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`US 20060240554Al
`
`(19) United States
`(12) Patent Application Publication
`Chen et al.
`
`(10) Pub. No.: US 2006/0240554 Al
`Oct. 26, 2006
`(43) Pub. Date:
`
`(54) LIPID NANOPARTICLE BASED
`COMPOSITIONS AND METHODS FOR THE
`DELIVERY OF BIOLOGICALLY ACTIVE
`MOLECULES
`
`(75)
`
`Inventors: Tongqian Chen, Longmont, CO (US);
`Chandra Vargeese, Broomfield, CO
`(US): Kurt Vagle, Longmont, CO (US);
`Weimin Wang, Superior, CO (US); Ye
`Zhang, Broomfield, CO (US)
`
`Correspondence Address:
`MCDONNELL BOEHNEN HULBERT &
`BERGHOFF LLP
`300 S. WACKER DRIVE
`32ND FLOOR
`CHICAGO, IL 60606 (US)
`
`(73) Assignee: Sirna Therapeutics, Inc., Boulder, CO
`
`(21) Appl. No.:
`
`11/353,630
`
`(22) Filed:
`
`Feb. 14, 2006
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/652,787, filed on Feb.
`14, 2005. Provisional application No. 60/678,531,
`filed on May 6, 2005. Provisional application No.
`60/703,946, filed on Jul. 29, 2005. Provisional appli(cid:173)
`cation No. 60/737,024, filed on Nov. 15, 2005.
`
`Publication Classification
`
`(51)
`
`Int. Cl.
`C12N 5102
`C07C 229110
`
`(2006.01)
`(2006.01)
`
`(52) U.S. Cl ............................................. 435/375; 554/110
`
`(57)
`
`ABSTRACT
`
`The present invention relates to novel cationic lipids, trans(cid:173)
`fection agents, microparticles, nanoparticles, and short inter(cid:173)
`fering nucleic acid (siNA) molecules. The invention also
`features compositions, and methods of use for the study,
`diagnosis, and treatment of traits, diseases and conditions
`that respond to the modulation of gene expression and/or
`activity in a subject or organism. Specifically, the invention
`relates to novel cationic lipids, microparticles, nanoparticles
`and transfection agents that effectively transfect or deliver
`biologically active molecules, such as antibodies (e.g.,
`monoclonal, chimeric, humanized etc.), cholesterol, hor(cid:173)
`mones, antivirals, peptides, proteins, chemotherapeutics,
`small molecules, vitamins, co-factors, nucleosides, nucle(cid:173)
`otides, oligonucleotides, enzymatic nucleic acids, antisense
`nucleic acids, triplex forming oligonucleotides, 2,5-A chi(cid:173)
`meras, dsRNA, allozymes, aptamers, decoys and analogs
`thereof, and small nucleic acid molecules, such as short
`interfering nucleic acid (siNA), short interfering RNA
`(siRNA), double-stranded RNA (dsRNA), micro-RNA
`(miRNA), and short hairpin RNA (shRNA) molecules, to
`relevant cells and/or tissues, such as in a subject or organ(cid:173)
`ism. Such novel cationic lipids, microparticles, nanopar(cid:173)
`ticles and transfection agents are usefl.Jl, for example, in
`providing compositions to prevent, inhibit, or treat diseases,
`conditions, or traits in a cell, subject or organism. The
`compositions described herein are generally referred to as
`formulated molecular compositions (FMC) or lipid nano(cid:173)
`particles (LNP).
`
`Moderna Ex 1005-p. 1
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`

`Patent Application Publication Oct. 26, 2006 Sheet 1 of 45
`
`US 2006/0240554 Al
`
`Figure 1
`
`A
`
`B
`
`c
`
`D
`
`k}o
`to
`~H~oy---oJ~H,
`
`~
`
`n
`
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`

`Patent Application Publication Oct. 26, 2006 Sheet 2 of 45
`
`US 2006/0240554 Al
`
`)
`
`(
`
`<
`0 <
`0~)
`\
`
`- z
`\
`
`- z
`
`u
`
`Moderna Ex 1005-p. 3
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`

`Patent Application Publication Oct. 26, 2006 Sheet 3 of 45
`
`US 2006/0240554 Al
`
`Figure 3
`
`A
`
`B
`
`c
`
`D
`
`0
`
`0
`
`0
`
`J0~0
`
`'N
`/
`
`0
`
`0
`
`0
`
`0
`
`J0~0
`
`'N
`/
`
`0
`
`0 o0o
`
`0
`
`0 0
`NH~OY'o}~H3
`0
`
`Moderna Ex 1005-p. 4
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q =
`a'
`e::
`== --· Q =
`.....
`
`~
`
`Figure4
`
`A
`
`B
`
`c
`
`D
`
`E
`
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`

`FIGURE 5
`Cationic Lipids
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
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`

`FIGURE 6
`
`Cationic lipid
`
`/
`
`""d
`
`"C
`
`~
`
`~ -("!> = -
`>
`"C -.....
`~ -.....
`Q = ""d = e::
`~ --· Q =
`
`.....
`
`~
`
`0
`~
`:""'"
`N
`~a-,
`N
`0
`0 a-,
`171 =-("!>
`
`("!> -a-,
`Q -""" u.
`
`d
`171
`N
`0
`0
`
`a-, -0
`""" 0 u. u.
`""" > .....
`
`N
`
`Moderna Ex 1005-p. 7
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`

