US20050288246A1 - Peptide conjugated, inosine-substituted antisense oligomer compound and method - Google Patents

Peptide conjugated, inosine-substituted antisense oligomer compound and method Download PDF

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US20050288246A1
US20050288246A1 US11/136,245 US13624505A US2005288246A1 US 20050288246 A1 US20050288246 A1 US 20050288246A1 US 13624505 A US13624505 A US 13624505A US 2005288246 A1 US2005288246 A1 US 2005288246A1
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conjugate
subunits
compound
bases
inosine
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Patrick Iversen
Dwight Weller
Jed Hassinger
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Sarepta Therapeutics Inc
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Priority to US11/136,245 priority Critical patent/US20050288246A1/en
Priority to CA2565103A priority patent/CA2565103C/en
Priority to HK07109442.8A priority patent/HK1101314B/en
Priority to AU2005247460A priority patent/AU2005247460B2/en
Priority to ES05754332T priority patent/ES2375631T3/es
Priority to JP2007527563A priority patent/JP4974890B2/ja
Priority to EP05754332A priority patent/EP1765414B1/en
Priority to PCT/US2005/018213 priority patent/WO2005115479A2/en
Priority to AT05754332T priority patent/ATE529513T1/de
Assigned to AVI BIOPHARMA, INC. reassignment AVI BIOPHARMA, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HASSINGER, JED N., IVERSEN, PATRICK L., WELLER, DWIGHT D.
Publication of US20050288246A1 publication Critical patent/US20050288246A1/en
Priority to US12/060,135 priority patent/US8053420B2/en
Priority to JP2011130577A priority patent/JP2011172596A/ja
Priority to US13/243,341 priority patent/US8877725B2/en
Assigned to SAREPTA THERAPEUTICS, INC. reassignment SAREPTA THERAPEUTICS, INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: AVI BIOPHARMA, INC.
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Definitions

  • This invention relates to an antisense oligomer compound (i) conjugated to an arginine rich-peptide effective to enhance the uptake of the oligomer into cells, and (ii) in which strings of G bases are broken by one of more inosine bases, and methods of using such compound.
  • Antisense oligomers offer great potential as pharmaceutical drugs, as evidenced by the number of antisense drugs currently in clinical development, and aided by the fact that a number of potential limitations of antisense oligomers have been successfully addressed over the past several years (Devi, Stein).
  • Novel uncharged oligomer backbones have been developed to improve uptake into cells, and to increase resistance to nuclease degradation (Hudziak, Iversen, Summerton).
  • the modified backbone has been found to give enhanced binding affinity to its target nucleic acid (Iversen, Summerton).
  • This discovery has the potential to significantly increase the therapeutic potential of a variety of antisense oligomers, including those intended to block expression of selected proteins, those aimed at blocking certain donor or acceptor splice sites in pre-processed mRNA, and those designed to treat viral infection by blocking expression of viral genes or replication of single-stranded viral genomes.
  • the optimal targeting sequence against which the oligomer antisense is directed may include a run of four of more cytosine bases, in which case the oligomer will contain a corresponding string of four or more complementary guanine bases.
  • an optimal target sequence for the c-myc protein is a region containing the AUG start site of the c-myc RNA that includes a run of four cytosine bases.
  • Anti-sense oligomers directed against the start-codon region of c-myc have a number of important therapeutic applications, including the treatment of cancer, polycistic kidney disease (see, for example, co-owned U.S. Pat. No.
  • the method includes, in one aspect, an improvement in a method for enhancing the cellular uptake of a substantially uncharged oligonucleotide analog compound, by forming a conjugate of the compound and an arginine-rich peptide effective to enhance the uptake of the compound into target cells, where the compound includes a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind.
  • the improvement includes substituting an inosine base for at least one guanine base in the string of bases in the compound so as to limit the number of contiguous guanine bases in the string to three or fewer, preferably two or fewer.
  • the improvement may be effective to enhance the water solubility of the conjugate during a purification step involving conjugate binding to and release from an cationic ion exchange resin, relative to the same conjugate in the absence of the inosine substitution.
