(19) United States
`(12) Patent Application Publication (10) Pub. No.: US 2009/0088562 A1
`Weller et al.
`(43) Pub. Date:
`Apr. 2, 2009
`
`US 20090088562A1
`
`(54) OLIGONUCLEOTIDE ANALOGS HAVING
`CATIONIC INTERSUBUNIT LINKAGES
`
`60/800,120, filed on May 11, 2006, provisional appli
`cation No. 60/800,145, filed on May 11, 2006.
`
`(76) Inventors:
`
`Dwight D. Weller, Corvallis, OR
`(US); Jed N. Hassinger, Philomath,
`OR (US)
`Correspondence Address:
`King & Spalding LLP
`P.O. BOX 889
`Belmont, CA 94002-0889 (US)
`
`22) Filed:
`(22) File
`
`(21) Appl. No.:
`y x- - -
`
`11F8O1885
`9
`Maw 10, 2007
`ay U,
`O
`O
`Related U.S. Application Data
`(60) Provisional application No. 60/800,076, filed on May
`11, 2006, provisional application No. 60/799,976,
`filed on May 11, 2006, provisional application No.
`
`Publication Classification
`
`(51) Int. Cl.
`(2006.01)
`C7H 2L/00
`52) U.S. Cl. ....................................................... 536/24.5
`(52)
`(57)
`ABSTRACT
`Morpholino oligomers containing both uncharged and cat
`ionic intersubunit linkages are provided. The oligomers are
`oligonucleotide analogs containing predetermined sequences
`of base-pairing moieties. The presence of the cationic inter
`Subunit linkages in the oligomers, typically at a level of about
`10-50% of total linkages, provides enhanced antisense activ
`ity, in various antisense applications, relative to the corre
`sponding uncharged oligomers. Also provided are such oli
`gomers conjugated to peptide transporter moieties, where the
`transporters are preferably composed of arginine subunits, or
`arginine dimers, alternating with neutral amino acid subunits.
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 1 of 31
`
`US 2009/0088562 A1
`
`
`
`9
`
`s
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 2 of 31
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`US 2009/0088562 A1
`
`[]
`
`
`
`ap?dad quodsue]]
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 3 of 31
`
`US 2009/0088562 A1
`
`y
`
`1a-e, where P=
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 4 of 31
`
`US 2009/0088562 A1
`
`HO
`
`H
`
`HO
`2a-df
`
`N
`{
`
`rvy
`
`O
`
`NH
`
`e
`
`f P
`
`HO
`
`1. NaiO4; aqueous methanol
`2. (NH4)2B407
`3, Borane-triethylamine
`4. Methanolic acid (p-TsOH or HCI)
`
`HO
`
`P
`
`O
`
`H. H 8
`3a-df, where X:
`
`O O
`a - Ho ||
`|| o?
`2.
`
`b, C d, f =
`
`1. Triethylamine? DMF
`2. Trity Chloride
`3. Diethylamine
`
`HO
`
`O P
`
`N
`
`N
`
`{
`
`NH
`e
`
`1. ImidazoleITBDMS-CII DCM
`2. K2CO3/Chloromethyl
`pivalate/THF
`3. Et3N-3HF THF
`
`N
`{
`w
`
`NO
`N -k
`e
`
`Fig. 2B
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 5 of 31
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`US 2009/0088562 A1
`
`
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 6 of 31
`
`US 2009/0088562 A1
`
`2Succinate
`
`N C)
`
`H
`
`d)
`
`C
`
`Piperazine
`
`13a CFCOOCHCH
`13b HCFCOOCH
`13c. CHCFCOOCHCH
`13d (CF32CHCOOCH3
`
`a Y = COCF3
`b Y=COCHF
`cY =COCFCH3
`d Y = COCH(CF).
`
`Fig. 2D
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 7 of 31
`
`US 2009/0088562 A1
`
`HO
`
`o,
`
`H
`
`CO Swen Oxidation
`
`1are
`
`NH
`CFCON 1n 11n 10-1No.1a-NH2
`H
`
`Pyridine-borane
`
`16a-e
`
`So
`pyridine-Dorane
`
`Y
`
`P
`O 1n 10 N-1No1a- y
`
`Y
`N
`H3C y
`
`P
`
`N
`
`H
`
`CFacso
`
`20a-e Y = H
`21a-e YE FMOC
`20a-e Y = POCOEt
`
`17a-e Y = H
`18a-e YE FMOC
`19a-e Y = POCIOEt
`
`Fig. 2E
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 8 of 31
`
`US 2009/0088562 A1
`
`HO
`
`P
`
`Y
`
`TSO
`
`P
`
`O
`
`N
`
`Tosyl chloride
`N-methylimidazole
`
`1a-e
`
`26
`cfco-N1,N1 NH
`
`CFsco
`
`23a-e
`
`H
`2
`
`Y
`
`cfco-N1Nn1.
