United States Patent [19J
`Feigner et al.
`
`[75]
`
`[54] CATIONIC LIPIDS FOR INTRACELLULAR
`DELIVERY OF BIOLOGICALLY ACfiVE
`MOLECULES
`Inventors: Philip L. Feigner, Rancho Santa Fe;
`Raj Kumar; Channa Basava, both of
`San Diego; Richard C. Border,
`Poway; Jiin-Yu Hwang-Felgner,
`Rancho Santa Fe, all of Calif.
`[73] Assignee: Vical, Inc., San Diego, Calif.
`[21] Appl. No.:
`686,746
`Apr. 16, 1991
`[22] Filed:
`
`[56]
`
`(63]
`
`Related U.S. Application Data
`Continuation of Ser. No. 563,444, Aug. 7, 1990, aban(cid:173)
`doned, which is a continuation of Ser. No. 511,219,
`Apr. 19, 1990, abandoned.
`Int. Cl.s .............................................. C07C 69/52
`[51]
`[52] U.S. Cl ..................................... 560/224; 560/155;
`560/252; 530/323; 554/227; 554/224; 554/223;
`564/292; 574/549; 574/552
`[58] Field of Search ................ 560/224; 554/227, 226,
`554/223; 530/323; 574/292, 549, 552
`References Cited
`U.S. PATENT DOCUMENTS
`3,931,397 7/1976 Harnden ................................ 424/85
`4,897,355 3/1990 Eppstein et al. .................... 424/427
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`OTHER PUBLICATIONS
`CA 112(2):86804 1988.
`CA 107(25):236676d 1987.
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`Mannino, R. J., et al. Biotechniques 6:682-690 (1988).
`Itani, T., et al. Gene 56:267-276 (1987).
`Nicolau, C., et al. Meth. Enz. 149:157-176 (1987).
`Straubinger, R., et al. Meth. Enz. 101:512-527 (1983).
`Feigner, P., et al., Proc. Nat/. Acad. Sci. USA
`84:7413-7417 (1987).
`
`lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
`US005264618A
`5,264,618
`[11] Patent Number:
`[45] Date of Patent: Nov. 23, 1993
`
`Ballas, N., et al., Biochim. Biophys. Acta 939:8-18 (1988).
`Pinnaduwage, P., et al., Biochim. Biophys. Acta
`985:33-31 (1989).
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`89:2928-2933 (1985).
`Rupert, L., et al., J. Amer. Chern. Soc. 108:3920-3925
`(1986).
`
`(List continued on next page.)
`
`Primary Examiner-Paul J. Killos
`Attorney, Agent, or Firm-Knobbe, Martens, Olson &
`Bear
`
`ABSTRACf
`[57]
`Disclosed are cationic lipids capable of facilitating
`transport of biologically active agents into cells, includ(cid:173)
`ing the transfection of cells by therapeutic polynucleo(cid:173)
`tides, the delivery of antiviral drugs, and the introduc(cid:173)
`tion of immunogenic peptides. The cationic lipids, com(cid:173)
`prising an ammonium group, have the general structure
`
`(I)
`
`Also disclosed are adducts of these compounds com(cid:173)
`prising additional cationic sites that enhance the trans(cid:173)
`fective or transport activity. Structure-activity correla(cid:173)
`tions provide for the selection of preferred compounds
`to be synthesized for this purpose. Compositions dis(cid:173)
`closed for use of these cationic lipid include formula(cid:173)
`tions for in vitro transfection and pharmaceutical for(cid:173)
`mulations for parenteral and topical administration of
`therapeutic agents.
`
`12 Claims, 18 Drawing Sheets
`
`Moderna Ex 1017-p. 1
`Moderna v Arbutus
`
`

