`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
`FOREIGN PATENT DOCUMENTS
`0187702 1/1986
`3102682 7/1988 Japan .
`OTHER PUBLICATIONS
`CA 112(2):86804 1988.
`CA 107(25):236676d 1987.
`Duzgunes, N., Subcellular Biochemistry 11:195-286
`(1985).
`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).
`Behr, J.-P., et al., Proc. Nat/. Acad. Sci. USA
`86:6982-6986 (1989).
`Eibl, H., et al., Biophys. Chern. 10:261-271 (1979).
`Stamatotos, L., et al. Biochemistry 27:3917-3925 (1988).
`J. Phys. Chern.
`et al.,
`Carmona-Ribeiro, A.,
`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
`~c;
`~ ~1000
`-c:
`z
`
`.......... 500 --
`VI
`0
`
`20 CONTROL
`15
`1 0
`5
`0
`% OF SERUM DURING COMPLEX FORMATION
`
`FIG. 2
`
`f&\\\~ NO SERUM
`-5% SERUM
`~10% SERUM
`KKM 15% SERUM
`t?Z///420% SERUM
`TRANSFECTION CONDITION ANALYSIS
`80:20 DOTMA:DOPE
`E1s~-------------------------------.
`n .,,4
`-fTl
`~::0
`5?;;12
`c:fTl
`~ c10
`zC>
`:I: 8
`0
`(/)~
`-c:
`z 6
`=i
`(/) 4
`..........
`VI
`0 2 -----
`(/)
`
`~ 0 "------"~
`
`5
`0
`MINUTES OF COMPLEX
`
`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
`
`. .
`
`.
`..J
`..J
`..J
`u
`u u
`~ ~ ~
`0
`0
`0
`N
`IX)
`It)
`
`~ ~ I
`
`a..
`<(
`1-
`0
`Cl
`
`<(
`~
`1-
`
`0 c
`
`<(
`~
`1-
`0
`Cl
`
`a..
`<(
`1-
`0
`<(CI
`~
`1-
`0
`Cl
`
`a..
`<(
`1-
`0
`Cl
`
`<(
`~
`1-
`0
`Cl
`
`....J
`
`I..&J >
`ZI..&J o-'
`1-L&.J
`UV>
`I..&J<(
`L&...o:::
`.(/) I..&J
`ZL&...
`< ( (cid:173)
`o:::U
`t-=> __.
`<(::=.::: z<
`o:::I..&J
`a..
`
`0
`0
`0
`It)
`
`0
`0
`0
`0
`0
`0
`-.:t
`...,.,
`......
`LUCIFERASE LIGHT UNITS (30 SEC)
`(THOUSANDS)
`
`0
`
`Moderna Ex 1017-p. 4
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 3 of 18
`
`5,264,618
`
`:
`
`I
`I
`I
`I
`I
`l
`l
`
`I
`I
`
`--~
`
`~
`....J
`0
`~0
`....J~
`O....J
`0:::
`LaJ L&..
`t-O
`(/)
`La.IO')
`....J::s
`0
`:I:Lf)
`UN
`
`I
`
`L&Jo
`o..O
`0..-
`0
`
`M
`
`M
`
`Lf)
`
`'" <
`
`M
`
`~0
`1--Lf)
`0
`0
`
`0
`0
`0 ....,..
`
`r
`
`I /
`
`I
`I
`I
`I
`
`:
`
`I
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`!
`0
`0
`0
`0
`0
`0
`0
`0
`0
`N
`1'1')
`LUCIFERASE LIGHT UNITS (30 SEC)
`(THOUSANDS)
`
`I
`I
`I
`I
`I
`I
`I
`
`!
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`
`:
`
`I
`I
`I
`I
`I
`
`I
`
`I
`I
`!
`i
`
`I
`I
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`!
`I
`I
`!
`
`[ /
`
`V/
`
`V/
`
`""""
`
`0
`1'1')
`...........
`~
`0
`...........
`
`0 "
`
`~
`
`0
`1'1')
`...........
`co
`Lf)
`...........
`...-
`...-
`
`0
`1'1')
`...........
`Lf)
`1'1')
`...........
`Lf)
`1'1')
`
`0
`...........
`0
`...........
`0
`0 ...-
`
`z
`0
`!:::
`(/)
`0
`0..
`~
`
`0 u
`w
`....J u
`(/) w
`>
`<
`
`1----
`
`-t! 1--
`
`t.
`
`I
`
`~
`
`~
`0
`...........
`0
`N
`...........
`0 co
`~
`
`•
`
`'//LL
`
`~
`
`0
`...........
`0
`Lf)
`...........
