.
`cell lines.
`45 Examples of such anions include chloride, bromide, sulfate,
`What is needed in the art are new compositions and
`phosphate, acetate, benzoate, citrate, glutamate, and lactate.
`methods which are both more effective at transfection and
`The preparation of pharmaceutically acceptable salts is
`more affordable. Surprisingly, the present invention pro(cid:173)
`described in Berge, et al., J. Pharm. Sci. 66:1-19 (1977),
`vides such compositions and methods.
`inc01porated herein by reference.
`As used herein, the term "polyanion" refers to materials
`SUMMARY OF THE INVENTION
`having more than one anionic group. For example, polyan-
`The present invention provides compositions and meth-
`ion is used to refer to nucleic acids, both DNA and RNA
`ods which are useful for the introduction of polyanionic
`which are present in their polyanionic form having more
`materials into cells. The compositions are mixtures of cat-
`than one anionic phosphodiester group along the nucleic
`ionic compounds and neutral lipids which are typically 55 acid backbone. The term "polyanion" also refers to those
`formulated as liposomes. The cationic compounds are qua-
`pharmaceutical agents which have more than one anionic
`ternary ammonium compounds wherein the nitrogen has two
`group at neutral pH. Such pharmaceutical agents include
`attached long chain alkyl groups, at least one of which is
`peptides having multiple carboxylic acid functionalities
`unsaturated. The methods for transfecting cells involve (a)
`present (i.e., Glu, Asp).
`contacting the polyanionic materials with a liposomal for- 60
`The term "neutral lipid" refers to any of a number of lipid
`mulation of the above compositions to generate a complex,
`species which exist in an uncharged or neutral zwitterionic
`and (b) contacting the complex with the cells to be trans-
`form at physiological pH. Such lipids include, for example
`fected.
`d i a c y 1 p h o s p h a t i d y 1 c h o 1 i n e ,
`diacylphosphatidylethanolarnine, cerarnide, sphingomyelin,
`65 cephalin, cardiolipin, and cerebrosides.
`The terms "transfection" and ''transformation" are used
`herein interchangeably, and refer to the introduction of
`
`50
`
`BRIEF DESCR1PTION OF THE DRAWINGS
`FIG. 1 illustrates the fusion of DOTMA:DOPE and
`DODAC:DOPE vesicles induced by plasmid DNA.
`
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`

`5,753,613
`
`10
`
`5
`
`3
`polyanionic materials, particularly nucleic acids, into cells.
`The term "lipofection" refers to the introduction of such
`materials using liposome complexes. The polyanionic mate(cid:173)
`rials can be in the form of DNA or RNA which is linked to
`expression vectors to facilitate gene expression after entry
`into the cell. Thus the polyanionic material used in the
`present invention is meant to include DNA having coding
`sequences for structural proteins, receptors and hormones, as
`well as transcriptional and translational regulatory elements
`(i.e., promoters, enhancers, terminators and signal
`sequences) and vectors. Methods of incorporating particular
`nucleic acids into expression vectors are well known to
`those of skill in the art, but are described in detail in, for
`example, Sambrook et al., Molecular Cloning: A Laboratory
`Manual (2nd ed.), Vols. 1-3, Cold Spring Harbor
`Laboratory, (1989) or Current Protocols in Molecular
`Biology, F. Ausubel et al., ed. Greene Publishing and Wiley(cid:173)
`Interscience, New York (1987), both of which are incorpo(cid:173)
`rated herein by reference.
`"Expression vectors", "cloning vectors", or ''vectors" are
`often plasmids or other nucleic acid molecules that are able
`to replicate in a chosen host cell. Expression vectors may
`replicate autonomously, or they may replicate by being
`inserted into the genome of the host cell, by methods well
`known in the art. Vectors that replicate autonomously will 25
`have an origin of replication or autonomous replicating
`sequence (ARS) that is functional in the chosen host cell(s).
`Often. it is desirable for a vector to be usable in more than
`one host cell, e.g., in E. coli for cloning and construction,
`and in a mammalian cell for expression.
`Detailed Description
`The present invention provides compositions and meth(cid:173)
`ods for the introduction of polyanionic materials into cells.
`The compositions comprise a cationic compound of formula
`I and at least one neutral lipid.
`
`4
`a colipid in liposomal compositions with other detergents
`(see, Takahashi, et al., GB 2147243).