`CH
`N
`3
`
`H
`
`Y
`
`1N1N1N1
`CFCO-N H
`
`P
`Ol'
`
`N
`
`O P
`
`N
`
`27a-e Y =POCIOEt
`
`24a-eY = FMOC
`25a-eY =POCIOEt
`
`Fig. 2F
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 9 of 31
`
`US 2009/0088562 A1
`
`CHNH
`
`P.
`
`Y
`
`O
`
`N
`
`Sulfur trioxideloyridine
`Triethylamine
`DMF
`
`EtNH OSN
`
`2.Y.
`
`o,
`
`N
`
`17a-e
`
`28a
`8a-e
`
`SOC
`
`CFCO-N
`
`1N1N1N/
`H
`
`if a P.
`Y
`
`SOC
`
`is of
`H3C Y.
`
`N
`
`N
`
`30a-e
`
`29a-e
`
`Fig. 2G
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 10 of 31
`
`US 2009/0088562 A1
`
`O O
`Ho 1-1S as 1a- 1. Carbonyldimidazde K)
`N -o- S
`2, 12
`31
`O 32
`
`O
`
`Triphenylphosphine
`C
`NaOH, 31
`( )
`( \ls---s-n-
`O 3 1,1-Carbonyldiimidazole
`
`\ /
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 11 of 31
`
`US 2009/0088562 A1
`
`O By chloroformale
`K) K): S6io colo
`O 11
`O 35
`
`HO
`
`NaHINMP
`Triethylene glycol
`
`Succinic anhydride? DMAP O 36
`THF, 55 C
`
`O ().C.-
`cks' HO-N
`EDC cos-3
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 12 of 31
`
`US 2009/0088562 A1
`
`8
`
`Cz º 6 TJ
`
`
`
`
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 13 of 31
`
`US 2009/0088562 A1
`
`y
`
`N
`Tr 1c
`
`
`
`TIPS-C
`
`Triethylamine
`DMAP
`
`--
`
`inal
`
`TBDMS-C
`Dichloromethane
`
`C
`
`Tr
`
`41
`
`N-methylpyrrolidine; DBU
`O
`
`HO
`
`-()-o
`43
`
`h
`
`NS
`
`s
`
`(
`
`N
`r
`
`44
`
`Triethylamine - 3 HF
`
`o
`
`N
`7/
`
`NS
`N
`
`c 1.
`c N.
`
`N
`
`t
`
`45
`
`O
`
`NH
`
`O
`
`O
`
`Dichloronethane
`2,6-lutidine
`N-methylimidazole
`N,N-dimethylphosphoramidodichloridate
`
`N
`(
`N
`
`NS
`N
`els
`N.
`NH
`
`O
`
`3.
`MegN
`
`Tr
`
`Fig. 2K
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 14 of 31
`
`US 2009/0088562 A1
`
`| | /ºOO?eN ‘be W GO-}
`
`~~~~~~~~~~~~
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 15 of 31
`
`US 2009/0088562 A1
`
`
`
`@————. "€.
`
`N
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 16 of 31
`
`US 2009/0088562 A1
`
`HN
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 17 of 31
`
`US 2009/0088562 A1
`
`OO
`
`Oz • 6TH
`
`

`

`US 2009/0088562 A1
`
`Patent Application Publication
`
`Apr. 2, 2009 Sheet 18 of 31
`
`as
`
`{{Ç
`
`
`Q – pºdWN JOSWOo ? NON8-?
`* ——————~~~~—~~~~–------- {?,º
`
`O* I
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 19 of 31
`
`US 2009/0088562 A1
`
`* 6 TOEI
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 20 of 31
`
`US 2009/0088562 A1
`
`(Iz º 67,5
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 21 of 31
`
`US 2009/0088562 A1
`
`2000
`
`
`
`E 1000
`.
`-
`
`O 537-AUG
`A 164-AUG+
`V 165-5'-term
`0 166-5'-term
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 22 of 31
`
`US 2009/0088562 A1
`
`L-AUG (SEQID NO:11)
`No Charge (EC50=203.6 nM)
`L-2+ (SEQID NO.12)
`+2 Charge (EC50=76.6 nM)
`L-4a (SEQ D NO:13)
`+4 Charge (EC50=21.7 nM)
`L-4b+ (SEQID NO:14)
`+4 Charge (EC50=7.1 nM)
`
`S.
`
`1700
`1600
`1500
`1400
`1300
`1200
`1100
`1000
`900
`800
`700
`SOO
`500
`400
`300
`200
`100
`
`0.0
`
`2.5
`
`3.0
`
`1.0
`
`1.5
`2.0
`logPMOnM
`Fig. 4
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 23 of 31
`
`US 2009/0088562 A1
`
`--L-AUG (SEQID NO:11)
`No Charge (EC50=203.6 nM)
`--L-2+ (SEQID NO.12)
`+2 Charge (EC50=76.6 nM)
`-Y-L-4a- (SEQ ID NO:13)
`+4 Charge (EC50=21.7 nM)
`--L-4b+ (SEQID NO.14)
`+4 Charge (EC50=7.1 nM)
`
`7000 - se-s: d
`
`Q
`
`8000
`
`E 6000
`D
`E 5000
`9.