`FIGURE 7
`
`Lamella Structure
`
`Inverted Hexagonal Structure
`
`Lipid
`bilayer
`
`--+
`
`Nucleic--.
`acid
`
`Nucleic
`acid
`
`""d
`
`"C
`
`~
`
`~ -("!> = ->
`"C -.....
`~ -.....
`Q = ""d = e::
`~ --· Q =
`
`.....
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171 =-("!>
`
`("!> --.l
`Q -""" u.
`
`d
`rJ)
`N
`0
`0
`
`0'1 -0
`""" 0 u. u.
`""" > .....
`
`N
`
`Moderna Ex 1005-p. 8
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`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 8
`
`Lipid Composition in L051
`
`0
`
`CLinDMA
`
`DSPC
`
`Cholesterol
`
`2KPEG-DMG
`
`Moderna Ex 1005-p. 9
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 9
`Lipid Structures in L073
`
`Me
`-o~o
`Me~N"+
`'p'
`Me' ........,......o .. "'o
`
`Joo
`
`0
`
`CLinDMA
`
`DMOBA.
`
`DSPC
`
`Cholesterol
`
`2KPEG-DMG
`
`Moderna Ex 1005-p. 10
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 10
`Lipid Composition in L069
`
`0
`
`ClinDMA
`
`DSPC
`
`Cholesterol
`
`0
`Cholesterol-PEG M•~o---f.o)(~-../'~CJ..../'-.~Jlo
`0
`
`Moderna Ex 1005-p. 11
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`

`FIGURE 11
`
`1.5
`
`~L065
`
`-o--f2
`
`--t:r-l051
`
`-<!>-l073
`
`0.0-r-----~~----~------~------~------~------~
`24
`20
`0
`4
`12
`16
`8
`
`(hours)
`
`""d
`
`"C
`
`~
`
`~ -("!> = ->
`"C -.....
`~ -.....
`Q = ""d = e::
`~ --· Q =
`
`.....
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171 =-("!>
`
`("!> -1-o'
`Q -"""' u.
`
`1-o'
`
`d
`171
`N
`0
`0
`
`0'1 -0
`"""' 0 u. u.
`"""' > .....
`
`N
`
`Moderna Ex 1005-p. 12
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`

`FIGURE 12
`
`pH-dependent phase transition at 30min
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`-11- L065
`
`-Jr- L051
`_._F2
`
`-+-L073
`
`3
`
`3.5
`
`4
`
`4.5
`
`5
`
`5.5 6
`
`6.5 7
`
`7.5
`
`8
`
`8.5
`
`9
`
`9.5
`
`pH
`
`1.08
`
`1.00
`
`0.96
`
`(§)
`·-"'C'J
`~ .1.04
`:CE
`.. ..,
`... c
`::Jo
`Q)CW') > ;;
`C'G -Q,)
`
`~
`
`Moderna Ex 1005-p. 13
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 13
`
`pH-dependent phase transition at 30min
`
`E
`c
`0
`U')
`(¥')
`@)
`
`:::J
`
`~ ·--c ·-.c ....
`....
`(I) > ·-....,
`C'O -(I)
`0::
`
`1.05
`
`1.02
`
`0.99
`
`0.96
`
`0.93
`
`0.90 +--r-----r--r----r----.--.-----.--.......----.--....--or-..------.-----,
`3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5
`
`pH
`
`t
`
`L069.2A
`
`Moderna Ex 1005-p. 14
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = :=:
`== --· Q =
`
`.....
`
`~
`
`FIGURE 14
`
`HBV site 263 siRNA
`5'- B G G A C U U C U C U C A A U U U U C U T T B - 3' Sense
`I I I I I I I I I I I I I I I I I I I
`3'- T5T C C U GAAGAGAG U U AAAA GA- 5'
`
`Antisense
`
`(SEQ ID NO: 6)
`
`(SEQ ID NO: 7)
`
`HBV site 1583 siRNA
`5'- B G C A C U U C G C U U C A C C U C U G T T 8 - 3' Sense
`t I I I I I I I I I I I
`t t t t
`t t
`I
`3'- TsT C G U GAAG C GAAG U GGAGA C -5'
`
`Antisense
`
`(SEQ ID NO: 8)
`
`(SEQ IDNO: 9)
`
`AGT= deoxy A, G &T
`AGCU = ribo A, G, C, U
`AG = 2 '-0-methy/ A & G
`cu = 2"-ffuoro C & U
`3 ',5' inverted deoxy abasic
`B
`= phosphorothioate
`s
`
`Moderna Ex 1005-p. 15
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`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 15: In vitro Analysis of siNA Formulation L051
`Activity in HBV Expressing Hep G2 Cells
`
`1
`
`E 0.8
`s::
`0
`l.t)
`"'':t
`:::J 0.6
`c
`0
`
`0.4
`
`0.2
`
`0
`
`Untreated
`
`2SnM
`
`lOnM SnM
`
`lnM
`
`25nM
`
`lOnM SnM
`
`lnM
`
`Active
`
`Control
`
`Moderna Ex 1005-p. 16
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 16: In vitro Analysis of siNA Formulation L053
`·and L054 activity in HBV Expressing Hep G2 Cells
`2 .5 , - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - .
`
`2
`
`Formulation L053
`
`Formulation L054
`
`a 1.s
`= 0
`...,.
`~
`0
`
`lO
`
`1
`
`0.5
`
`Active
`
`Control
`
`Active
`
`Control
`
`Moderna Ex 1005-p. 17
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 17
`
`HBV/siRNA KJ#137 (E389Z) - L069 - HBsAg ELISA
`
`0.9
`
`0.6
`
`CD
`en
`
`> 0.7
`"'CC - 0.6
`• -+
`E 0.5
`r::
`0
`~ 0.4
`::i 0.3
`d
`0
`
`0.2
`
`0.1
`
`0
`
`]i g
`
`c:
`::l
`
`::::'E
`
`8 :;;
`~
`:2
`~
`
`:a:
`~
`
`::::'E
`c:
`U')
`
`:E.
`....
`c:
`
`::::'E
`c
`U')
`d
`
`::::'E
`.:=
`d
`
`FMC L069
`
`::::'E
`§
`
`::::'E
`Ji
`
`::::'E
`.:=
`
`::::'E
`.Iii
`d
`
`::::'E
`c .,...
`d
`
`Irrelevant Control
`
`Concentration
`
`Moderna Ex 1005-p. 18
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`