`

`5,264,618
`
`Page 2
`
`OTHER PUBLICATIONS
`Rosenthal, A., et a!., J. Bioi. Chern. 235(8):2202-2206
`(1960).
`Wilschut, J., eta!., Biochemistry 199:6011-6021 (1980).
`Mayer, L., et a!., Biochim. Biophys. Acta 858:161-168
`(1986).
`Bangham, A., et a!., J. Mol. Bioi. 23:238-252 (1965).
`Olson, F., eta!., Biochim. Biophys. Acta 557.·9-23 (1979).
`Szoka F., eta!., Proc. Nat/. Acad. Scz: USA 75:4194-4198
`(1978).
`Mayhew E., et a!. Biochim. Biophys. Acta 775:169-175
`(1984).
`Kim, S., et a!. Biochim. Biophys. Acta 728:339-348
`(1983).
`Fukunaga M., et al., EndrocrinoL 115:757-761 (1984).
`J., et a!., Biochim. Biophys. Acta
`Israelachvili,
`470:185-201 (1977).
`Kunkel, L., et al. Brit. Med. Bull. 45(3):630-643 (1989).
`Goodfellow, P. Nature 341(6238): 102-3 (Sep. 14, 1989).
`Ts'o P., et a!., Annals New York Acad. Sci. 570:220-241
`(1987).
`
`Hampel, et al., Nucleic Acids Research 18(2): 299-304
`(1990).
`Cech, T., et al., Annual Rev. Biochem. 55:5499-629
`(1986).
`Matsukura, M., et a!., Proc. Nat'/ Acad. Sci.
`86:4244-4248 ( 1989).
`Kumar, R., et a!., Biochim. Biophys. Acta 917.·33-41
`(1987).
`Brigham, K., et a!. Amer. J. of the Med. Sci.
`298(4):278-281 (1989).
`Berge, S., et a!., J. of Pharmaceutical Sciences 66:1-19
`(1977).
`Feigner, P., et a!., Focus 11(2): (Spring 1989).
`Malone, R., et al., Proc. Nat' I A cad. Sci. USA
`86:6077-6081 (1989).
`Chiang, M.-L., eta!., J. Bioi. Chern. 268:1-10 (1991).
`Feigner, P., eta!. Nature 349(6307): 351-352 (1991).
`R. Elbert et a!., J. Amer. Chern. Soc., 107: 4134-4141
`(1985).
`K. L. Meyer, et al., J. Med. Chern., 34: 1377-1383
`(1991 ).
`
`Moderna Ex 1017-p. 2
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 1 of 18
`
`5,264,618
`
`FIG. I
`
`TRANSFECTION CONDITION ANALYSIS
`80:20 DOTMA:DOPE
`~2000~------------------------------~
`.,
`-fTl
`~::0
`6 ~ 1500
`c:
`( / ) I
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`~ ~1000
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`15
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`% OF SERUM DURING COMPLEX FORMATION
`
`FIG. 2
`
`f&\\\~ NO SERUM
`-5% SERUM
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`KKM 15% SERUM
`t?Z///420% SERUM
`TRANSFECTION CONDITION ANALYSIS
`80:20 DOTMA:DOPE
`E1s~-------------------------------.
`n .,,4
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`
`10
`INCUBATION
`
`20
`IN SERUM
`
`Moderna Ex 1017-p. 3
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 2 of 18
`
`5,264,618
`
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`
`Moderna Ex 1017-p. 4
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 3 of 18
`
`5,264,618
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`Moderna Ex 1017-p. 5
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 4 of 18
`
`5,264,618
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`Moderna Ex 1017-p. 6
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 5 of 18
`
`5,264,618
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`Moderna Ex 1017-p. 7
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 6 of 18
`
`5,264,618
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`Moderna Ex 1017-p. 8
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 7 of 18
`
`5,264,618
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`Moderna v Arbutus
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`

`

`U.S. Patent
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`Nov. 23, 1993
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`Sheet 8 of 18
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`5,264,618
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`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 9 of 18
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`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
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`Sheet 10 of 18
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`5,264,618
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`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 11 of 18
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`5,264,618
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`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
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`Sheet 12 of 18
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`5,264,618
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`Moderna Ex 1017-p. 14
`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
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`Sheet 13 of 18
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`5,264,618
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`Moderna v Arbutus
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`

`

`U.S. Patent
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`Nov. 23, 1993
`
`Sheet 14 of 18
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`5,264,618
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`Moderna Ex 1017-p. 16
`Moderna v Arbutus
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`