`- C 4000
`2
`is 3000
`E. 2000
`1000
`
`O
`10
`
`100
`PMO, nM
`Fig. 5
`
`1000
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 24 of 31
`
`US 2009/0088562 A1
`
`0-VP24-AUG; SEQ ID NO:5
`-O-VP24-2+; SEQ ID NO:6
`mAVP24-4+; SEQ ID NO:7
`HVP24-4+;SEQID NO:8
`m) (PBS
`
`* 100 ug total
`
`\
`\
`
`
`
`
`
`O
`100%
`
`75%
`
`50%
`
`25%
`
`O%
`
`O
`
`1
`
`7 8 9 10 11 12 13 14
`2 3 4 5 6
`Days Post Challenge
`Fig. 6
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 25 of 31
`
`US 2009/0088562 A1
`
`30
`
`25
`
`20
`S
`ar 15
`
`10
`
`5
`
`r
`2
`E. 2.
`O-
`O
`
`25
`
`-O-P-Polar
`P-Disp
`-V- P-Disp
`-0- P-Disp
`II
`II P-Disp
`m
`P-Cent
`H NOT
`
`al"tuit III III'
`
`II
`
`A7
`
`se-P
`H
`75
`
`50
`
`100
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 26 of 31
`
`US 2009/0088562 A1
`
`30
`
`25
`
`20
`
`15
`
`10
`
`-- G-Polar
`- Go-Disp
`-v- Go-Disp
`Ga-Disp
`-o-Gs-Cent
`-- 705F
`Im NOT
`
`75
`
`100
`
`25
`
`50
`uM
`Fig. 8
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 27 of 31
`
`US 2009/0088562 A1
`
`35
`
`30
`
`25
`S. 20
`or 15
`
`
`
`10
`
`5
`
`O
`
`-O-G-Cent
`-O- SCR-G-Cent
`-O-P-Cent
`in SCR-P-Cent
`-H PMO
`H NOT
`
`O
`
`25
`
`50
`uM
`
`75
`
`100
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 28 of 31
`
`US 2009/0088562 A1
`
`80
`78
`76
`74
`72
`
`
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 29 of 31
`
`US 2009/0088562 A1
`
`
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 30 of 31
`
`US 2009/0088562 A1
`
`35
`
`30
`25
`g 20
`D
`15
`Y
`
`10
`
`5
`
`O
`O
`
`-o- G3-Cent
`-- P-Cent
`a PPMO
`H. PM
`O
`m
`NOT
`
`25
`
`75
`
`50
`uM
`Fig. 11B
`
`100
`
`

`

`Patent Application Publication
`
`Apr. 2, 2009 Sheet 31 of 31
`
`US 2009/0088562 A1
`
`3000
`
`
`
`4472 (SEQID NO:35) (EC-295.1 nM)
`A 0136-3a+ (SEQID NO:36) (EC-74.1 nM)
`v 0137-3b+ (SEQID NO:37) (EC50=114.8nM)
`0 0138-3C+ (SEQID NO:38) (EC-128.8nM)
`0 0139-3d (SEQID NO:39) (EC50=66.1 nM)
`D 0140-3e+ (SEQID NO.40) (EC=70.8nM)
`A 0141-4ft (SEQID NO41)(EC=63.1 nM)
`v O142-6g+(SEQID NO42) (EC50=26.3 nM)
`
`2.75
`2.50
`100 125 150 1.75 200 2.25
`log (PMOnM
`Fig. 12
`
`3.00
`
`3.25
`
`3.50
`
`3.75 400
`
`

`

`US 2009/0088,562 A1
`
`Apr. 2, 2009
`
`OLGONUCLEOTDE ANALOGS HAVING
`CATIONIC INTERSUBUNIT LINKAGES
`
`0001. This application claims priority to U.S. Patent
`Application Nos. 60/800,076 filed May 11, 2006, 60/799,976
`filed May 11, 2006, 60/800,120 filed May 11, 2006, 60/800,
`145 filed May 11, 2006, Ser. No. 1 1/431,968 filed May 10,
`2006, Ser. No. 11/432,031 filed May 10, 2006, Ser. No.
`11/432,155 filed May 10, 2006, Ser. No. 1 1/432.216 filed
`May 10, 2006, Ser. No. 11/433,033 filed May 11, 2006, Ser.
`No. 11/433,213 filed May 11, 2006, Ser. No. 11/433,214 filed
`May 11, 2006, Ser. No. 11/433,257 filed May 11, 2006, Ser.
`No. 11/433,724 filed May 11, 2006, Ser. No. 11/433,840 filed
`May 11, 2006, Ser. No. 11/517,757 filed Sep. 8, 2006, Ser.
`No. 11/518,058 filed Sep. 8, 2006, Ser. No. 1 1/595,161 filed
`Nov. 8, 2006, and Ser. No. 1 1/715,572 filed Mar. 7, 2007, all
`of which are incorporated herein in their entirety by refer
`CCC.