`0
`Lf)
`
`J
`
`----LL
`
`0
`
`Moderna Ex 1017-p. 5
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 4 of 18
`
`5,264,618
`
`I
`I
`I
`I
`~'\.'\.'\.'\.'\.'\.'\.''''\': \.'\.'\.'\.'\.'\':
`
`~'\':
`
`I
`
`I
`
`I
`I
`I
`I
`I
`I
`
`I
`
`I
`
`L.
`
`L
`
`I
`
`I
`
`I
`I
`L
`... ""'""""'""""'''''''''''"''''\': "'''''''''''\':>
`I
`L
`I
`
`t77.7DT~
`
`I
`
`t..'\."-'''"''"'''''''''""'''''''''''''''"''''~
`!
`I
`I
`L
`
`"'~~~~,,~
`
`1Zf
`
`0
`
`0
`
`0 ,., .
`,....
`N .
`N . 0
`
`If')
`
`0
`
`0
`If')
`
`If')
`N
`N
`0
`
`If')
`
`0
`0
`N
`
`Lr..l
`1-
`
`0
`
`0
`
`~
`T
`I
`
`~
`
`0
`
`-
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`r
`I
`I
`I
`I
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`
`I
`
`I
`I
`I
`
`I
`
`~''~
`I
`I
`
`I
`
`I
`
`I
`I
`I
`
`I
`
`I
`I
`
`I
`
`-
`
`L.a.-
`
`tf) -tf)
`~~
`<t~ zw <(/')
`0~ -u
`0..
`-w
`_Jz -u> _o
`za:l
`0~
`-
`l[')
`1-
`<t
`(.)
`
`I
`
`I
`I
`
`N
`
`I
`I
`I
`
`0
`
`I
`
`I
`I
`
`I
`
`I
`co
`LUCIFERASE LIGHT UNITS (30 SEC)
`(THOUSANDS)
`
`I
`
`I
`
`N
`
`0
`
`~
`
`I
`
`~
`
`~
`1777/7///.
`L
`
`~ 1--
`~ 1--
`
`- j 1-
`
`. 0
`:5
`c..
`,....
`::,: - u .
`0 -
`If') - .........
`0 - 0
`. ~
`,.... Lr..l
`. 0
`0
`.....J
`0
`::,:
`0
`a:::
`u
`::,:
`
`If') 0
`Lr..l
`N
`
`0
`0
`
`0
`
`0 ~
`0
`c..
`
`If') V')
`
`0
`If')
`0
`0
`
`If')
`N
`
`0 .
`
`0
`
`N
`
`0
`0
`
`0
`
`1.()
`•
`(:)
`~
`
`Moderna Ex 1017-p. 6
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 5 of 18
`
`5,264,618
`
`0 ' It) ' It)
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`~~~~--~~~
`\
`0 0 0 0 0
`o o o o o
`0\..QNt:()~
`~~~
`
`\
`\
`\
`\
`\
`
`(.)
`Q_
`I
`0
`C/)
`~ /
`I
`.....J \
`>-' o'
`__.
`UJ
`\
`\
`0
`\
`\
`I
`'
`..--
`\
`UJ
`Q_
`0
`'
`0
`<(
`~
`..-:;.
`t-
`0
`0
`
`,.......
`
`(/')
`
`0 u
`z
`0
`
`N
`0
`0
`I
`
`t......
`0
`z
`0
`(/')
`
`(/') w
`0::
`a..
`X w
`
`Moderna Ex 1017-p. 7
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 6 of 18
`
`5,264,618
`
`L()
`N
`•
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`-
`
`,.....
`
`(/)
`
`0 u
`z
`0
`N
`() -o
`I
`
`...:..i
`<(
`(.!)
`I
`<(
`1-w
`
`CD
`
`L.....
`0
`z
`0
`(/)
`
`(/) w
`0::: a..
`X w
`0'1 a..
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`
`\
`
`\
`\
`
`' \
`
`' \
`' \
`
`00000
`00000
`O~NCC~
`....
`....
`....
`N~~
`
`Moderna Ex 1017-p. 8
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 7 of 18
`
`5,264,618
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`"' . (/')
`0 u
`z
`0
`
`...:_j
`<(
`(.!)
`I
`<(
`~ w
`CD
`u....
`0
`z
`0
`(/')
`
`(/') w
`Q::
`a..
`X w
`O'l a..
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`
`00000
`ooooo
`--
`0\..CNt:C~
`....
`....
`N~~
`
`Moderna Ex 1017-p. 9
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 8 of 18
`
`5,264,618
`
`c::;1 c::;1 c:;:1
`c::;1 ~ c:;:1 ~ c:;:1
`c.q r-::! ~ c:>
`q
`""0'-
`a:::;1
`t:'"'V -
`
`/ - /
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`-
`
`t--
`\.C)~
`Q")Q')
`
`"
`
`(/)
`0
`(.)