  • the improvement may be effective to enhance the ability of the conjugate to block translation of the protein encoded by the mRNA, relative to the same conjugate in the absence of the inosine substitution.
  • the improvement may be effective to enhance the ability of the conjugate to mask mRNA splicing at the target region, relative to the same conjugate in the absence of the inosine substitution.
  • the improvement may be effective to enhance the ability of the conjugate to block viral replication, relative to the same conjugate in the absence of the inosine substitution.
  • the arginine-rich peptide comprises 8 to 16 subunits selected from X subunits, Y subunits, and optional Z subunits, including at least six X subunits, at least two Y subunits, and at most three Z subunits, where >50% of said subunits are X subunits, and where
  • the oligonucleotide compound is a morpholino oligomer composed of morpholino subunits linked by phosphorus-containing linkages between the morpholino nitrogen of one subunit and an exocyclic carbon at the morpholino 3-position of an adjacent subunit.
  • the invention includes a therapeutic oligomer-peptide conjugate composed of (a) a substantially uncharged oligonucleotide analog compound having a base sequence that includes a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind, and (b) conjugated to the compound, an arginine-rich peptide effective to enhance the uptake of the compound into target cells.
  • the string of bases in the compound includes at least one inosine base, positioned in the string so as to limit the number of contiguous guanine bases in said string to three or fewer, preferably two or fewer. Exemplary embodiments of the conjugate are as described above.
  • the compound's targeting sequence may include one of the sequences identified as SEQ ID NOS: 2-10.
  • the arginine-rich peptide conjugated to the compound may include the sequence identified as SEQ ID NOS: 16, 17 or 18.
  • a conjugate of the type just described is administered to the subject, in a therapeutically effective amount.
  • the conjugate has greater cellular uptake than the antisense compound alone, in the absence of the arginine-rich peptide, and is more active in blocking c-myc translation than the same conjugate in the absence of the one or more inosine bases.
  • Exemplary embodiment of the conjugate used in the method are as described above.
  • the conjugate may be administered by transurethral delivery, and the method may further include administering a cis-platin anti-cancer compound to the patient.
  • the conjugate may be delivered by intravascular delivery, for example, via a drug-releasing stent or via intravenous injection of microbubbles carrying the drug.
  • the conjugate may be administered by exposing the vein to the conjugate prior to its surgical placement.
  • the conjugate may be administered to the subject by oral or parenteral administration.
  • FIGS. 1A-1D show several preferred morpholino-type subunits having 5-atom (A), six-atom (B) and seven-atom (C-D) linking groups suitable for forming polymers;
  • FIGS. 2 A-D show the repeating subunit segment of exemplary morpholino oligonucleotides, constructed using subunits A-D, respectively, of FIG. 1 ;
  • FIGS. 3 A-G show exemplary X side chain structures, for use in various embodiments of the arginine-rich peptides employed in the invention
  • FIGS. 4 A-D show oligomer-peptide conjugates and methods of their preparation, where FIG. 4C shows preparation of an in vivo cleavable conjugate and FIG. 4D shows preparation of a conjugate with a 6-aminohexanoic acid/beta-alanine linker;
  • FIG. 5 is a schematic representation of G-quartet base pairs where PMO represents either the intermolecular or intramolecular morpholine residue of the oligomer backbone;
  • FIG. 6 shows graphically the results of cell-free rabbit reticulocyte lysate translation inhibition using various unconjugated inosine substituted and nonsubstituted c-myc oligomers
  • FIG. 7 represents the inhibition of cell-free translation using arginine-rich peptide conjugated c-myc PMOs with or without inosine substitutions.
  • Alkyl refers to a fully saturated monovalent radical containing carbon and hydrogen, which may be branched, linear, or cyclic (cycloalkyl). Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl, n-heptyl, isopropyl, cyclopropyl, cyclopentyl, ethylcyclopentyl, and cyclohexyl.