`
`FIELD OF THE INVENTION
`0002 The present invention relates to oligonucleotide
`analogs (oligomers) useful as antisense compounds, and
`more particularly to oligomers containing cationic linkages,
`and the use of such oligomers in antisense applications. Par
`ticularly preferred are morpholino oligomers containing both
`uncharged linkages and cationic linkages, where both can be
`phosphorodiamidate linkages, and exemplary cationic link
`ages include a (1-piperazino) phosphinylideneoxy linkage
`and a (1-(4-(co-guanidino-alkanoyl))-piperazino) phosphi
`nylideneoxy linkage.
`
`REFERENCES
`0003) Arya, D. P. and T. C. Bruice (1999). “Triple-helix
`formation of DNA oligomers with methylthiourea-linked
`nucleosides (DNmt): a kinetic and thermodynamic analy
`sis.” Proc Natl AcadSci USA 96(8): 4384-9.
`0004 Bailey, C. P. J. M. Dagle et al. (1998). “Cationic
`oligonucleotides can mediate specific inhibition of gene
`expression in Xenopus oocytes.' Nucleic Acids Res 26(21):
`4860-7.
`0005 Barawkar, D. A. and T. C. Bruice (1998). “Synthe
`sis, biophysical properties, and nuclease resistance prop
`erties of mixed backbone oligodeoxynucleotides contain
`ing cationic internucleoside guanidinium linkages:
`deoxynucleic guanidine/DNA chimeras.” Proc Natl Acad
`Sci USA 95(19): 11047-52.
`0006 Bray, M., K. Davis et al. (1998). “A mouse model for
`evaluation of prophylaxis and therapy of Ebola hemor
`rhagic fever.”J Infect Dis 178(3): 651-61.
`0007 Connolly, B. M., K. E. Steele et al. (1999). “Patho
`genesis of experimental Ebola virus infection in guinea
`pigs.”J Infect Dis 179 Suppl 1: S203-17.
`0008 Crooke, S. T. (2001). Antisense Drug Technology:
`Principles, Strategies, and Applications. New York, Mar
`cel Dekker.
`0009. Dempcy, R. O. O. Almarsson et al. (1994). “Design
`and synthesis of deoxynucleic guanidine: a polycation ana
`logue of DNA” Proc Natl AcadSci USA 91(17): 7864-8.
`0010 Dempcy, R. O. J. Luo et al. (1996). “Design and
`synthesis of ribonucleic guanidine: a polycationic analog
`of RNA” Proc Natl AcadSci USA 93(9): 4326-30.
`
`Egli, M., G. Minasov et al. (2005). “Probing the
`(0011
`influence of stereoelectronic effects on the biophysical
`properties of oligonucleotides: comprehensive analysis of
`the RNA affinity, nuclease resistance, and crystal structure
`often 2'-O-ribonucleic acid modifications.” Biochemistry
`44(25): 9045-57.
`(0012 Linkletter, B. A. and Bruice, T. C. (2000). “Solid
`phase synthesis of positively charged deoxynucleic guani
`dine (DNG) modified oligonucleotides containing neutral
`urea linkages: Effect of charge deletions on binding and
`fidelity.” Bioorg. Med. Chem. 8(11): 1893-1901.
`(0013 Linkletter, B. A., I. E. Szabo et al. (2001). “Solid
`phase synthesis of oligopurine deoxynucleic guanidine
`(DNG) and analysis of binding with DNA oligomers.”
`Nucleic Acids Res 29(11): 2370-6.
`(0014) Micklefield, J. (2001). “Backbone modification of
`nucleic acids: Synthesis, structure and therapeutic applica
`tions.” Curr Med Chem 8(10): 1157-79.
`(0015 Moulton, H. M., M. H. Nelson et al. (2004). “Cel
`lular uptake of antisense morpholino oligomers conjugated
`to arginine-rich peptides. Bioconjug Chem 15(2): 290-9.
`(0016. Nelson, M. H., D. A. Stein et al. (2005). “Arginine
`rich peptide conjugation to morpholino oligomers: effects
`on antisense activity and specificity. Bioconjug Chem
`16(4): 959-66.
`(0017 Summerton, J. and D. Weller (1997). “Morpholino
`antisense oligomers: design, preparation, and properties.”
`Antisense Nucleic Acid Drug Dev 7(3): 187-95.
`(0018 Warfield, K. L., D. L. Swenson, et al. (2006). “Gene
`Specific Countermeasures against Ebola Virus Based on
`Antisense Phosphorodiamidate Morpholino Oligomers.”
`PLOS Pathog2(1): el.