`~
`
`""d a ("!> = -> :a -.....
`== --· Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 18: Activity. of siNA Formulation L051
`compositions in HBV Mouse Model, Serum HBV DNA
`
`G.oo,.----------------1--------------.
`
`Day3
`
`Day7
`
`4.00
`
`2.50
`
`~ w
`en
`i -...J
`E
`U;
`(L) ·c.
`0 u
`<(
`z
`c
`> m
`:I:
`E
`....
`:::J
`(L) en
`
`0 -C)
`
`.2
`
`2.00
`
`PBS
`3mg!I,g/day X 3 days
`
`Active
`
`Control
`
`PBS
`
`Active
`
`Control
`
`Moderna Ex 1005-p. 19
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`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 19: Activity of siNA siNA Formulation L051
`compositions in HBV Mouse Model, Serum HBsAg
`
`Day3
`
`Day7
`
`~1
`(/) m
`:r:
`E
`::l
`lo...
`Q)
`(/)
`..J
`
`100.0
`
`E -C) c
`
`10.0
`
`1.0
`
`PBS
`
`Active
`
`Control
`
`PBS
`
`Active
`
`Control
`
`0.1~------------------------~--------------------------~
`
`3mglkglday X 3 days
`
`Moderna Ex 1005-p. 20
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`

`~
`
`""d a ("!> = -> :a -.....
`== --· Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 20: In Vitro Analysis of siNA Formulation
`L051 Activity in HCV Replicon System
`
`1.~------------------------------------------------~
`
`<(
`z
`0:
`:I:
`c
`~
`C) < z
`0:::
`> 0
`
`:I:
`
`Untreated
`
`25nM
`
`10nM
`
`5nM
`
`1nM
`
`25nM
`
`10nM
`
`5nM
`
`1 nM
`
`Moderna Ex 1005-p. 21
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 21: In Vitro Analysis of siNA Formulation
`L053 and L054 Activity in HCV Replicon System
`
`1~~--------------------------------------------------~
`
`Formulation L053
`
`Formulation L054
`
`1
`
`Active
`
`Control
`
`Active
`
`Control
`
`Moderna Ex 1005-p. 22
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`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 22
`Concentration of siRNA in the Mouse Lung after IT Administration (20 pg)
`
`• Unfomrulated
`.. Formulation 18.1A
`• Unformulated
`• &IRNA • Cholesterol
`• Formulation 19.1A
`
`T1/2- 3-4 h
`
`T1/2- 9 h
`
`0
`
`10
`
`20
`
`30
`
`40
`
`50
`
`60
`
`70
`
`80
`
`Time (h)
`
`100
`
`10
`
`1
`
`0.1
`
`Moderna Ex 1005-p. 23
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`

`

`FIGURE 23A
`
`Toluenesulfonyl chloride
`
`Pyridine
`
`MsCI, TEA, 0 •c
`
`DCM
`
`2
`
`Mso,
`R-0
`
`Diol,£\
`
`Dioxane
`
`Ho,
`R-Q
`
`3a R- -C4H8-
`3b R- -(CH1 hO(CHzh-
`
`4a R- -C•Hs-
`4b R- ·(CH2) 20(CHz)z-
`
`~
`
`""d a ("!> = -> :a -......
`== -......
`Q = ""d = e::
`== --· Q =
`
`......
`
`~
`
`!
`fODMTr +
`/" ~OH
`5
`
`..
`
`NaH,£\
`
`Toluene
`
`NaH,a
`
`Toluene
`
`7
`
`Cia R= -c.H,-
`Cib R= -(CH 1)!0(CH 1h·
`
`,~o-R-o
`
`I
`
`l._0_......(CH2>e~c·H"
`
`8a R= -C4 He·
`8b R= ·(CH1 )tO(CH~)2·
`
`Moderna Ex 1005-p. 24
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`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`Figure 23B
`
`HO~C>«> +
`HO~
`
`R-OMs
`
`CsCOJ, 100 "C
`
`diglyme
`
`1
`
`2a, R Cl8H34, oleyl
`2b, R=C18H32, linoleyl
`
`R-Ox::rCHO
`
`R-0
`
`3a, R=C18H34, oleyl
`3b, R=Cl8H32, linoleyl
`
`l) Me2NH·HCI, Ti(OFr')4, TEA,
`BtOH
`l)NaB.U., NH,.OH
`
`I
`R-Ol(YN'
`R-0~
`4a, R=Cl8H34, oleyl
`4b, R=Cl8H32, linoleyl
`
`Moderna Ex 1005-p. 25
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`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== --· Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 24
`
`H
`Me1o/'-tOI(N~O~O~NH2
`0
`
`n-45
`
`DMAP, DIPEA
`
`THF, 6.
`
`Cholesterol-PEG
`
`DMB-Peg
`
`Moderna Ex 1005-p. 26
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 25
`Lipid Composition in LNP 083
`
`pCLinDMA (48%)
`
`DSPC (40%)
`
`Cholesterol(1 0°/o)
`
`2KPEG-Chol (2%)
`
`Moderna Ex 1005-p. 27
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`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 26
`Lipid Composition in LNP 077
`
`0
`
`eCLinDMA (48°/u)
`
`DSPC (40°/o)
`
`Cholesterol{1 Oo/o)
`
`2KPEG-Chol (2o/o)
`
`H
`0
`Me(o~O"M"N.........,...0..............,o ............. NJ.lO
`n g
`
`H
`
`Moderna Ex 1005-p. 28
`Moderna v Arbutus
`
`