`The neutral lipids which are part of the present compo(cid:173)
`sitions are not critical and can be any of a variety of neutral
`lipids which are typically used in detergents, or for the
`formation of micelles or liposomes. Examples of neutral
`lipids which are useful in the present compositions are
`d i a c y l p h o s p h a t i d y l c h o l i n e ,
`diacylphosphatidylethanolamine, ceramide, sphingomyelin,
`cephalin, cardiolipin, and cerebrosides. In preferred
`embodiments, the present compositions will include one or
`more neutral lipids which are diacylphosphatidylcholine,
`diacylphosphatidylethanolamine, ceramide or sphingomy(cid:173)
`elin. The acyl groups in these neutral lipids are preferably
`acyl groups derived from fatty acids having C 10-C24 carbon
`15 chains. More preferably the acyl groups are lauroyl,
`myristoyl, palmitoyl, stearoyl or oleoyl. In particularly pre(cid:173)
`ferred embodiments, the neutral lipid will be 1,2-sn(cid:173)
`dioleoylphosphatidylethanolamine.
`The anion, x-, can similarly be any of a variety a
`20 pharmaceutically acceptable anions. These anions can be
`organic or inorganic, including for example. Br-, Cl-, F", r,
`sulfate, acetate and nitrate. In preferred embodiments, x-is
`Cl- or AcO-.
`In the compositions of the present invention, the ratio of
`cationic compound to neutral lipid is not critical but will be
`within a range of from about 25:75 (cationic compound(cid:173)
`:neutrallipid) to 75:25 (cationic compound:neutrallipid),
`preferably about 50:50.
`The cationic compounds which are used in the above
`30 compositions can be prepared by methods known to those of
`skill in the art using standard synthetic reactions (see March,
`Advanced Organic Chemistry, 4th Ed., Wiley-Interscience,
`New York, N.Y. (1992), incorporated herein by reference).
`For example. the synthesis of OSDAC can be carried out by
`35 first treating oleylamine with formaldehyde and sodium
`cyanoborohydride under conditions which result in the
`reductive alkylation of the amine. This provides dimethyl
`oleylamine, which can then be alkylated with stearyl bro(cid:173)
`mide to form the corresponding ammonium salt. Anion
`40 exchange results in the formation of OSDAC. For cationic
`compounds in which both fatty acid chains are unsaturated
`(i.e., DODAC), the following general procedure can be used.
`An unsaturated acid (i.e., oleic acid) can be converted to its
`corresponding acid chloride with such reagents as oxalyl
`45 chloride, thionyl chloride, PC13 or PC15 • The acid chloride
`can be treated with an unsaturated amine (i.e., oleylamine)
`to provide the corresponding amide. Reduction of the amide
`with, for example, lithium aluminum hydride provides a
`secondary amine wherein both alkyl groups are unsaturated
`50 long chain alkyl groups. The secondary amine can then be
`treated with alkyl halides such as methyl iodide to provide
`a quaternary ammonium compound. Anion exchange can
`then be carried out to provide cationic compounds having
`the desired pharmaceutically acceptable anion.
`In one group of embodiments, the pharmaceutical com(cid:173)
`positions of the present invention will be formulated as
`micelles or liposomes.
`Micelles containing the cationic compounds and neutral
`lipids of the present invention can be prepared by methods
`60 which are well known to one of skill in the art. In addition
`to the micellar formulations of the present compositions, the
`present invention also provides micellar formulations which
`include other species such as lysophosphatidylcholine,
`lysophosphatidylethanolamine, lysophosphatidylserine,
`65 lysophosphatidylglycerol, phosphatidylethanolamine(cid:173)
`polyoxyethylene conjugate, ceramide-polyoxyethylene con(cid:173)
`jugate or phosphatidic acid-polyoxyethylene conjugate. The
`
`Rl
`I
`H3C-(CHz),-Y -(CHz).,-f+-Rl
`
`x-
`
`(I)
`
`H3C-(CHz)4 -Z-(CHz)p
`In formula I, R 1 and R 2 are each independently C 1 to C3
`alkyl. Y and Z are alkyl or alkenyl chains and are each
`independently
`-CH 2 CH 2 CH 2 CH 2 CH 2 - ,
`-CH=CHCH2CH2CH2- , -CH2CH=CHCH2CH 2- .
`-CH2 CH2CH=CHCH2- , -CH2CH2CH2CH=CH-,
`-CH=CHCH=CHCH2- . -CH=CHCH2CH=CH-,
`or -CH2CH=CHCH=CH-, with the proviso that Y and
`Z are not both -CH2CH2CH2CH2CH2- . The letters n and
`q denote integers of from 3 to 7, while. the letters m and p
`denote integers of from 4 to 9, with the proviso that the sums
`n+m and q+p are each integers of from 10 to 14. The symbol
`x- represents a pharmaceutically acceptable anion. In the
`above formula, the orientation of the double bond is not
`critical, however the cis isomers are generally preferred.