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`U.S. Patent
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`Nov. 23, 1993
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`Sheet 15 of 18
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`Moderna v Arbutus
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`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 16 of 18
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`5,264,618
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`Moderna Ex 1017-p. 18
`Moderna v Arbutus
`
`

`

`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 17 of 18
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`5,264,618
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`Moderna Ex 1017-p. 19
`Moderna v Arbutus
`
`

`

`U.S. Patent
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`Nov. 23, 1993
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`Sheet 18 of 18
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`(/) w
`0::
`a..
`X w
`0> a..
`
`0 0 0 0 0
`o o o o o
`O~NC:C...q-
`....
`....
`...
`N ~ -.:---
`
`Moderna Ex 1017-p. 20
`Moderna v Arbutus
`
`

`

`1
`
`5,264,618
`
`CATIONIC LIPIDS FOR INTRACELLULAR
`DELIVERY OF BIOLOGICALLY ACTIVE
`MOLECULES
`
`This application is a continuation-in-part of U.S. ap(cid:173)
`plications Ser. No. 07/563,444, filed Aug. 7, 1990 aban(cid:173)
`doned; and Ser. No. 07/511,219, filed Apr. 19, 1990
`abandoned.
`
`2
`form hollow lipid vesicles, or liposomes, in aqueous
`systems. This property can be used to entrap the sub(cid:173)
`stance to be delivered within the liposomes; in other
`applications, the drug molecule of interest can be incor-
`5 porated into the lipid vesicle as an intrinsic membrane
`component, rather than entrapped into the hollow aque(cid:173)
`ous interior.
`Intracellular delivery of beneficial or interesting pro(cid:173)
`teins can be achieved by introducing expressible DNA
`10 and mRNA into the cells of a mammal, a useful tech(cid:173)
`BACKGROUND OF THE INVENTION
`nique termed transfection. Gene sequences introduced
`The present invention relates to cationic lipids which
`in this way can produce the corresponding protein
`are used to enhance delivery of biologically active
`coded for by the gene by using endogenous protein
`agents, particularly polynucleotides, proteins, peptides,
`synthetic enzymes. The therapy of many diseases could
`and drug molecules, by facilitating transmembrane 15 be enhanced by the induced intracellular production of
`transport or by encouraging adhesion to biological sur-
`peptides which could remain inside the target cell, be
`faces. It relates particularly to cationic lipids compris-
`secreted into the local environment of the target cell, or
`ing ammonium groups.
`be secreted into the systemic circulation to produce
`Some bioactive substances do not need to enter cells
`their effect.
`to exert their biological effect, because they operate 20 Various techniques for introducing the DNA or
`either by acting on cell surfaces through cell surface
`mRNA precursors of bioactive peptides into cells in-
`receptors or to by interacting with extracellular compo-
`elude the use of viral vectors, including recombinant
`nents. However, many natural biological molecules and
`vectors and retroviruses, which have the inherent abil-
`their analogues, including proteins and polynucleotides,
`ity to penetrate cell membranes. However, the use of
`or foreign substances, such as drugs, which are capable 25 such viral agents to integrate exogenous DNA into the
`of influencing cell function at the subcellular or molecu-
`chromosomal material of the cell carries a risk of dam-
`Jar level are preferably incorporated within the cell in
`age to the genome and the possibility of inducing malig-
`order to produce their effect. For these agents the cell
`nant transformation. Another aspect of this approach
`membrane presents a selective barrier which is imper-
`which restricts its use in vivo is that the integration of
`meable to them.
`30 DNA into the genome accomplished by these methods
`Just as the plasma membrane of a cell is a selective