  • lower alkyl alkyl groups having one to six carbon atoms
  • lower alkyl alkyl groups having one to six carbon atoms
  • lower alkyl refers to C 1 to C 4 alkyl.
  • Alkenyl refers to an unsaturated monovalent radical containing carbon and hydrogen, which may be branched, linear, or cyclic.
  • the alkenyl group may be monounsaturated or polyunsaturated. Generally preferred are alkenyl groups having one to six carbon atoms, referred to as “lower alkenyl”.
  • Aryl refers to a substituted or unsubstituted monovalent aromatic radical, generally having a single ring (e.g., benzene) or two condensed rings (e.g., naphthyl). This term includes heteroaryl groups, which are aromatic ring groups having one or more nitrogen, oxygen, or sulfur atoms in the ring, such as furyl, pyrrole, pyridyl, and indole.
  • substituted is meant that one or more ring hydrogens in the aryl group is replaced with a halide such as fluorine, chlorine, or bromine; with a lower alkyl group containing one or two carbon atoms; nitro, amino, methylamino, dimethylamino, methoxy, halomethoxy, halomethyl, or halo-ethyl.
  • Preferred substituents include halogen, methyl, ethyl, and methoxy.
  • aryl groups having a single ring are preferred.
  • Alkyl refers to an alkyl, preferably lower (C 1 -C 4 , more preferably C 1 -C 2 ) alkyl, substituent which is further substituted with an aryl group; examples are benzyl (—CH 2 C 6 H 5 ) and phenethyl (—CH 2 CH 2 C 6 H 5 ).
  • a “heterocycle” refers to a non-aromatic ring, preferably a 5- to 7-membered ring, whose ring atoms are selected from the group consisting of carbon, nitrogen, oxygen and sulfur.
  • the ring atoms include 3 to 6 carbon atoms.
  • Such heterocycles include, for example, pyrrolidine, piperidine, piperazine, and morpholine.
  • substituted refers to replacement of a hydrogen atom with a heteroatom-containing substituent, such as, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esters such as carboxylic, sulfonic, or phosphonic.
  • a heteroatom-containing substituent such as, for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino, alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esters such as carboxylic, sulfonic, or phosphonic.
  • an antisense oligomer compound” or “antisense oligomer analog” or antisense compound” or oligomer analog compound” all refer to a substantially uncharged nucleic acid analog, typically having a length between 8 and 40 bases, and having a base sequence that is complementary or substantially complementary to a single-stranded target nucleic acid, e.g., a processed or preprocessed mRNA transcript, or a single-stranded viral genomic RNA or DNA.
  • the compound may be in an unconjugated or conjugated form, e.g., conjugated to an arginine-rich peptide.
  • a “morpholino oligomer” is an oligonucleotide analog composed of morpholino subunit structures of the form shown in FIG. 1 , where (i) the structures are linked together by phosphorus-containing linkages, one to three atoms long, preferably two atoms long, and preferably uncharged, joining the morpholino nitrogen of one subunit to the 5′ exocyclic carbon of an adjacent subunit, and (ii) P i and P j are purine or pyrimidine base-pairing moieties effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide.
  • the purine or pyrimidine base-pairing moiety is typically adenine, cytosine, guanine, uracil or thymine.
  • the synthesis, structures, and binding characteristics of morpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506, 5,166,315, 5,521,063, and 5,506,337, all of which are incorporated herein by reference.
  • the subunit shown FIG. 1B having a two-atom linkage, is used for 6-atom repeating-unit backbones, as shown in FIG. 2B .
  • the atom Y 1 linking the 5′ morpholino carbon to the phosphorus group may be sulfur, nitrogen, carbon or, preferably, oxygen.
  • the X moiety pendant from the phosphorus is any stable group which does not interfere with base-specific hydrogen bonding. Preferred groups include alkyl, alkoxy, thioalkoxy, and alkyl amino, including cyclic amines, all of which can be variously substituted, as long as base-specific bonding is not disrupted. Alkyl, alkoxy and thioalkoxy preferably include 1-6 carbon atoms.