`In one group of preferred embodiments, the cationic 55
`compounds are of formula I, wherein R 1 and R2 are methyl
`and Y
`and Z
`are
`each
`independently
`-CH=CHCH2 CH2CH2- , -CH2CH=CHCH2CH2- ,
`or -CH2CH2CH2CH=CH-.
`-CH2CH2CH=CHCH2-
`In particularly preferred embodiments of the present
`invention, R 1 and R 2 are methyl; Y and Z are each
`-CH...:...CHCH2CH2CH2- ; n and q are both 7; and m and
`pare both 5. In the most preferred embodiment, the cationic
`compound
`is DODAC
`(N,N-dioleyl-N,N(cid:173)
`dimethylammonium chloride). DODAC is a known com(cid:173)
`pound that has been used extensively as an additive in
`detergents and shampoos. There is also a report of its use as
`
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`5,753,613
`
`15
`
`6
`Several techniques are available for sizing liposomes to a
`desired size. One sizing method is described in U.S. Pat. No.
`4,737 323, incotporated herein by reference. Sonicating a
`liposome suspension either by bath or probe sonication
`5 produces a progressive size reduction down to small unila(cid:173)
`mellarvesicles less than about 0.05 microns in size. Homog(cid:173)
`enization is another method which relies on shearing energy
`to fragment large liposomes into smaller ones. In a typical
`homogenization procedure, multilamellar vesicles are recir-
`10 culated through a standard emulsion homogenizer until
`selected liposome sizes, typically between about 0.1 and 0.5
`microns, are observed. In both methods, the particle size
`distribution can be monitored by conventional laser-beam
`particle size discrimination.
`Extrusion of liposome through a small-pore polycarbon-
`ate membrane or an asymmetric ceramic membrane is also
`an effective method for reducing liposome sizes to a rela(cid:173)
`tively well-defined size distribution. Typically, the suspen(cid:173)
`sion is cycled through the membrane one or more times until
`20 the desired liposome size distribution is achieved. The
`liposomes may be extruded through successively smaller(cid:173)
`pore membranes, to achieve a gradual reduction in liposome
`size. For use in the present inventions, liposomes having a
`size of from about 0.05 microns to about 0.15 microns are
`25 preferred.
`The compositions of the present invention are useful for
`the introduction of polyanionic materials into cells.
`Accordingly, the present invention also provides methods
`for introducing a polyanionic material into a cell. The
`30 methods are carried out in vitro by first contacting the
`polyanionic material with a composition of at least one
`neutral lipid and a cationic compound of formula I, to form
`a polyanionic material-liposome complex. After contacting
`the polyanionic material with the liposomal formulations to
`35 form a complex, the complex is then contacted with the cells
`for a period of time sufficient for transfection to occur.
`
`5
`polyoxyethylene conjugates which are used in the compo(cid:173)
`sitions of the present invention can be prepared by combin(cid:173)
`ing the conjugating group (i.e. phosphatidic acid or
`phosphatidylethanolarnine) with an appropriately function(cid:173)
`alized polyoxyethylene derivative. For example, phosphati(cid:173)
`dylethanolamine can be combined with polyoxyethylene
`bis(p-toluenesulfonate)
`to
`provide
`a
`phosphatidylethanolarnine-polyoxyethylene conjugate. See,
`Woodle, et al., Biochim. Biophys. Acta 1105:193-200
`(1992), incotporated herein by reference.
`Uposomes can also be formed from the cationic com(cid:173)
`pounds and neutral lipids of the present pharmaceutical
`compositions. The selection of neutral lipids is generally
`guided by consideration of, e.g., liposome size and stability
`of the liposomes in the bloodstream.
`As noted above, the neutral lipid component in the
`liposomes is a lipid having two acyl groups, (i.e., dia(cid:173)
`cylphosphatidylcholine and diacylphosphatidyl(cid:173)
`ethanolamine). lipids having a variety of acyl chain groups
`of varying chain length and degree of saturation are avail(cid:173)
`able or may be isolated or synthesized by well-known
`techniques. In general, less saturated lipids are more easily
`sized, particularly when the liposomes must be sized below
`about 0.3 microns, for pUiposes of filter sterilization. In one
`group of embodiments, lipids containing saturated fatty
`acids with carbon chain lengths in the range of C14 to C22 are
`preferred. In another group of embodiments, lipids with
`mono or diunsaturated fatty acids with carbon chain lengths
`in the range of C14 to C22 are used. Additionally, lipids
`having mixtures of saturated and unsaturated fatty acid
`chains can be used. Uposomes useful in the present inven(cid:173)
`tion may also be composed of sphingomyelin or phospho(cid:173)
`lipids with other head groups, such as serine and inositol.