`implies a loss of control over the expression of the pep-
`barrier preventing random introduction of potentially
`tide it codes for, so that transitory therapy is difficult to
`toxic substances into the cell, the human body is sur-
`achieve and potential unwanted side effects of the treat-
`rounded by protective membranes which serve a similar
`ment could be difficult or impossible to reverse or halt.
`defensive function to the whole organism. These mem- 35
`Liposomes have been discussed as possible in vivo
`branes include skin, gastric mucosa, nasal mucosa and
`delivery vehicles and some encouraging results using
`the like. While these membranes serve a protective
`this approach to the intracellular expression of DNA
`function preventing entry of toxic substances, they can
`have been obtained (Mannino, R. J. Fould-Fogerite, S.,
`also prevent passage of potentially beneficial therapeu-
`Biotechniques 6, 682-690 (1988); Itani, T., Ariga, H.,
`tic substances into the body. The complex composition 40 Yamaguchi, N., Tadakuma, T. & Yasuda, T. Gene 56
`of the cell membrane comprises phospholipids, glyco-
`267-276 (1987); Nicolau, C. Legrand, A. & Grosse, G.
`lipids, and cholesterol, as well as intrinsic and extrinsic
`E. Meth. Enz. 149 157-176 (1987); Straubinger, R. M. &
`proteins, and its functions are influenced by cytoplasmic
`Papahadjopou!os, D. Meth. Enz. 101 512-527 (1983);
`components which include Ca + + and other metal ions, Wang, C. Y. & Huang, L. Proc Nat/. A cad. Sci. USA 84
`anions, ATP, microfilaments, microtubules, enzymes, 45 7851-7855 (1987)); however, the methodology has fun-
`and Ca ++-binding proteins. Interactions among struc-
`damental problems. Chief among the difficulties is the
`tural and cytoplasmic cell components and their re-
`failure of liposomes to fuse with the target cell surface,
`sponse to external signals make up transport processes
`but to be taken up phagocytically instead. Phagocytized
`responsible for the membrane selectivity exhibited
`liposomes are delivered to the lysosomal compartment,
`within and among cell types.
`50 where polynucleotides are subjected to the action of
`Successful intracellular delivery of agents not natu-
`digestive enzymes and degraded, leading to low effi-
`rally taken up by cells has been achieved by exploiting
`ciency of expression.
`the natural process of intracellular membrane fusion, or
`A major advance in this area was the discovery that
`by direct access of the cell's natural transport mecha-
`a positively charged synthetic cationic lipid, N-[1-(2,3-
`nisms which include endocytosis and pinocytosis (Duz- 55 dioleyloxy)propyl]-N,N,N-trimethylammonium chlo-
`gunes, N., Subcellular Biochemistry 11:195-286 (1985).
`ride (DOTMA), in the form of Iiposomes, or small vesi-
`The membrane barrier can be overcome in the first
`cles, could interact spontaneously with DNA to form
`instance by associating these substances in complexes
`lipid-DNA complexes which are capable of fusing with
`with lipid formulations closely resembling the lipid
`the negatively charged lipids of the cell membranes of
`composition of natural cell membranes. These lipids are 60 tissue culture cells, resulting in both uptake and expres-
`able to fuse with the cell membranes on contact, and in
`sion of the DNA (Feigner, P. L. et a!. Proc. Nat/. A cad.
`the process, the associated substances are delivered
`Sci., USA 84:7413-7417 (1987) and U.S. Pat. No.
`intracellularly. Lipid complexes can not only facilitate
`4,897,355 to Eppstein, D. et a!.). Others have success-
`intracellular transfers by fusing with cell membranes
`fully used a DOTMA analogue, 1,2-bis(oleoyloxy)-3-
`but also by overcoming charge repulsions between the 65 (trimethylammonio)propane (DOT AP) in combination
`cell membrane and the molecule to be inserted. The
`with a phospholipid to form DNA-complexing vesicles.
`lipids of the formulations comprise an amphipathic
`The Lipofectin TM reagent (Bethesda Research Labo-
`lipid, such as the phospholipids of cell membranes, and
`ratories, Gaithersburg, Md.), an effective agent for the
`
`Moderna Ex 1017-p. 21
`Moderna v Arbutus
`
`