  • Alkyl amino preferably refers to lower alkyl (C 1 to C 6 ) substitution, and the cyclic amines are preferably 5- to 7-membered nitrogen heterocycles optionally containing 1-2 additional heteroatoms selected from oxygen, nitrogen, and sulfur.
  • Z is sulfur or oxygen, and is preferably oxygen.
  • a preferred morpholino oligomer is a phosphorodiamidate-linked morpholino oligomer, referred to herein as a PMO.
  • PMO phosphorodiamidate-linked morpholino oligomer
  • Desirable chemical properties of the morpholino-based oligomers include the ability to selectively hybridize with a complementary-base target nucleic acid, including target RNA, with high Tm, even with oligomers as short as 8-14 bases, the ability to be actively transported into mammalian cells, and the ability of an oligomer:RNA heteroduplex to resist RNAse degradation.
  • a “substantially uncharged” morpholino oligomer includes at most one charged intersubunit linkage for every four, preferably for every ten, and more preferably for every twenty, uncharged intersubunit linkages.
  • Any charged linkages are preferably charged phosphoramidate (or thiophosphoramidate) linkages, e.g. a linkage as shown in FIG. 2B where X is O ⁇ or S ⁇ .
  • the morpholino oligomers are fully uncharged.
  • An “amino acid subunit” is preferably an ⁇ -amino acid residue (i.e. —CO—CHR—NH—); it may also be a ⁇ - or other amino acid residue (e.g. —CO—CH 2 CHR—NH—), where R is a side chain.
  • G-quartet consists of stacked planar hydrogen-bonded guanine tetramers that can cause guanine-rich nucleic acids to adopt intermolecular and intramolecular quadruplex structures that are stabilized by the presence of the G-quartets.
  • non-natural amino acids refers to those amino acids not present in proteins found in nature such as beta-alanine (i-Ala) or 6-aminohexanoic acid (Ahx).
  • the present invention includes, in one aspect, a therapeutic oligomer-peptide conjugate composed of a substantially uncharged oligonucleotide analog compound and, conjugated thereto, an arginine-rich peptide effective to enhance the uptake of the compound into target cells.
  • the compound contains a string of bases that are complementary to four or more contiguous cytosine bases in a target nucleic acid region to which the compound is intended to bind, and this string includes at least one inosine base, positioned in the string so as to limit the number of contiguous guanine bases in the string to three or fewer.
  • the string of bases includes at least two inosine bases, and the number of contiguous guanines in the string is two or fewer.
  • the inosine base(s), which are complementary to the target cytosine bases, but form a less stable Watson-Crick base pair than the usual G-C base pair, serve to enhance the solubility of the conjugate during a purification step involving conjugate binding to and release from an cationic ion exchange resin, relative to the same conjugate in the absence of the inosine substitution.
  • This enhancement is important in obtaining a purified conjugate by practical purification methods.
  • the inosine-base substitution(s) are also effective to enhance the activity of the compound with respect to its target nucleic acid, as evidenced by:
  • Methods for demonstrating the enhanced antisense activity of the inosine-base substituted oligomer-peptide conjugates are typically cell-free translation assays and tissue culture-based assays designed to measure the inhibition of mRNA translation, preprocessed mRNA splice accuracy or viral replication.
  • Cell-free translation assays consist of a translation competent cell lysate (e.g., rabbit reticulocyte lysate) and input mRNA containing a reporter gene such as firefly luciferase with antisense oligomer target sequences placed immediately upstream.
  • a variety of plasmid constructs can be used to generate the reporter mRNA.
  • Antisense oligomers are added to the cell-free translation reaction and the relative inhibition of the reporter gene signal is a measure of the antisense activity. More detailed descriptions of cell-free translation assays used to describe the present invention are presented in Examples 4 and 5.
  • Tissue culture-based assays designed to demonstrate inhibition of mRNA translation use antisense oligomers targeted to either native cellular genes whose translation products (e.g. proteins) can be quantitatively measured or reporter genes that have been stably transfected into a cell line. Measurement of the degree of translation inhibition of the target mRNA can be performed using a variety of analytical methods including reporter gene signal output or quantitation of protein expression using immunological methods.