`
`z
`0
`
`~
`0
`z
`0
`(/)
`(/)
`w
`0::::
`a..
`X
`w
`en
`a..
`
`Cl
`(0
`•
`~
`L:::
`
`Q')cc
`t-----
`t:C~
`\.{')\.C)
`t-.
`o---
`ol-f)\.C)
`o---
`~l-f)
`0
`~
`~
`
`1.(')
`
`1.(') '
`"
`
`1.(')
`u
`a..
`I
`0
`(/)
`~
`I
`_J
`>-
`0
`w
`_J
`0
`I
`.....
`w
`a..
`0
`Cl
`<
`::E
`1-
`0
`Cl
`
`"
`"
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`-
`
`0 0 0 0 0
`o o o o o
`-
`Ot..CNCC~
`....
`....
`N ~ .__.
`
`Moderna Ex 1017-p. 10
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 9 of 18
`
`5,264,618
`
`to\
`
`L() ' L()
`0 ' <(
`
`~
`
`t(cid:173)o
`0
`
`/
`/
`/
`/
`/
`/
`L..U f--+---f---+-~~-(
`a..
`\
`\
`\
`0
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`-0
`
`O'l
`I
`
`(/)
`0
`1-u
`:5
`
`<(
`(!)
`I
`<(
`1-
`L..J
`CD
`
`t......
`0
`O'l a.
`
`Moderna Ex 1017-p. 11
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 10 of 18
`
`5,264,618
`
`-
`
`I
`
`I
`
`I
`
`/
`
`I
`
`/
`
`I
`
`I
`
`I
`
`I
`
`/
`
`I
`
`I
`
`I
`
`/
`
`I
`
`I
`
`I
`
`I
`
`/
`
`I
`
`/
`
`/
`
`/
`
`I
`
`I
`
`/
`
`I
`
`/
`
`I
`
`w
`a_
`I
`I
`/
`0
`I
`I
`/
`0 ~-+---+-+---+-_,..,
`"\ U\
`~ \
`
`L() ' L()
`
`/
`
`I
`
`I
`
`\
`\
`\
`\
`\
`
`0
`0 0 -0 0
`ooooo
`o~~cc~
`....
`....
`....
`~~~
`
`I'(cid:173)
`(/)
`0
`u
`z
`0
`
`0
`O'l
`I
`<(
`1-(cid:173)w
`
`(I)
`u...
`0
`z
`0
`(/)
`(/)
`w
`0::: c...
`X w
`O'l
`Q.
`
`Moderna Ex 1017-p. 12
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 11 of 18
`
`5,264,618
`
`-
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`L{) ' L{)
`w
`a.. f---+---+-+--+----<
`o'
`'\ u'
`0
`\
`• w -0:::
`
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`
`<:(
`
`0
`0
`
`oaaoo
`oaooo
`...
`OU)~C:C~
`....
`...
`~~~
`
`-.
`0
`Ol
`I
`0
`N
`I
`........,
`(.0
`
`La.J
`(/)
`<(
`c
`(/)
`0
`1-u
`<( __.
`
`<(
`(.!)
`I
`<(
`1-
`La..J
`CD
`u...
`0
`z
`0
`(/)
`(/)
`La.J
`a:::
`0..
`X
`La.J
`
`Moderna Ex 1017-p. 13
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 12 of 18
`
`5,264,618
`
`-
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`0 ' 0
`
`Cl ' a::
`
`0
`Cl
`
`/
`/
`/
`/
`/
`/
`/
`/
`L..a.J f---f--+-f---f---<
`c..
`\
`\
`\
`\
`0
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\ \,~,. ..
`
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`
`00000
`aoooa
`- ...
`O~Nt:C~
`...
`N~~
`
`" (/')
`0 u
`z
`0
`
`N
`0 c
`I
`N
`
`l.&..
`0
`z
`0
`(/')
`
`(/') w
`a:::
`0..
`X w
`0'1
`CL
`
`Moderna Ex 1017-p. 14
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 13 of 18
`
`5,264,618
`
`' \
`
`\
`\
`\
`
`"' U')
`0 u
`z
`0
`N
`()
`0
`I
`
`t......
`0
`z
`0
`U')
`U')
`w
`0::
`a..
`X w
`C) a.
`
`00000
`oaooo
`Ou::>NCC~
`....
`....
`....