`
`BACKGROUND OF THE INVENTION
`0019 Requirements for successful implementation of
`antisense therapeutic molecules, which are generally
`designed to bind to DNA or RNA of disease-causing proteins
`to prevent the production of Such proteins, include (a) stabil
`ity in vivo, (b) sufficient membrane permeability and cellular
`uptake, and (c) a good balance of binding affinity and
`sequence specificity. Many oligonucleotide analogs have
`been developed in which the phosphodiester linkages of
`native DNA are replaced by other linkages that are resistant to
`nuclease degradation (see e.g. Barawkar and Bruice 1998:
`Linkletter, Szabo et al. 2001; Micklefield 2001). Antisense
`oligonucleotides having various backbone modifications
`other than to the internucleoside linkage have also been pre
`pared (Crooke 2001; Micklefield 2001). In addition, oligo
`nucleotides have been modified by peptide conjugation in
`order to enhance cellular uptake (Moulton, Nelson et al. 2004:
`Nelson, Stein et al. 2005).
`0020. The performance of such nucleic acid analogs as
`antisense or antigene drugs has been hampered by certain
`characteristics of the various analogs. For example, analogs
`with negatively charged linkages, including phosphorothio
`ate-linked analogs, Suffer from considerable electrostatic
`repulsion between the negative charges of the oligomer and
`the DNA or RNA target. The phosphorothioates also exhibit
`non-specific binding to other cellular components such as
`proteins. These attributes limit the usefulness of antisense
`oligomers comprised of native RNA, native DNA, and nega
`tively charged analogs as therapeutic agents (Crooke 2001).
`The nonionic methylphosphonate-linked oligonucleotide
`analogs can be transported into cells by passive diffusion
`
`

`

`US 2009/0088,562 A1
`
`Apr. 2, 2009
`
`and/or fluid phase endocytosis, but their use is hampered by
`stereoisomeric complexity and poor solubility (Crooke 2001;
`Micklefield 2001).
`0021 Several groups have reported the synthesis of posi
`tively charged oligonucleotides (Bailey, Dagle et al. 1998:
`Micklefield 2001; Egli, Minasov et al. 2005). For example, a
`class of guanidinium linked nucleosides (designated DNG).
`formed by replacement of the phosphate linkages in DNA and
`RNA by achiral guanidino groups, has been reported
`(Dempcy. Almarsson et al. 1994: Dempcy, Luo et al. 1996;
`Barawkar and Bruice 1998: Linkletter, Szabo et al. 2001).
`Oligomers linked with positively charged methylated thio
`urea linkages have also been reported (Arya and Bruice
`1999). Replacement of some of these linkages with neutral
`urea linkages is reported to reduce the tendency of Such
`positively charged oligomers towards non-sequence-specific
`binding (Linkletter and Bruice, 2000). However, there
`remains a need for oligonucleotide analogs with improved
`antisense orantigene performance, particularly in the area of
`stronger affinity for DNA and RNA, without compromising
`sequence selectivity.
`
`SUMMARY
`
`0022. The invention provides, in one aspect, an oligomer
`comprising a backbone consisting of a sequence of mor
`pholino ring structures joined by intersubunit linkages, where
`each Such ring structure Supports a base-pairing moiety. Such
`that said oligomer can bind in a sequence-specific manner to
`a target nucleic acid, and where at least one intersubunit
`linkage between two consecutive such ring structures con
`tains a pendant cationic group. The pendant group bears a
`distal nitrogen atom that can bear a positive charge at neutral
`or near-neutral (e.g. physiological) pH.
`0023 The intersubunit linkages are preferably phospho
`rus-containing linkages, having the structure:
`
`where
`W is S or O, and is preferably O,
`
`Y—O or NR7,
`0024 and each said linkage in the oligomer is selected
`from:
`0025 (a) uncharged linkage (a), where each of R. R. R.
`and R is independently selected from hydrogen and lower
`alkyl:
`(0026 (b1) cationic linkage (b1), where X=NR'R'' and
`Y=O, and NR'R' represents an optionally substituted
`piperazino group, such that RR = CHRCHRN(R)
`(R)CHRCHR , where
`each R is independently H or CH,
`0027
`R" is H, CH, or an electron pair, and
`0028
`
`0029 R is selected from H. lower alkyl, C(=NH)NH2,
`Z-L-NHC(=NH)NH, and C(O)CHR'NHH, where: Z is
`C(O) or a direct bond, L is an optional linker up to 18 atoms
`in length, preferably upt to 12 atoms, and more preferably up
`to 8 atoms in length, having bonds selected from alkyl,
`alkoxy, and alkylamino, R is a side chain of a naturally
`occurring amino acid or a one- or two-carbon homolog
`thereof, and m is 1 to 6, preferably 1 to 4:
`0030) (b2) cationic linkage (b2), where X=NR'R'' and
`Y—O, R'—H or CH, and R=LNRRR, where L, R, and
`R" are as defined above, and R is H. lower alkyl, or lower
`(alkoxy)alkyl, and
`0031 (b3) cationic linkage (b3), where Y—NR7 and
`X-OR, and R=LNRRR, where L, R, R and Rare as
`defined above, and R is H or lower alkyl:
`0032 and at least one said linkage is selected from cat
`ionic linkages (b1), (b2), and (b3).