  • Inhibition of preprocessed mRNA splicing can be demonstrated by measuring the level of mis-spliced mRNA by northern blots or quantitative polymerase chain reaction in cells treated with antisense oligomers targeted to splice donor or acceptor sites.
  • An alternative approach (see e.g. (Kang, Cho et al. 1998) utilizes a cell line stably transfected with a plasmid that has a luciferase gene interrupted by a mutated human beta-globin intron that causes incorrect splicing.
  • Antisense oligomers targeted to the mutation within the intron results in splice correction and up-regulation of functional luciferase reporter protein.
  • Enhanced activity of antisense oligomers targeted to cis-acting elements involved in viral replication can be demonstrated using standard tissue culture-based viral replication assays or viral replicons. Inhibition of viral replication in the presence of antisense oligomers is measured by determining the titer of virus that has replicated in the presence of the antisense oligomer.
  • Viral replicon systems utilize derivatives of full length infectious viral clones where the viral structural genes have been replaced either in part or completely with a reporter gene. The replicons are introduced, usually by transfection, into cells that are infected with replication competent virus. The replicons encode the essential cis-acting replication elements that are recognized by the replication machinery of the virus resulting in amplification of the reporter gene and an increase in the reporter signal, e.g. luciferase activity.
  • the arginine-rich peptide used in the invention for enhancing the uptake of a substantially uncharged antisense oligomer compound across a biological membrane generally comprises a moiety consisting of 10-15 subunits selected from X and Y, including 8-13 X subunits, 2-4 contiguous Y subunits, Y subunits interspersed singly amidst the X subunits, and an optional linker subunit to which the agent is linked.
  • X represents an amino acid subunit comprising a side chain moiety of the structure R 1 N ⁇ C(NH 2 )R 2 (see FIG.
  • R 1 is H or R
  • R 2 is R, NH 2 , NHR, or NR 2 , where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen
  • R 1 and R 2 may together form a ring
  • the side chain moiety is linked to the amino acid subunit via R 1 or R 2 .
  • the side chain moiety is independently selected from the group consisting of guanidyl (HN ⁇ C(NH 2 )NH—), amidinyl (HN ⁇ C(NH 2 )C ⁇ ), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl (FIGS. 3 B-G, respectively, with possible linkage sites indicated). Note that, in structures 3 D, 3 E, and 3 G, linking of the side chain to the amino acid subunit could take place via any of the ring —NH— groups as well as via any of the carbon atoms indicated.
  • the side chain moiety is guanidyl, as in the amino acid subunit arginine (Arg).
  • Y represents a hydrophobic amino acid subunit having a side chain selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, aralkyl, and alkaryl, any of which may be substituted or unsubstituted; when the side chain is selected from substituted alkyl, substituted alkenyl, and substituted alkynyl, it includes at most one heteroatom for every six carbon atoms.
  • Y represents a hydrophobic amino acid subunit having a side chain selected from the group consisting of substituted or unsubstituted aryl, aralkyl, and alkaryl.
  • each Y is selected from the group consisting of phenylalanine, tyrosine, leucine, isoleucine, and valine.
  • each Y is a phenylalanine (Phe or F) subunit.
  • the Y subunit is the non-natural amino acid 6-aminohexanoic acid (Ahx).
  • the X and Y subunits may be referred to herein as “cationic subunits” and “hydrophobic subunits”.
  • the Y subunits are either contiguous, in that no X subunits intervene between Y subunits, or interspersed singly between X subunits.
  • the linking subunit may be between Y subunits.
  • the Y subunits are at a terminus of the transporter; in other embodiments, they are flanked by X subunits.
  • the transporter is a peptide, where the amino acids are joined by peptide linkages.
  • the amino acids may be d-amino acids, 1-amino acids, non-natural amino acids or a combination thereof.