`

`FIGURE 27
`Lipid Composition in LNP 080
`
`0
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`0
`
`eCLinDMA (48°/o)
`
`DSPC (40%)
`
`Cholesterol(1 0%)
`
`2KPEG-DMG (2%)
`
`M•~o-"'t~~......-"o...._a.......---)l}a 0
`
`n O
`
`H
`
`Moderna Ex 1005-p. 29
`Moderna v Arbutus
`
`

`

`FIGURE 28
`Lipid Composition in LNP 082
`
`0
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`0
`
`0 Jo o
`
`pCLinDMA (48°/o)
`
`DSPC (40o/o)
`
`Cholesterol(1 0%)
`
`2KPEG-DMG (2o/o)
`
`II
`H
`Mei>~O)(N~0~o..........,..-.N~O
`n O
`H
`
`0
`
`Moderna Ex 1005-p. 30
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 29
`
`Serum HBV DNA in SCID mice: siRNA formulation comparison
`
`3 mg/kg/d IV x 3 days; siRNA formulated HBV site 263 7/25
`
`3 days post last treatment
`
`6.5
`
`6.0
`
`5.5
`
`5.0
`
`4.5
`
`4.0
`
`3.5
`
`3.0
`LOQ= 2.9
`2.5
`
`PBS
`Control
`
`069.2A
`
`069.1
`Control
`
`077.2A
`
`080.1A
`
`082.1A
`
`083.1A
`
`060.1A
`
`061.1A
`
`051.2A
`1 mg/kg/d
`
`Treatment
`
`~
`
`.
`+
`
`0 -g»
`
`:;J
`L.,
`
`E
`Q) en _.
`~ z
`c
`> m
`:::t:
`
`Moderna Ex 1005-p. 31
`Moderna v Arbutus
`
`

`

`FIGURE 30
`HBV/siRNA Sirna 083 and 084-Site 263 stab 7/25-
`HBsAg ELISA
`
`(
`
`1.8
`
`1.6
`
`1.4
`
`1
`
`e 1.2
`c
`0
`~ -::; 0.8
`
`c:i
`0 0.6
`
`0.4
`
`0.2
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`0
`
`]1
`B
`
`1:
`:::>
`
`:e
`1:
`0 ......
`t.b
`~
`HepG2 45,000/well
`6.5 hour vector tranafectJon
`3 day formulation transfectlon
`
`:e c
`0 ....
`
`L082.1A
`
`L083.1A
`
`Treatment
`
`Moderna Ex 1005-p. 32
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== --· Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 31
`
`HBV/siRNA Sirna Formulation 077 -HBsAg ELISA
`
`1.8
`1.6
`1.4
`
`E
`c
`0
`
`- 1.2
`!:f -
`1
`~ 0.8
`c
`0.6
`0
`0.4
`0.2
`
`0
`
`Untreated
`
`7/25-10nM
`
`10nM
`
`5nM
`
`1nM
`
`0.5nM
`
`0.1nM
`
`HepG2 45,000/well
`7 .5hr vector tfn
`3 day fonnulatlon lfn
`
`Treatment
`
`Sirna 077.2A
`HBV 263 stab 7/25
`
`Moderna Ex 1005-p. 33
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== --· Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`FIGURE 32
`
`HBV/siRNA Sirna Formulation 080-HBsAg ELISA
`
`Untreated
`
`263 7/25-10nM
`
`10nM
`
`5nM
`
`1nM
`
`0.5nM
`
`0.1nM
`
`0.9
`0.8
`0.7
`
`1:
`0
`ll)
`
`0.5
`
`-E 0.6
`"':t -::::) 0.4
`c:i 0.3
`0
`0.2
`0.1
`0
`
`HepG2 45,000/well
`7 .5hr vector tfn
`3 day formulation tfn
`
`Treatment
`
`Sirna 080.1A
`HBV 263 stab 7/25
`
`Moderna Ex 1005-p. 34
`Moderna v Arbutus
`
`

`

`FIGURE 33
`
`The serum stability of Upld nano particles
`
`L077.2A
`
`L080.1A
`
`L082.1A
`
`L083.1A
`
`-+-
`-+-
`--D-
`
`----.----
`
`2
`
`1.5
`
`1
`
`0.5
`
`0
`
`&;-
`;;
`:a ...
`::::ll ...
`!l
`~
`Qj a:
`
`0
`
`4
`
`8
`
`12
`
`16
`
`20
`
`24
`
`Time (hrs)
`
`""d
`
`"C
`"C
`
`~
`
`Q
`
`~ -("!> = ->
`-.....
`~ -.....
`=
`""d = e::
`~ --· Q =
`.....
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171
`
`("!> -w
`=-("!>
`...
`"""' ... Ul
`
`Q
`
`d
`171
`N
`0
`0
`
`0
`N
`
`0'1 -
`... 0
`Ul ...
`>
`.....
`
`Ul
`
`Moderna Ex 1005-p. 35
`Moderna v Arbutus
`
`