`0033 Preferably, the oligomer includes at least two con
`secutive linkages of type (a) (i.e. uncharged linkages). In
`further embodiments, at least 5% of the linkages in the oli
`gomer are cationic linkages (i.e. type (b1), (b2), or (b3)); for
`example, 10% to 80%, 10% to 50%, or 10% to 35% of the
`linkages may be cationic linkages.
`0034 Preferably, all cationic linkages in the oligomer are
`of the same type; i.e. all of type (b1), all of type (b2), or all of
`type (b3).
`0035. In one embodiment, at least one linkage is of type
`(b1), where, preferably, each R is H. R. is H, CH, or an
`electronpair, andR is selected from H. lower alkyl, e.g. CHs.
`C(=NH)NH, and C(O)-L-NHC(=NH)NH. The latter two
`embodiments of R provide a guanidino moiety, either
`attached directly to the piperazine ring, or pendant to a linker
`group L. respectively. For ease of synthesis, the variable Z in
`R is preferably C(O) (carbonyl), as shown.
`0036. The linker group L, as noted above, contains bonds
`in its backbone selected from alkyl (e.g. —CH2—CH2—).
`alkoxy ( C-O ), and alkylamino (e.g. —CH2—NH ),
`with the proviso that the terminal atoms in L (e.g., those
`adjacent to carbonyl or nitrogen) are carbon atoms. Although
`branched linkages (e.g. —CH2—CHCH ) are possible, the
`linker is preferably unbranched. In one embodiment, the
`linker is a hydrocarbon linker. Such a linker may have the
`structure —(CH), , where n is 1-12, preferably 2-8, and
`more preferably 2-6.
`0037. The morpholino subunits have the structure:
`
`(i)
`
`r P
`
`0038 where Pi is a base-pairing moiety, and the linkages
`depicted above connect the nitrogen atom of (i) to the 5'
`carbon of an adjacent Subunit. The base-pairing moieties Pi
`may be the same or different, and are generally designed to
`provide a sequence which binds to a target nucleic acid.
`
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`

`US 2009/0088,562 A1
`
`Apr. 2, 2009
`
`0039. The use of embodiments of linkage types (b1), (b2)
`and (b3) above to link morpholino subunits may be illustrated
`graphically as follows:
`
`0041. In further embodiments, the cationic linkages are
`selected from linkages (b1) and (b1") as shown below, where
`(b1") is referred to herein as a “Pip” linkage and (b1") is
`referred to herein as a "GuX' linkage:
`
`(b1)
`
`H
`
`(a)
`
`A //\
`\ /
`
`NH2
`
`WEP-N
`
`|
`
`m
`
`(b1")
`
`A
`
`NH2
`1s
`N
`WEP-
`N
`w ~~~ NH,
`
`(b2)
`
`O Pi
`
`o=-\ NR3RR5
`n 1 L
`
`O Pi
`
`..
`
`O Pi
`
`-
`o=-or
`R5R4R3N
`N
`n 1 L
`
`O Pi
`
`(b3)
`
`0040 Preferably, at least 5% of the linkages in an oligomer
`are selected from cationic linkages (b1), (b2), and (b3); in
`further embodiments, 10% to 35% of the linkages are selected
`from cationic linkages (b1), (b2), and (b3). As noted above,
`all of the cationic linkages in an oligomerare preferably of the
`same type or structure.
`
`0042. In the structures above, W is S or O, and is prefer
`ably O; each of R' and R is independently selected from
`hydrogen and lower alkyl, and is preferably methyl; and A
`represents hydrogenora non-interfering substituent on one or
`more carbon atoms in (b1") and (b1"). Preferably, each A is
`hydrogen; that is, the nitrogen heterocycle is preferably
`unsubstituted. In further embodiments, at least 10% of the
`linkages are of type (b 1') or (b1"); for example, 20% to 80%,
`20% to 50%, or 20% to 30% of the linkages may be of type
`(b1") or (b1").
`0043. In other embodiments, the oligomer contains no
`linkages of type (b1"). Alternatively, the oligomer contains no
`linkages of type (b1) where each R is H, R is H or CH, and
`R" is H, CH, or an electron pair.
`0044. In still further embodiments, the cationic linkages
`are of type (b2), where L is a linker up to 12 atoms in length
`having bonds selected from alkyl (e.g. —CH2—CH2—),
`alkoxy ( C-O ), and alkylamino (e.g. —CH2—NH ),
`with the proviso that the terminal atoms in L (e.g., those
`adjacent to carbonyl or nitrogen) are carbon atoms.
`0045. The morpholino subunits may also be linked by
`non-phosphorus-based intersubunit linkages, as described
`further below, where at least one linkage is modified with a
`pendant cationic group as described above. For example, a
`5"nitrogenatom on a morpholino ring could be employed in a
`Sulfamide linkage (see e.g. FIG. 2G) or a urea linkage (where
`phosphorus is replaced with carbon or Sulfur, respectively)
`and modified in a manner analogous to the 5'-nitrogenatom in
`structure (b3) above.