  • the compound to be delivered may be a compound employed for detection, such as a fluorescent compound, but it is preferably a biologically active agent, e.g. a therapeutic or diagnostic agent.
  • agents include nucleic acids or nucleic acid analogs, particularly antisense oligonucleotides.
  • the transport moieties as described above greatly enhance cell entry of attached uncharged oligomer compounds, relative to uptake of the compound in the absence of the attached transport moiety, and relative to uptake by an attached transport moiety lacking the hydrophobic subunits Y.
  • Such enhanced uptake is preferably evidenced by at least a two-fold increase, and preferably a four-fold increase, in the uptake of the compound into mammalian cells relative to uptake of the agent by an attached transport moiety lacking the hydrophobic subunits Y.
  • Uptake is preferably enhanced at least twenty fold, and more preferably forty fold, relative to the unconjugated compound.
  • a further benefit of the transport moiety is its expected ability to stabilize a duplex between an antisense oligomer and its target nucleic acid sequence, presumably by virtue of electrostatic interaction between the positively charged transport moiety and the negatively charged nucleic acid.
  • the number of charged subunits in the transporter is less than 14, as noted above, and preferably between 8 and 11, since too high a number of charged subunits may lead to a reduction in sequence specificity.
  • the transport moiety also lowers the effective concentration of an antisense oligomer to achieve antisense activity as measured in both tissue culture and cell-free systems.
  • Cell-free translation systems provide an independent means to assess the enhanced effect of the transport moiety on the antisense oligomer's ability to bind to its target and, through steric blocking, inhibit translation of downstream sequences.
  • Cell-free translation assays designed to test the antisense activity of arginine-rich peptide-PMO conjugates demonstrate between 10 fold and 500 fold improvement in antisense activity compared to the unconjugated PMO (see Example 5 and FIGS. 6 and 7 ).
  • the antisense oligomer compound is a synthetic oligomer capable of base-specific binding to a target sequence of a polynucleotide, e.g. an antisense oligonucleotide analog.
  • a polynucleotide e.g. an antisense oligonucleotide analog.
  • Such analogs in which the backbone structure, ring structure, or, less frequently, base structure of natural polynucleotides is modified, are well known and include charged analogs, such as phosphorothioate-linked oligonucleotides, and uncharged analogs, such as methylphosphonates and peptide nucleic acids.
  • Some analogs, such as N3′ ⁇ P5′ phosphoramidates may be charged or uncharged, depending on the substation on the linking moiety.
  • the polymer is a morpholino oligomer, as defined above, which is about 840 subunits in length. More typically, the oligomer is about 10-30, or about 12-25, subunits in length. For some applications, such as antibacterial, short oligomers, e.g. from about 8-12 subunits in length, can be especially advantageous, particularly when attached to a peptide transporter as disclosed herein.
  • the oligomer is an uncharged phosphorodiamidate-linked morpholino oligomer (PMO), also defined above.
  • the PMO can be of any sequence, where the supported base pairing groups include standard or modified A, T, C, G, I and U bases.
  • the target nucleic acid sequence against which the oligomer compound is directed includes a region of four or more contiguous cytosine bases.
  • This target region may be part of the AUG start site in an mRNA, where it is desired to inhibit or block expression of a selected protein encoded by the mRNA.
  • it may include or be adjacent to a donor or acceptor splice site in a preprocessed mRNA, where it is desired to block correct splicing at that site, either for purposes of creating splice mutation polypeptides, or incomplete or inactive peptides.
  • the target may be a cis-acting element in a viral genome, where binding of the oligomer (which may be targeted against either the + or ⁇ viral genome strand), is effective to block viral replication in virus-infected cells.
  • Exemplary target sequences containing a string of four or more guanine bases in each of these three target types can be found from public sequence databases well know to those of skill in the Art.
  • One exemplary target sequence described below includes the AUG start site in the human c-myc mRNA. It will be appreciated, however, that this sequence, and the various inosine-for guanine substitutions made in the targeting oligomer compound are illustrative of how the oligomer compound may be modified, when targeting any target sequence with a string of four or more cytosine bases, to achieve the advantages of the invention.