`

`J!'
`'5
`
`:e :::J
`
`1-
`~
`:;:J
`111 &
`
`FIGURE 34
`
`Phase transition of lipid nano particles
`
`•
`•
`'
`
`L077.2A
`
`L080.1A
`
`L082.1A
`
`L083.1A
`
`1.05
`
`1.00
`
`0.95
`
`0.90
`
`3.5
`
`4
`
`4.5
`
`5
`
`5.5
`
`6
`
`6.5
`
`7
`
`7.5
`
`8
`
`8.5
`
`9
`
`9.5
`
`pH
`
`""d
`
`"C
`.....
`
`~
`
`~ -("!> = ->
`"C -
`~ --· Q =
`--· Q =
`
`""d = e::
`.....
`
`~
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171
`
`("!> -
`
`=-
`w
`tJI
`Q
`
`("!>
`
`....
`"""'
`
`tJI
`
`d
`171
`N
`0
`0
`
`0'1 -0
`
`0
`tJI
`tJI
`
`N ....
`....
`> .....
`
`Moderna Ex 1005-p. 36
`Moderna v Arbutus
`
`

`

`1.0
`0.9
`0.8
`0.7
`0.6
`0.5
`0.4
`0.3
`0.2
`0.1
`0.0
`
`Figure 35
`
`48h post-treatment
`
`1.1')
`
`.
`1.1')
`"'
`N
`~ T"'
`"' ~
`u.
`N
`u.
`..J
`..J
`
`1.1')
`
`"'
`
`-.::1"
`L()
`0
`..J
`
`.
`
`1.1')
`N
`"'r
`-.::1"
`L()
`0
`..J
`
`It)
`N
`CX)
`en
`0
`...J
`
`1.1') ..
`N
`T"'
`
`CX) en
`0
`..J
`
`1.1')
`N
`;::
`.....
`C.)
`c.
`z
`...J
`
`..
`I()
`N
`-
`...
`T"'
`.....
`0
`a.
`z
`...J
`
`LNP#/siNA (nM)
`
`""d
`
`"C
`
`~
`
`~ -("!> = ->
`"C -.....
`~ -.....
`Q = ""d = e::
`~ --· Q =
`
`.....
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`("!> -
`
`171 =-("!>
`w
`0'1
`Q
`
`....
`"""'
`
`Ul
`
`d
`171
`N
`0
`0
`
`0'1 -0
`N .... 0
`....
`> .....
`
`Ul
`Ul
`
`Moderna Ex 1005-p. 37
`Moderna v Arbutus
`
`

`

`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Figure 36
`
`48h post-treatment
`
`U')
`N
`:::.::::
`N
`LL.
`..J
`
`.
`
`U')
`N
`or-
`:::.::::
`N
`LL.
`..J
`
`1.()
`N
`CX)
`CD
`0
`..J
`
`.
`
`U')
`N
`or-
`CX)
`CD
`0
`..J
`
`LNP number/siNA {nM)
`
`U')
`-
`N
`....
`......
`(.)
`0..
`z
`..J
`
`.
`U')
`N
`-
`or-
`....
`.....
`(.)
`0..
`z
`..J
`
`""d
`
`"C
`
`~
`
`~ -("!>
`= ->
`"C -.....
`~ -.....
`Q =
`""d = :=:
`~ -
`.....
`-· Q =
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171
`
`=-("!>
`
`("!> -w
`
`........
`Q
`....
`"""'
`
`Ul
`
`d
`171
`N
`0
`0
`
`0
`N
`
`0'1 -
`.... 0
`....
`> .....
`
`Ul
`Ul
`
`Moderna Ex 1005-p. 38
`Moderna v Arbutus
`
`

`

`0 .
`
`0
`0
`
`0 .
`
`Ul
`0
`
`MapK14/3684 RNA
`. .
`.
`.
`.
`.
`N w w ~
`•
`0
`0
`Ul
`Ul
`0
`0
`0
`0
`
`~ ~ N
`0
`0
`Ul
`0
`0
`0
`
`LF2K 25
`
`LF2K 12.5
`
`L054 25
`
`L05412.5
`
`L097 25
`
`L09712.5
`
`L098 25
`
`L09812.5
`
`~
`00
`':J"
`"C
`0
`.....
`UJ
`~ ..,
`CD
`.....
`DJ
`3
`CD
`.....
`::l
`
`l1 CQ'
`s:
`<a
`w
`""'
`
`r-z .,
`
`::s
`c
`3
`C"
`CD
`:t
`
`tA -· z
`)>
`..-.
`::s
`35: -
`
`LNP ctrl25
`
`LNP ctrl12.5
`
`IV t'SSOt'Z0/90oz sa
`
`St' Jo 8£ Jaaqs 9ooz '9Z ·po uonuJnqnd uo!JBJ!fddv JU<tJBd
`
`Moderna Ex 1005-p. 39
`Moderna v Arbutus
`
`