`0046. The subject oligomer may also be conjugated to a
`peptide transport moiety which is effective to enhance trans
`port of the oligomer into cells. The transport moiety is pref
`erably attached to a terminus of the oligomer, as shown, for
`example, in FIG. 1D, as well as FIGS. 2P-2O.
`0047 Preferably, the transport moiety comprises 6 to 16
`amino acids and is composed of Subsequences selected from
`the group consisting of (XYX'), (XY), (X,Z), and (XZZ),
`0048 where
`0049 (a) each X subunit independently represents argin
`ine or an arginine analog, said analog being a cationic
`
`

`

`US 2009/0088,562 A1
`
`Apr. 2, 2009
`
`C.-amino acid comprising a side chain of the structure
`RN=C(NH)R’, where R is Hor R; R is R, NH, NHR, or
`NR, where R is lower alkyl or lower alkenyland may further
`include oxygen or nitrogen; R' and R may together form a
`ring; and the side chain is linked to said amino acid via R' or
`R2;
`0050 (b) each Y subunit independently represents a neu
`tral linear amino acid—C(O)—(CHR), NH , where n is 1
`to 7 and each R is independently H or methyl; and
`0051
`(c) each Z subunit independently represents an
`C.-amino acid having a neutral aralkyl side chain.
`0052. In selected embodiments, the peptide comprises a
`sequence which consists of at least two, or at least three,
`repeats of a single subsequence selected from (X'Y'X'),
`(XY), (X,Z), and (XZZ). For example, the peptide may
`comprise a sequence represented by one of (XY'X'), (XY)
`and (XZZ), where p is 2 to 5 and m is 2 to 8.
`0053 Preferably, for each X', the side chain moiety is
`guanidyl; each Y is —CO (CH), NH , where n is 1 to
`7; and each Z is phenylalanine. In preferred embodiments of
`Y", n is 2 or 5, such thatY" is selected from a B-alanine subunit
`and a 6-aminohexanoic acid subunit.
`0054 Preferred peptides of this type include those com
`prising arginine dimers alternating with single Y subunits,
`where Y' is preferably Ahx. Examples include peptides hav
`ing the formula (RYR) or the formula (RRY), where Y is
`preferably Ahx. In one embodiment, Y is a 6-aminohexanoic
`acid subunit and p is 4. In a further embodiment, each Z is
`phenylalanine, and m is 3 or 4.
`0055. The conjugated peptide is preferably linked to a
`terminus of the oligomer via a linker AhX-B, where Ahx is a
`6-aminohexanoic acid subunit and B is a 3-alanine subunit, as
`shown, for example, in FIG. 1D.
`0056. In a related aspect, the invention provides a method
`of enhancing antisense activity of an oligomer having a
`sequence of morpholino subunits, joined by intersubunit link
`ages, Supporting base-pairing moieties, by modifying said
`oligomer to contain at least one cationic intersubunit linkage
`as disclosed herein. In one embodiment, said cationic inter
`Subunit linkage(s) do not include linkages of type (b1") as
`depicted above. Enhancement of antisense activity may be
`evidenced by:
`0057 (i) a decrease in expression of an encoded protein,
`relative to that provided by a corresponding unmodified oli
`gomer, when binding of the antisense oligomer to its target
`sequence is effective to block a translation start codon for the
`encoded protein, or
`0058 (ii) an increase in expression of an encoded protein,
`relative to that provided by a corresponding unmodified oli
`gomer, when binding of the antisense oligomer to its target
`sequence is effective to block an aberrant splice site in a
`pre-mRNA which encodes said protein when correctly
`spliced. Assays suitable for measurement of these effects are
`described further below. In one embodiment, modification
`provides this activity in a cell-free translation assay, or a
`splice correction translation assay in cell culture, as described
`herein. Preferably, activity is enhanced by a factor of at least
`two, more preferably by a factor of at least five, and most
`preferably by a factor of at least ten.
`0059. The compounds described herein may be used in
`methods of inhibiting production of a protein. Accordingly, a
`nucleic acid encoding Such a protein is exposed to an anti
`sense oligomer containing at least one cationic intersubunit
`linkage, and preferably containing 20% to 50% such cationic
`
`linkages, as disclosed herein, where the base pairing moieties
`Pi form a sequence effective to hybridize to a portion of the
`nucleic acid at a location effective to inhibit production of the
`protein. The location may be, for example, an ATG start
`codon of an mRNA, a splice site of a pre-mRNA, or a viral
`target sequence as described below.
`0060 Preferably, the oligomer has a T, with respect to
`binding to the target sequence of greater than about 50° C.
`and it is actively taken up by mammalian cells. The oligomer
`may be conjugated to a transport moiety as described herein
`to facilitate such uptake.