`Still other liposomes useful in the present invention will
`include cholesterol, diglycerides, ceramides,
`phosphatidylethanolarnine-polyoxyethylene conjugates or
`phosphatidic acid-polyoxyethylene conjugates. Methods
`used in sizing and filter-sterilizing liposomes are discussed
`below.
`A variety of methods are available for preparing lipo(cid:173)
`somes as described in, e.g., Szoka et al., Ann. Rev. Biophys. 40
`Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728,
`4,837,028, the text Liposomes, Marc J. Ostro, ed., Marcel
`Dekker, Inc., New York, 1983, Chapter 1, and Hope, et al.,
`Chem. Phys. Lip. 40:89 (1986), all of which are incorporated
`herein by reference. One method produces multilamellar 45
`vesicles of heterogeneous sizes. In this method, the vesicle(cid:173)
`forming lipids are dissolved in a suitable organic solvent or
`solvent system and dried under vacuum or an inert gas to
`form a thin lipid film. If desired. the film may be redissolved
`in a suitable solvent, such as tertiary butanol, and then 50
`lyophilized to form a more homogeneous lipid mixture
`which is in a more easily hydrated powder-like form. This
`film is covered with an aqueous buffered solution and
`allowed to hydrate, typically over a 15-60 minute period
`with agitation. The size distribution of the resulting multi- 55
`lamellar vesicles can be shifted toward smaller sizes by
`hydrating the lipids under more vigorous agitation condi(cid:173)
`tions or by adding solubilizing detergents such as deoxy(cid:173)
`cholate.
`Following liposome preparation, the liposomes may be 60
`sized to achieve a desired size range and relatively narrow
`distribution of liposome sizes. A size range of about 0.2-0.4
`microns allows the liposome suspension to be sterilized by
`filtration through a conventional filter, typically a 0.22
`micron filter. The filter sterilization method can be carried 65
`out on a high through-put basis if the liposomes have been
`sized down to about 0.2-0.4 microns.
`
`(I)
`
`x-
`Rl
`I
`H3C-(CH2)n-Y-(CH2),.-N+-R2
`I
`H3C-(CH2)q -Z-(CH2)p
`In formula I, the symbols R 1
`, R2
`, Y, Z, n, m, p, q. andx(cid:173)
`are as described above for the compositions of the present
`invention. In one group of preferred embodiments, the
`cationic compounds used in the present methods are of
`formula I, wherein R 1 and R2 are methyl andY and Z are
`each independently -CH=CHCH 2 CH 2 CH 2 - ,
`-CH2CH=CHCH2CH2- , -CH2CH2CH=CHCH2-
`or
`-CH2 CH2 CH2 CH=CH-. In particularly preferred
`embodiments, R1 and R2 are methyl; Y and Z are each
`-CH=HCH2CH2CH2- ; nand q are both 7; and m and p
`are both 5. More preferably, the cationic compound is
`DODAC (N,N-dioleyl-N,N-dimethylammonium chloride).
`Other preferred embodiments for the method of the present
`invention will encompass the preferred compositions dis(cid:173)
`cussed above.
`As noted above, the polyanionic material is first contacted
`with a composition comprising neutral lipids and cationic
`compounds to provide a polyanionic material-liposome
`complex. The contact can be made either prior to liposome
`formation (from the neutral lipids and cationic compounds)
`or subsequent to an initialliposome formation. In a preferred
`embodiment, liposomes of neutral lipids and the cationic
`compounds are formed first, then brought into contact with
`the polyanionic materials. The polyanionic materials will
`typically bind to the surface of the liposome as a result of the
`ionic attraction between the negatively charged polyanionic
`
`Moderna Ex 1012-p. 8
`Moderna v Arbutus
`
`

`

`5,753,613
`
`7
`material and the positively charged surface of the liposome.