`

`Figure 38
`
`48h post-treatment
`
`0.25
`
`0.20
`
`0.15
`
`0.10
`
`0.05
`
`It)
`N
`~
`N
`LL
`....I
`
`L() ..
`N
`..:-
`:::.:::
`N
`LL
`...1
`
`It)
`N
`00
`0)
`0
`...1
`
`L() .
`N
`..::-
`00
`0)
`0
`...1
`
`LNP number/siNA (nM}
`
`It)
`
`N -....
`.... (.)
`D.. z
`
`-1
`
`.
`It)
`C"'
`..:--....
`.....
`(.)
`D..
`z
`....I
`
`""d
`
`"C
`
`~
`
`~ -("!> = ->
`"C -.....
`~ -.....
`Q = ""d = :=:
`~ --· Q =
`
`.....
`
`~
`
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`171 =-("!>
`
`("!> -w
`
`~
`Q
`
`....
`"""'
`
`Ul
`
`d
`171
`N
`0
`0
`0'1
`
`-0
`N .... 0
`....
`> .....
`
`Ul
`Ul
`
`Moderna Ex 1005-p. 40
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = :=:
`== --· Q =
`
`.....
`
`~
`
`Figure 39
`
`1.2
`
`L054
`IC50 = 5.6nM
`
`L098
`IC50 = 3.5nM
`
`< 1.0
`z
`0!:
`0.8
`..q m co
`-..q
`
`("')
`
`0.6
`
`..:-
`~
`c. 0.4
`tU
`:E
`
`0.2
`
`0.0
`
`LC')
`N,
`
`. . ~ ...:-
`.
`
`LC')
`N,
`...:-
`
`LC')
`
`CD
`
`N,
`
`("')
`
`CD
`00
`0)
`~ ""': ~
`...:-
`0
`0
`
`0
`
`CD
`
`LC') .
`
`0
`
`0)
`("')
`0
`
`siNA concentration (nM)
`
`Moderna Ex 1005-p. 41
`Moderna v Arbutus
`
`

`

`2.00
`1.80
`1.60
`1.40
`1.20
`
`--=t
`al
`CD
`
`<C
`z a::
`C") - 1.00
`
`"'¢
`~
`~ 0.80
`c.
`CG
`:e
`0.60
`0.40
`0.20
`0.00
`
`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`Figure 40
`
`L098
`IC50 = 4.5nM
`
`II)
`N
`
`II)
`4:'i
`or-
`
`II)
`N
`u)
`
`N
`or-
`C"'i
`
`CD
`It!
`or-
`
`CIO
`I':
`c
`
`0)
`
`~ c
`
`c
`
`siNA concentration (nM)
`
`Moderna Ex 1005-p. 42
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`CQ
`
`<( z
`0:::
`-.::t co
`M -~
`~ c.
`C'G :e
`
`~
`
`3.5
`
`3.0
`
`2.5
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`l.t)
`N
`
`Figure 41
`
`L097
`IC50 = 2.1nM
`
`L098
`IC50 = 1.8nM
`
`0')
`
`M .
`
`0
`
`0
`
`CX)
`
`I':
`0
`
`en
`M .
`
`0
`
`0
`
`siNA concentration (nM)
`
`Moderna Ex 1005-p. 43
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = e::
`== --· Q =
`
`.....
`
`~
`
`<
`z
`c:::
`"¢
`aJ
`U)
`('I')
`-...
`~
`T"'"
`~
`c.
`cu
`==
`
`0.20
`0.18
`0.16
`0.14
`0.12
`0.10
`0.08
`0.06
`0.04
`0.02
`0.00
`
`Figure 42
`
`L098
`IC50 = 3.4nM
`
`L()
`N
`
`L()
`•
`N
`T"'"
`
`.
`
`L()
`N
`U)
`
`N
`T"'"
`•
`('I')
`
`U)
`In
`•
`T"'"
`
`CIO
`.......
`•
`0
`
`en
`('I') .
`
`0
`
`0
`
`siNA concentration (nM)
`
`Moderna Ex 1005-p. 44
`Moderna v Arbutus
`
`

`

`(I)
`
`::i:'
`c
`a..
`c
`0
`;:
`0
`·;::
`C/)
`
`-
`-
`
`s:::
`0
`0
`-~
`.s::
`(.)
`s:::
`.._
`0
`ID
`
`Figure 43
`
`"'ill-1 mg/kg Formulated
`
`........ 0.1 mg/kg Formulated
`-o-o.o1 mgfkg Formulated
`
`-t-Vehlcle
`
`-.-Naive
`
`12
`
`10
`
`8
`
`6
`
`4
`
`2
`
`0
`
`0
`
`10
`Concentration of Methacholine (mg/ml)
`
`25
`
`""d
`
`"C
`
`~
`
`~ -("!> = -
`>
`"C -.....
`~ -.....
`Q =
`""d
`=
`e::
`--·
`.....
`=
`0
`~
`:""'"
`N
`-?'
`N
`0
`0
`0'1
`
`~
`~
`
`Q
`
`171 =-
`
`("!>
`("!>
`
`-
`
`.&;;...
`.&;;...
`Q
`
`"""'
`
`.&;;...
`Ul
`
`d
`171
`N
`0
`0
`
`0'1 -0
`
`N
`.&;;...
`0
`Ul
`Ul
`.&;;...
`
`> .....
`
`Moderna Ex 1005-p. 45
`Moderna v Arbutus
`
`

`

`~
`
`""d a ("!> = -> :a -.....
`== -.....
`Q = ""d = :=:
`== --· Q =
`
`.....
`
`~
`
`Figure 44: Relative expression of HD RNA with active LNP-98 and
`LNP-61 formulated active siNA compared to LNP-61 mismatch
`· control
`
`<( z
`0::::
`c
`::I:
`<P
`>
`;::
`cu e
`<P
`> <(
`
`1
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`LNP-98
`
`LNP-61
`
`LNP61 mismatch
`
`Uninjected
`
`Treatment
`
`Moderna Ex 1005-p. 46
`Moderna v Arbutus
`
`