`0061. In one embodiment, the oligomer can be used in a
`method of reducing the risk of restenosis in a region of a
`patient's coronary vessel which has been treated by coronary
`angioplasty using a catheter with a distal-end expandable
`balloon, or which is at a junction formed in a coronary bypass
`operation. The method includes administering to the patient,
`by local administration directly to the vessel site of injury, an
`oligomeras described herein, containing at least one cationic
`intersubunit linkage, and preferably containing 20% to 50%
`Such cationic linkages, having from 12 to 40 subunits, includ
`ing a targeting base sequence that is complementary to a
`target sequence of at least 12 contiguous bases within the
`AUG start site region of human c-myc mRNA defined by SEQ
`ID NO. 59, in an amount effective to reduce the risk of
`restenosis in the patient. The compound is administered by
`one of:
`0062 (a) contacting the region of the vessel with a reser
`voir containing the antisense compound, and introducing the
`compound from the reservoir into the vessel by iontophoresis
`or electroporation;
`0063 (b) injecting the compound from the catheter
`directly into the region of the vessel, under pressure, through
`injectors contained on the Surface of the catheter balloon,
`where said injectors are capable of penetrating the tunica
`media in the vessel;
`0064 (c) injecting into or contacting the region of the
`vessel, microparticles containing the antisense compound in
`entrapped form;
`0065 (d) contacting the region of the vessel with a hydro
`gel coating contained on the Surface of the catheter balloon,
`and containing the antisense compound is diffusable form;
`0.066
`(e) contacting the region of the vessel with a stent
`having an outer Surface layer containing the antisense com
`pound in diffusable form; and
`0067 (f) injecting the compound by intravascular admin
`istration, resulting in systemic delivery to the vascular tissues.
`0068. The antisense compound may have a targeting
`sequence having at least 90% homology to the sequence
`identified by SEQID NO:43, and alternatively, at least 90%
`homology to a sequence selected from SEQID NOs: 60 and
`61.
`0069. In another embodiment, the oligomer can be tar
`geted againstan mRNA sequence having its 5' end 1 to about
`25 base pairs downstream of a normal splice acceptor junc
`tion in a preprocessed mRNA. Such targeting is effective to
`inhibit natural mRNA splice processing and produce splice
`variant mRNAs.
`0070 Suitable target proteins include, for example, tran
`Scription factors, particularly oncogenic or proto-oncogenic
`proteins such as myc, my b, rel, fos, jun, abl. bcl, and p53;
`matrix proteins, such as integrins and cathedrins; other
`tumor-expressed proteins, such as hCG, telomerases; recep
`tor proteins; viral proteins, such as those expressed from the
`
`

`

`US 2009/0088,562 A1
`
`Apr. 2, 2009
`
`subgenomic spliced mRNAs of HIV, human papilloma virus,
`and human parvovirus B19; and immunomodulatory proteins
`such as, for example, CTLA-4, B7-2, PD-1, Foxp3. TGFbeta,
`and TNF receptor.
`0071. In another embodiment, the oligomer can be used
`for inhibiting replication of an RNA virus from the picornavi
`rus, calicivirus, togavirus or flavivirus families, having a
`single-stranded, positive sense genome of less than 12 kb, and
`a first open reading frame that encodes a polyprotein contain
`ing multiple functional proteins. Accordingly, the virus, or,
`typically, a cell infected with the virus, is exposed to an
`oligomeras disclosed herein, containing at least one cationic
`intersubunit linkage, and preferably containing 20% to 50%
`Such cationic linkages, and having a sequence of Subunits
`Supporting a targeting base sequence that is Substantially
`complementary to a viral target sequence which spans the
`translation initiation region of the first open reading frame.
`0072 Exemplary targeting sequences have at least 90%
`homology to a sequence selected from the group consisting
`of:
`0073 (i) SEQID NO. 62, for a polio virus of the Mahoney
`and Sabin Strains,
`0074 (ii) SEQID NO. 63, for a hepatitis A virus,
`0075 (iii) SEQID NO. 64, for a rhinovirus 14,
`0076 (iv) SEQID NO. 65, for a rhinovirus 16,
`0.077
`(v) SEQID NO. 66, for a rhinovirus 1B,
`0078. Other exemplary targeting sequences, directed
`against a calcivirus, have at least 90% homology to a
`sequence selected from the group consisting of
`0079 (i) SEQID NOS. 67,68, and 69, for a serotype Pan-1
`VeS1V1rus,
`0080 (ii) SEQID NO. 70, for a porcine calicivirus,
`I0081
`(iii) SEQID NO. 71, for a Norwalk virus, and
`I0082 (iv) SEQID NO. 72, for a feline calicivirus.
`0083. For use in inhibition of hepatitis E virus, the target
`ing sequence has at least 90% homology to a sequence
`selected from the group consisting of SEQID NOs: 73 and
`74. For use in inhibition of a hepatitis C flavivirus, the target
`ing sequence is complementary to a sequence of at least 12
`contiguous bases of the HCV AUG start-site region identified
`by SEQ ID NO: 75. Exemplary targeting sequences include
`those havi