  • the transporter can be linked to the compound to be delivered by a variety of methods available to one of skill in the art. Exemplary methods are provided in Example 1 below and illustrated in FIGS. 4 A-D.
  • the transporter is a peptide containing a single cysteine residue whose side chain thiol is used for linking.
  • the linkage point can be at various locations along the transporter. In selected embodiments, it is at a terminus of the transporter. Typically, it is adjacent to the hydrophobic residues of the transporter. Multiple transporters can be attached to a single compound if desired.
  • the linker can also be any combination of two ⁇ -Ala and/or Ahx residues attached to the 5′ end of the PMO and the C-terminus of the peptide transporter.
  • a preferred embodiment is to attach the Ahx residue to the C terminus of the peptide transporter and the ⁇ -Ala residue to the 5′ terminus of the PMO as shown in FIG. 4D .
  • the transporter can be attached at the 5′ end of the PMO, e.g. via the 5′-hydroxyl group, or via an amine capping moiety, as described in Example 3 and illustrated in FIG. 4A .
  • the transporter may be attached at the 3′ end, e.g. via a morpholino ring nitrogen, as shown in FIG. 4B , or via the side chain of an intersubunit linkage, either at a terminus or an internal linkage.
  • the linker may also comprise a direct bond between the carboxy terminus of a transporter peptide and an amine or hydroxy group of the PMO, formed by condensation promoted by e.g. carbodiimide.
  • Linkers can be selected from those which are non-cleavable under normal conditions of use, e.g., containing a thioether or carbamate bond. In some embodiments, it may be desirable to include a linkage between the transporter moiety and compound which is cleavable in vivo. Bonds which are cleavable in vivo are known in the art and include, for example, carboxylic acid esters, which are hydrolyzed enzymatically, and disulfides, which are cleaved in the presence of glutathione. It may also be feasible to cleave a photolytically cleavable linkage, such as an ortho-nitrophenyl ether, in vivo by application of radiation of the appropriate wavelength.
  • a conjugate having a disulfide linker using the reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or succinimidyloxycarbonyl ⁇ -methyl- ⁇ -(2-pyridyldithio) toluene (SMPT), is illustrated in FIG. 4C .
  • exemplary heterobifunctional linking agents which further contain a cleavable disulfide group include N-hydroxysuccinimidyl 3-[(4-azidophenyl)dithio]propionate and others described in (Vanin and Ji 1981).
  • Table 1 A Table of sequences of PMOs, peptide-conjugated PMOs and exemplary transporter peptides discussed in the following sections is provided below as Table 1.
  • the peptides include an N-terminal amino group and C-terminal amine (e.g., NH 2 —RRRRRRRRRFFC—CONH 2 SEQ ID NO:16). Inosine substituted guanine residues are shown in bold.
  • Examples 4 and 5 demonstrate that guanine-rich PMO sequences are involved in the aggregation observed in the strong cation exchange (SCX) HPLC of the c-myc PMO, and that substitution of inosine bases for one or more of the contiguous guanine bases reduced or eliminated this aggregation.
  • SCX strong cation exchange
  • one exemplary structure for AVI-5126 contains three inosine residues and forms no aggregated species even under conditions that strongly favor aggregation.
  • the aggregation phenomenon observed when arginine-rich transport peptides are conjugated to PMOs is not limited to this class of nucleic acid analogs.
  • Other uncharged nucleic acids or nucleic acid analogs, including other morpholino backbones, methylphosphonates, phosphorothioates and phosphodiesters (i.e., DNA and RNA) and PNAs all have the same potential for aggregation when conjugated to an arginine-rich polypeptide, and thus the potential for improvement by the inosine-to-guanine base substitution of the invention.
  • Inosine substituted PMOs that target the c-myc gene were tested in a cell free rabbit reticulocyte lysate (RRL) assay described in Example 4.
  • RTL cell free rabbit reticulocyte lysate
  • Inosine substituted PMOs that target the c-myc gene were tested in a cell free rabbit reticulocyte lysate (RRL) assay described in Example 4.