`

`4
`t~an previously known procedures, and permits tran(cid:173)
`sient as well as stable transfection and peptide expres(cid:173)
`sion, it is not understood what factors regulate the effi(cid:173)
`ciency of the transfection process and how it may be
`optimized. It would be desirable to determine these
`factors in order to develop an intracellular delivery
`system having the advantages of the above-described
`systems but without their inherent limitations.
`Accordingly, it is an object of the invention to pro(cid:173)
`vide cationic lipids which carry out both stable and
`transient transfections of polynucleotides such as DNA
`and mRNA into cells more effectively.
`It is also an object ofthe invention to provide cationic
`lipids which deliver other molecules of therapeutic
`interest, including proteins, peptides and small organic
`molecules, into cells more effectively.
`Further, it is an object of the invention to provide
`cationic lipids that are not only more effective in ac(cid:173)
`complishing intracellular delivery but are also metabo(cid:173)
`lizable so as to have reduced in vivo and in vitro toxic(cid:173)
`ity.
`It is another object of the invention to provide trans(cid:173)
`fection formulation, comprising novel cationic lipids,
`that are optimally effective in both in vivo and in vitro
`transfection.
`
`25
`
`5,264,618
`3
`delivery of highly anionic polynucleotides into living
`tissue culture cells comprises positively charged
`DOTMA liposomes which interact spontaneously with
`negatively charged polynucleotides to form complexes.
`When enough positively charged liposomes are used, 5
`the net charge on the resulting complexes is also posi(cid:173)
`tive. Positively charged complexes prepared in this way
`spontaneously attach to negatively charged cell sur(cid:173)
`faces, fuse with the plasma membrane, and efficiently
`deliver functional polynucleotide into, for example, 10
`tissue culture cells.
`Although the use of known cationic lipids overcomes
`many problems associated with conventional liposome
`technology for polynucleotide delivery in vitro, several
`problems related to both in vitro and in vivo applica- 15
`tions remain. First, although the efficiency of cationic
`lipid mediated delivery is relatively high compared to
`other methods, the absolute level of gene product pro(cid:173)
`duced is typically only several hundred copies per cell
`on average. Thus it would be desirable to improve de- 20
`livery and expression by a factor of 10 to 1000-fold to
`achieve useful methodologies. Secondly, known cati(cid:173)
`onic lipids such as DOTMA are toxic to tissue culture
`cells; thus, any improvements that reduce in vitro toxic-
`ity would strengthen the methodology.
`A significant body of information is emerging regard(cid:173)
`ing the use of other cationic lipids for the delivery of
`macromolecules into cells. Loyter prepared vesicles
`containing a quaternary ammonium surfactant that are
`capable of transferring functional tobacco mosaic virus 30
`into plant protoplasts. (Ballas, N., Zakai, N., Sela, I. and
`Loyter, A. Biochim. Biophys Acta 939 8-18 (1988)).
`Huang used cetyltrimethylammonium bromide to ob(cid:173)
`tain functional expression from the chloramphenicol
`acetyl transferase gene transfected into mouse fibro- 35
`blasts (Pinnaduwage, P., Schmitt, L. and Huang, L.
`Biochim. Biophys Acta 985 33-37 (1989)). Behr has
`shown that a novel lipophilic derivative of spermine can
`transfect primary pituitary cells (Behr, J-P, Demeneix,
`B., Loeffler, J-P and Perez-Mutul, J. Proc. Nat/. Acad. 40
`Sci. USA 86 6982-6986 (1989)). Finally, John Silvius has
`shown that a cationic lipid (DOT AP), originally syn(cid:173)
`thesized by Eibl (Eibl, H. and Woolley, P. Biophys.
`Chern. 10 261-271 (1979)) forms liposomes which can(cid:173)
`fuse with negatively charged liposomes and can deliver 45
`functional DNA and RNA into tissue culture fibroblasts
`(Stamatatos, L., Leventis, R., Zuckermann, M. J. &
`Silvius, J. R. Biochemistry 27 3917-3925 (1988)). Other
`laboratories have studies the physical properties of vesi(cid:173)
`cles formed from synthetic cationic amphophiles (Ru- 50
`pert, L.A. M., Hoekstra, D. and Engberts, J. B. F. N.
`Am. Chern. Soc. 108: 2628-2631 (1985); Carmona(cid:173)
`Ribeiro, A. M., Yoshida, L. S. and Chaimovich, H. J.
`Phys Chern 89 2928-2933 (1985); Rupert, L. A. M.,
`Engberts, J. B. F. N. and Hoekstra, D. J. Amer. Chern. 55
`Soc. 108:3920-3925 (1986)).
`It is not feasible to extend in vitro transfection tech(cid:173)
`nology to in vivo applications directly. In vivo, the
`diether lipids, such as DOTMA or Lipofectin the cur(cid:173)
`rent commercial standard, would be expected to accu- 60
`mulate in the body due to the poorly metabolized ether
`bonds. And finally, it has been reported that the cationic
`lipid transfection methodology is inhibited by serum;
`for in vivo applications conditions must be identified
`which allow transfection to occur in a complex biologi- 65
`cal milieu such as 100% serum.
`Therefore, while the known lipofection technique of
`transfection described is more efficient and satisfactory
`
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1 presents data showing the effect the presence
`of serum during lipid complex formation on subsequent
`RNA transfection.
`FIG. 2 demonstrates the effect of serum on the effec(cid:173)
`tiveness of RNA transfection.
`FIG. 3 demonstrates the effect of cationic lipid con(cid:173)
`centration on the effectiveness of RNA transfection
`using DOT AP and DOTMA as cationic lipids.
`Fl G. 4 demonstrates the effect of neutral lipids on the
`comparative effectiveness of a series cationic lipids in
`promoting RNA transfection.
`FIG. 5 demonstrates the comparative effectiveness of
`DPTMA, DOTMA and corresponding derivatives of
`the Rosenthal Inhibitor in RNA transfection.
`FIGS. 6a-6d demonstrate the effect of increasing
`relative concentrations of lysophosphatidylcholine in
`lipid formulations on DNA transfection efficiency as
`demonstrated by expression of gene product in cell
`culture.
`FIGS. 7a-7c demonstrate the comparative DNA
`transfection activity of various cationic lipid analogs.
`FIGS. 8a-8d demonstrate the effect of neutral phos(cid:173)
`pholipids in the transfection lipid formulation on the
`efficiency of DNA transfection.
`FIGS. 9a-9c demonstrate the effect of cholesterol in
`the transfection lipid formulation on the efficiency of
`DNA transfection.
`
`SUMMARY OF THE INVENTION
`The present invention provides compositions of
`novel cationic lipids, suitable for use in the intracellular
`delivery of bioactive agents, comprising polynucleo(cid:173)
`tides, proteins, small organic molecules and drugs, in
`both in vivo and in vitro applications, and into the cells
`of plants and animals.
`These compositions have the general structure
`
`Moderna Ex 1017-p. 22
`Moderna v Arbutus
`
`