`NT-~
`
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`' \
`
`\
`\
`\
`\
`\
`\
`
`Moderna Ex 1017-p. 15
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 14 of 18
`
`5,264,618
`
`/
`
`/
`
`/
`
`/
`
`I
`
`/
`
`I
`
`I
`
`/
`
`/
`
`I
`
`/
`
`I
`
`/
`
`/
`
`/
`
`/
`
`I
`
`/
`
`/
`
`/
`/
`I
`/
`1/ /1 /1 / /
`
`I
`
`1
`
`/
`
`I
`
`1
`
`/
`
`1
`
`1
`
`I
`
`/
`
`1
`
`/
`
`1
`
`1
`
`1
`
`/
`
`/
`/
`/
`/
`~ ~~~~~~~~u
`c...
`0
`
`U1
`UJ
`'
`
`0 ' 0::::
`
`0
`0
`
`....... . (/)
`0 u
`z
`0
`
`u....
`0
`z
`0
`(/)
`
`(/) w
`0:::: a..
`X
`w
`0'1 a..
`
`Moderna Ex 1017-p. 16
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 15 of 18
`
`5,264,618
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`// // //
`/
`/
`// // //
`/
`/
`/
`/
`/
`/
`/
`L.a..J f---f--+-f---+---(
`a_
`0
`0
`
`'
`
`co
`N
`
`"""
`
`0:::
`0
`0
`
`!"-(cid:173)
`(/)
`0
`u
`z
`0
`
`0
`O'l
`I
`
`< 1-(cid:173)w
`
`CD
`
`LL.
`0
`z
`0
`(/)
`
`(/) w
`a:::
`CL.
`X w
`O'l a..
`
`Moderna Ex 1017-p. 17
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 16 of 18
`
`5,264,618
`
`c::;;J
`c::;1 c::;1
`c::;;J
`c::;1 c::;;J c::;;J c;::l
`q "? r::f c;::l
`c::;1
`-.:::r
`
`r"V ..,....._ - DO
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`,.....
`
`(/)
`0
`u
`z
`0
`N
`0
`0
`I
`N
`>
`c::;;J (/)
`c;::l Q.
`ao
`co
`
`-
`0
`0)
`I
`<C
`t-
`w
`CD
`
`LL...
`0
`z
`0
`(/)
`(/)
`w
`0:::
`a..
`X
`w
`0)
`Q.
`
`t--
`\..{')~
`0')0')
`O')c:o
`t--~
`c:o~
`\..{')\..{')
`t--
`a~
`ot-t)\!')
`o~
`U)t-t")
`a
`U)
`
`<(
`0)
`•
`
`(!) c::
`
`\
`\
`\
`\
`\
`\
`
`a:::
`0
`Cl
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`
`' '
`
`\
`
`\
`
`\
`\
`\
`\
`\
`\
`\
`
`0000 0
`ooooa
`O~NC:O~
`....
`....
`....
`N~~
`
`Moderna Ex 1017-p. 18
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 17 of 18
`
`5,264,618
`
`~
`
`'
`
`['..
`
`_J
`0
`:::c
`u
`a::
`0
`Cl
`
`' \
`
`/
`
`/
`
`/
`
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`\
`
`c::;;, c::;:J
`c::;:J
`q u:: <;;( c::;:J
`c::;:J c::;:J
`c::;:J c::;:J
`c::;:J
`c::;:J
`r v - ..-- 00 ~
`
`......--
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`I"'--
`(/)
`0
`<->
`z
`0
`N
`0
`0
`I
`N
`>
`c::;:J (/)
`c.
`c::;:J
`00
`-
`C.£>
`0
`0>
`I
`< I-
`w
`ID
`
`L&..
`0
`z
`0
`(/)
`(/)
`w
`a:;
`a..
`X
`w
`
`0>
`c.
`
`t--
`\!')~
`cn<:n
`cnt:C
`t--._....
`cel-l')
`\!')\..{")
`t-.
`0._....
`al-l')\!)
`o---
`\.Ol'l')
`a
`\..0
`
`m
`Ch
`•
`~
`~
`
`oooaa
`ooooo
`.... - -
`0\.0~CC~
`._....
`~ ._....
`
`Moderna Ex 1017-p. 19
`Moderna v Arbutus
`
`
`
`U.S. Patent
`
`Nov. 23, 1993
`
`Sheet 18 of 18
`
`5,264,618
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`L()
`
`'
`
`/
`
`L()
`w /
`a.. \
`0
`\
`\
`0
`
`"" \
`
`\
`\
`\
`\
`\
`
`e::::
`0
`0
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`/
`
`r-... . (/)
`
`0
`(.)
`
`z
`0
`N
`()
`0
`I
`N >
`
`~(/)
`~ 0..
`DO
`..:.;
`c.o
`< (.!)
`I
`<(
`1-w
`CD
`
`LL...
`0
`z
`0
`(/)
`
`(/) w
`0::
`a..
`X w
`0> a..
`
`00 00 0
`ooooo
`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-Y1-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 -O-CH2-,
`-0-C(O)- or -0-;
`Rl a