`

`US 2006/0240554 AI
`
`Oct. 26, 2006
`
`1
`
`LIPID NANOPARTICLE BASED COMPOSITIONS
`AND METHODS FOR THE DELIVERY OF
`BIOLOGICALLY ACTIVE MOLECULES
`
`[0001] This application claims the benefit of U.S. Provi(cid:173)
`sional patent application No. 60/652,787, filed Feb. 14,
`2005, U.S. Provisional patent application No. 60/678,531,
`filed May 6, 2005, U.S. Provisional patent application No.
`60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent
`application No. 60/737,024, filed Nov. 15, 2005. These
`applications are incorporated by reference herein in their
`entirety including the drawings.
`
`FIELD OF THE INVENTION
`
`[0002] The present invention relates to novel particle
`forming delivery agents including cationic lipids, micropar(cid:173)
`ticles, and nanoparticles that are useful for delivering vari(cid:173)
`ous molecules to cells. The invention also features compo(cid:173)
`sitions, and methods of use for the study, diagnosis, and
`treatment of traits, diseases and conditions that respond to
`the modulation of gene expression and/or activity in a
`subject or organism. Specifically, the invention relates to
`novel cationic lipids, microparticles, nanoparticles and
`transfection agents that effectively transfect or deliver bio(cid:173)
`logically active molecules, such as antibodies (e.g., mono(cid:173)
`clonal, chimeric, humanized etc.), cholesterol, hormones,
`antivirals, peptides, proteins, chemotherapeutics, small mol(cid:173)
`ecules, vitamins, co-factors, nucleosides, nucleotides, oligo(cid:173)
`nucleotides, enzymatic nucleic acids, antisense nucleic
`acids, triplex forming oligonucleotides, 2,5-A chimeras,
`allozymes, aptamers, decoys and analogs thereof, and small
`nucleic acid molecules, such as short interfering nucleic acid
`(siNA), short interfering RNA (siRNA), double-stranded
`RNA (dsRNA), micro-RNA (miRNA), and short hairpin
`RNA (shRNA) molecules, to relevant cells and/or tissues,
`such as in a subject or organism. Such novel cationic lipids,
`microparticles, nanoparticles and transfection agents are
`usefbl, for example, in providing compositions to prevent,
`inhibit, or treat diseases, conditions, or traits in a cell, subject
`or organism.
`
`BACKGROUND OF THE INVENTION
`
`[0003] The present invention relates to the delivery of
`biologically active molecules to cells. Specifically, the
`invention relates to compounds, compositions and methods
`for delivering nucleic acids, polynucleotides, and oligo(cid:173)
`nucleotides such RNA, DNA and analogs thereof~ peptides,
`polypeptides, proteins, antibodies, honnones and small mol(cid:173)
`ecules, to cells by facilitating transport across cellular mem(cid:173)
`branes in, for example, epithelial tissues and endothelial
`tissues. The compounds, compositions and methods of the
`invention are useful in therapeutic, research, and diagnostic
`applications that rely upon the efficient transfer of biologi(cid:173)
`cally active molecules into cells, tissues, and organs. The
`discussion is provided only for understanding of the inven(cid:173)
`tion that follows. This sununary is not an admission that any
`of the work described below is prior art to the claimed
`invention.
`
`[0004] The cellular delivery of various therapeutic com(cid:173)
`pounds, such as antiviral and chemotherapeutic agents, is
`usually compromised by two limitations. First the selectivity
`of a number of therapeutic agents is often low, resulting in
`high toxicity to nom1al tissues. Secondly, the trafficking of
`
`many compounds into living cells is highly restricted by the
`complex membrane systems of the cell. Specific transporters
`allow the selective entry of nutrients or regulatory mol(cid:173)
`ecules, while excluding most exogenous molecules such as
`nucleic acids and proteins. Various strategies can be used to
`improve transport of compounds into cells, including the use
`of lipid carriers, biodegradable polymers, and various con(cid:173)
`jugate systems.
`
`[0005] The most well studied approaches for improving
`the transport of foreign nucleic acids into cells involve the
`use of viral vectors or cationic lipids and related cytofectins.
`Viral vectors can be used to transfer genes efficiently into
`some cell types, but they generally cal1llot be used to
`introduce chemically synthesized molecules into cells. An
`alternative approach is to use delivery fonnulations incor(cid:173)
`porating cationic lipids, which interact with nucleic acids
`through one end and lipids or membrane systems through
`another (for a review see Feigner, 1990, Advanced Drug
`Delivery Reviews, 5,162-187; Feigner 1993, J. Liposome
`Res., 3,3-16). Synthetic nucleic acids as well as plasmids can
`be delivered using the cytofectins, although the utility of
`such compounds is often limited by cell-type specificity,
`requirement for low serum during transfection, and toxicity.
`
`[0006] Another approach to delivering biologically active
`molecules involves the use of conjugates. Conjugates are
`often selected based on the ability of certain molecules to be
`selectively transported into specific cells, for example via
`receptor-mediated endocytosis. By attaching a compound of
`interest to molecules that are actively transported across the
`cellular membranes, the effective transfer of that compound
`into cells or specific cellular organelles can be realized.
`Alternately, molecules that are able to penetrate cellular
`membranes without active
`transport mechanisms, for
`example, various lipophilic molecules, can be used to
`deliver compounds of interest. Examples of molecules that
`can be utilized as conjugates include but are not limited to
`peptides, hormones, fatty acids, vitamins, fiavonoids, sug(cid:173)
`ars, reporter molecules, reporter enzymes, chelators, por(cid:173)
`phyrins, intercalcators, and other molecules that are capable
`of penetrating cellular membranes, either by active transport
`or passive transport.
`