`

`5
`
`H2C-Y 1-R 1
`I
`Hr-y2-;2
`
`(CH2)n-f+-R4
`
`x-
`R5-o-R6-R7
`
`5,264,618
`
`(I)
`
`6
`These
`dimethylaminopropyl-/3-hydroxyethylamine).
`cationic groups can in turn provide further hydropho(cid:173)
`bic regions to the cationic lipid composition through
`alkyl quaternizing groups on the attached lysine, sper-
`5 mine, or other amine-containing groups.
`Also included within the scope of the invention are
`analogues of known cationic lipids having ester linkages
`substituted for ether linkages between alkyl substituents
`and the glycerol moiety of the structure to provide less
`10 toxic, more easily metabolized compositions suitable for
`use in vivo. These analogues have the general structure
`
`wherein
`Yl and y2 are the same or different and are
`-O-CH2-, - 0 - C(O)-, or -0-;
`R 1 and R 2 are the same or different and are H, or C 1
`to C23 alkyl or alkenyl; and
`R3, R4, R5, R6, and R7 are as defined below. Preferred 15
`embodiments are compositions wherein R3 and R4 are
`individually C1 to C23 alkyl groups, R5 is -(CH2)m-,
`R6 is absent, R7 isH, and Rl and R2 individually have
`from 0 to 6 sites of unsaturation, and have the structure
`
`20
`
`CH3-(CH2)a-(CH=CH-CH2)b-(CH2)c-
`
`wherein the sum of a and c is from I to 23; and b is 0 to
`6.
`
`H2C-Y,-R1
`I
`HC-Y2-R2
`
`lr
`
`H2C-N+-R4
`~s
`
`(II)
`
`x-
`
`or an optical isomer thereof, wherein
`Yl and y2 are different and are either