`Typically, the contact between the polyanionic material and
`the liposome which results in formation of a complex will be
`carried out at temperatures of from about 15° C. to about 45°
`C., preferably about 25° C. The length of time required to
`complete the formation of a complex will depend on the
`temperature as well as the nature of the polyanionic material
`and the liposome itself. When contact temperatures of about
`25° C. are used, the length of time to form a complex
`between a liposome and a nucleic acid will be about 15
`minutes to about 2 hours, however in some instances longer
`times may be required. Alternatively, the polyanionic mate(cid:173)
`rials can be incorporated into the interior of the liposomes by
`methods used for loading conventional drugs which are
`known to those of skill in the art. One method for loading
`drugs into liposomes involves encapsulation and can be
`carried out by a variety of techniques.
`In one encapsulation technique, the drug and liposome
`components are dissolved in an organic solvent in which all
`species are miscible and concentrated to a dry film. A buffer
`is then added to the dried film and liposomes are formed
`having the polyanionic material incorporated into the vesicle
`walls. Alternatively, the polyanionic material can be placed
`into a buffer and added to a dried film of only lipid
`components. In this manner, the polyanionic material will
`become encapsulated in the aqueous interior of the lipo(cid:173)
`some. The buffer which is used in the formation of the
`biologically come any biologically compatible buffer solu(cid:173)
`tion of, for example, isotonic saline, phosphate buffered
`saline, or other low ionic strength buffers. Generally, the
`polyanionic material will be present in an amount of from
`about 0.01 ng/mL to about 50 mg/mL. The resulting lipo(cid:173)
`somes with the polyanionic material incorporated in the
`aqueous interior or in the membrane are then optionally
`sized as described above.
`In one group of preferred embodiments, the polyanionic
`material:liposome complexes will have charge ratios (±) of
`from about 0.5 to about 4.0. To achieve these charge ratios,
`the complexes are formed by preparing an aqueous liposome
`formulation of the cationic lipid and at least one neutral lipid
`in which the cationic lipid is present in from about 25% to
`about 75% of the total lipid concentration. After sizing the
`liposomes, as discussed above, an aqueous solution of the
`polyanionic material is treated with the liposome suspen(cid:173)
`sion. The resulting preparation is then diluted, preferably
`about 5-fold, with a biologically compatible buffer to pro(cid:173)
`vide a final concentration of 0.05 to 1.0 j.lg/mL of the
`polyanionic material:liposome complex having a charge
`ratio of from about 0.5 to about 4.0.
`Following formation of a polyanionic material:liposome
`complex, the complex is contacted with the cells to be
`transfected. Liposomes can be adsorbed to almost any cell
`type. Once adsorbed, the liposomes (including the com(cid:173)
`plexes previously described) can either be endocytosed by a
`portion of the cells, exchange lipids with cell membranes, or
`fuse with the cells. Transfer or incorporation of the polya(cid:173)
`nionic portion of the complex can take place via any one of
`these pathways. In particular, when fusion takes place, the
`liposomal membrane is integrated into the cell membrane
`and the contents of the liposome combine with the intrac(cid:173)
`ellular fluid. Contact between the cells and the polyanionic
`material-liposome complex, when carried out in vitro, will
`take place in a biologically compatible medium. The con(cid:173)
`centration of lipid can vary widely depending on the par(cid:173)
`ticular application, but is generally between about 1 j.IIDOl
`and about 10 mmol. Treatment of the cells with the polya(cid:173)
`nionic materical:liposome complex will generally be carried
`
`15
`
`8
`out at physiological temperatures (about 37° C.) for periods
`of time of from about 1 to 6 hours, preferably of from about
`2 to 4 hours. For in vitro applications, the delivery of
`polyanionic materials can be to any cell grown in culture,
`5 whether of plant or animal origin, vertebrate or invertebrate,
`and of any tissue or type. In preferred embodiments, the cells
`will be animal cells, more preferably mammalian cells, and
`most preferably human cells.
`In one group of preferred embodiments, the polyanionic
`material:liposome complex is added to 60-80% confluent
`10 plated cells having a cell density of from about 103 to about
`105 cells/mL, more preferably about 2xl04 cells/mL. The
`concentration of the complex added to the cells is preferably
`of from about 0.01 to 0.2 j.ig/mL, more preferably about 0.1
`j.lg/mL.
`Typical applications include using well known transfec-
`tion procedures to provide intracellular delivery of DNA or
`mRNA sequences which code for therapeutically useful
`polypeptides. However, the compositions can also be used
`for the delivery of the expressed gene product or protein
`20 itself. In this manner, therapy is provided for genetic dis(cid:173)
`eases by supplying deficient or absent gene products (i.e., for
`Duchenne's dystrophy, see Kunkel, et al., Brit. Med. BulL
`45(3):630-643 (1989), and for cystic