`[0007] The delivery of compounds to specific cell types,
`for example, cancer cells or cells specific to particular
`tissues and organs, can be accomplished by utilizing recep(cid:173)
`tors associated with specific cell types. Particular receptors
`are overexpressed in certain cancerous cells, including the
`high affinity folic acid receptor. For example, the high
`affinity folate receptor is a tumor marker that is overex(cid:173)
`pressed in a variety of neoplastic tissues, including breast,
`ovarian, cervical, colorectal, renal, and nasoparyngeal
`tumors, but is expressed to a very limited extent in nornml
`tissues. The use of folic acid based conjugates to transport
`exogenous compounds across cell membranes can provide a
`targeted delivery approach to the treatment and diagnosis of
`disease and can provide a reduction in the required dose of
`therapeutic compounds. Furthern10re, therapeutic bioavail(cid:173)
`ability, pharmacodynamics, and pharmacokinetic param(cid:173)
`eters can be modulated through the use of bioconjugates,
`including folate bioconjugates. Godwin eta!., 1972, J. Bioi.
`Chem., 247, 2266-2271, report the synthesis of biologically
`active pteroyloligo-L-glutamates. Habus et a!., 1998, Rio(cid:173)
`conjugate Chem., 9, 283-291, describe a method for the
`solid phase synthesis of certain oligonucleotide-folate con-
`
`Moderna Ex 1005-p. 47
`Moderna v Arbutus
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`US 2006/0240554 AI
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`Oct. 26, 2006
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`2
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`jugates. Cook, U.S. Pat. No. 6,721,208, describes certain
`oligonucleotides modified with specific conjugate groups.
`The use of biotin and folate conjugates to enhance trans(cid:173)
`membrane transport of exogenous molecules, including spe(cid:173)
`cific oligonucleotides has been reported by Low et a!., U.S.
`Pat. Nos. 5,416,016, 5,108,921, and International PCT pub(cid:173)
`lication No. WO 90/12096. Manoharan eta!, International
`PCT publication No. WO 99/66063 describe certain folate
`conjugates, including specific nucleic acid folate conjugates
`with a phosphoramidite moiety attached to the nucleic acid
`component of the conjugate, and methods for the synthesis
`of these folate conjugates. Nomura et a!., 2000, J. Org.
`Chem., 65, 5016-5021, describe the synthesis of an inter(cid:173)
`mediate, alpha-[2-(trimethylsilyl)ethoxycarbonyl]folic acid,
`usefl.Jl in the synthesis of ceratin types of folate-nucleoside
`conjugates. Guzaev eta!., U.S. Pat. No. 6,335,434, describes
`the synthesis of certain folate oligonucleotide conjugates.
`Vargeese et a!., International PCT Publication No. WO
`02/094185 and U.S. Patent Application Publication Nos.
`20030130186 and 20040110296 describe certain nucleic
`acid conjugates.
`[0008] The delivery of compounds to other cell types can
`be accomplished by utilizing receptors associated with a
`certain type of cell, such as hepatocytes. For example, drug
`delivery systems utilizing receptor-mediated endocytosis
`have been employed to achieve drug targeting as well as
`drug-uptake enhancement. The asialoglycoprotein receptor
`(ASGPr) (see for example Wu and Wu, 1987, J. Biol. Chem.
`262, 4429-4432) is unique to hepatocytes and binds
`branched galactose-terminal glycoproteins, such as asia(cid:173)
`loorosomucoid (ASOR). Binding of such glycoproteins or
`synthetic glycoconjugates to the receptor takes place with an
`affinity that strongly depends on the degree of branching of
`the oligosaccharide chain, for exan1ple, triatennary struc(cid:173)
`tures are bmmd with greater affinity than biatenarry or
`monoatem1ary chains (Baenziger and Fiete, 1980, Cell, 22,
`611-620; Connolly eta!., 1982, J. Biol. Chem., 257, 939-
`945). Lee and Lee, 1987, Glycoconjugate J., 4, 317-328,
`obtained this high specificity through the use of N-acetyl(cid:173)
`D-galactosamine as the carbohydrate moiety, which has
`higher affinity for the receptor, compared to galactose. This
`"clustering effect" has also been described for the binding
`and uptake of mannosyl-terminating glycoproteins or gly(cid:173)
`coconjugates (Ponpipom et a!., 1981, J. Med. Chem., 24,
`1388-1395). The use of galactose and galactosamine based
`conjugates to transport exogenous compounds across cell
`membranes can provide a targeted delivery approach to the
`treatment of liver disease such as HBV and HCV infection
`or hepatocellular carcinoma. The use of bioconjugates can
`also provide a reduction in the required dose of therapeutic
`compounds required for treatment. Furthermore, therapeutic
`bioavailability, pharmacodynan1ics, and pharn1acokinetic
`parameters can be modulated through the use of bioconju(cid:173)
`gates.
`[0009] A number of peptide based cellular transporters
`have been developed by several research groups. These
`peptides are capable of crossing cellular membranes in vitro
`and in vivo with high efficiency. Examples of such fusogenic
`peptides include a 16-amino acid fragment of the home(cid:173)
`odomain of ANTENNAPEDIA, a Drosophila transcription
`factor (Wang et a!., 1995, PNAS USA., 92, 3318-3322); a
`17 -mer fragment representing the hydrophobic region of the
`signal sequence of Kaposi fibroblast growth factor with or
`without NLS domain (Antopolsky et a!., 1999, Bioconj.
`
`Chem., 10, 598-606); a 17-mer signal peptide sequence of
`caiman crocodylus Ig(S) light chain (Chaloin et a!., 1997,
`Biochem. Biophys. Res. Comm., 243, 601-608); a 17-amino
`acid fusion sequence of HIV envelope glycoprotein gp4114,
`(Morris eta!., 1997, Nucleic Acids Res., 25, 2730-2736); the
`HIV-1 Tat49-57 fragment (Schwarze et a!., 1999, Science,
`285, 1569-1572); a transportan A~achimeric 27-mer con(cid:173)
`sisting of N -terminal fragment of neuropeptide galanine and
`membrane interacting wasp venom peptide mastoporan
`(Lindgren eta!.,