  • the PMO with four inosine for guanine substitutions (SEQ ID NO:10) was the least effective at inhibiting translation whereas the PMOs with only one I for G substitution (SEQ ID NOS:2 and 3) were the most effective of all the inosine containing PMOs.
  • the control c-myc PMO with no inosine substitutions (SEQ ID NO:1) had the highest level of inhibition in this assay.
  • the decreased inhibition of translation observed with inosine containing PMOs is consistent with the loss of one hydrogen bond between I:C base pairs as compared to G:C base pairs with three hydrogen bonds.
  • the lower Tm between the targeting PMO and its target may play a role in the relative loss of steric blocking that the RRL assay measures.
  • the conjugates of the present invention are useful in treatment of vascular proliferative disorders such as restenosis.
  • Areas of vessel injury include, for example, restenosis or renarrowing of the vascular lumen following vascular intervention, such as coronary artery balloon angioplasty, with or without stent insertion. Restenosis is believed to occur in about 30% to 60% of lesions treated by angioplasty and about 20% of lesions treated with stents within 3 to 6 months following the procedure. (See, e.g., Devi, N. B. et al., Cathet Cardiovasc Diagn 45(3):337-45, 1998).
  • Stenosis can also occur after a coronary artery bypass operation, wherein heart surgery is done to reroute, or “bypass,” blood around clogged arteries and improve the supply of blood and oxygen to the heart.
  • the stenosis may occur in the transplanted blood vessel segments, and particularly at the junction of replaced vessels.
  • Stenosis can also occur at anastomotic junctions created for dialysis.
  • a PMO conjugate preferably targeting c-myc
  • a coated stent or by an ex vivo soaking solution for treatment of saphenous veins, or otherwise delivered to the site of vascular injury.
  • Microbubble compositions have been found particularly useful in delivery of attached molecules, such as oligonucleotides, to areas of thrombosis or vessel injury, e.g. damaged endothelium (see e.g. PCT Pubn. No. WO 2000/02588) as well as to selected organs such as the liver and kidney.
  • a preferred antirestenotic composition is an inosine-substituted, anti-c-myc PMO conjugated to an (RAhxR) 4 transport peptide through an Ahx- ⁇ Ala linker (e.g., SEQ ID NO:12).
  • RhxR anti-c-myc PMO conjugated to an (RAhxR) 4 transport peptide through an Ahx- ⁇ Ala linker (e.g., SEQ ID NO:12).
  • the PMO conjugates targeting the c-myc gene described herein are also useful in the treatment of cancer in general and bladder cancer in particular.
  • Transurethral administration of a PMO conjugate, preferably targeting c-myc, in conjunction with chemotherapy provides enhanced anticancer activity as described (Knapp, Mata et al. 2003) and as described in co-owned and co-pending U.S. application Ser. No. 10/151,008 which is incorporated herein by reference in its entirety.
  • a preferred anticancer composition is an inosine-substituted, anti-c-myc PMO conjugated to an (RAhxR) 4 transport peptide through an Ahx- ⁇ Ala linker (e.g., SEQ ID NO:12).
  • Peptides were synthesized by Fmoc Solid Phase Peptide Synthesis, referred to herein as SPPS.
  • SPPS Fmoc Solid Phase Peptide Synthesis
  • a p-benzyloxybenzyl alcohol resin was used for synthesis of peptides with a C-terminal acid, while a Rink Amide MBHA resin was used for peptide amides. Both resins are available from Novabiochem (San Diego, Calif.).
  • a typical synthesis cycle began with N-terminal deprotection via 20% piperidine.
  • N- ⁇ -Fmoc-protected amino acids were coupled to the growing peptide chain by activation with 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) in the presence of N,N-diisopropylethylamine (DIEA).
  • HBTU 2-(1H-Benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
  • DIEA N,N-diisopropylethylamine
  • Arginine side chains were protected with the 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting group, cysteine with trityl, and lysine side chains with t-butoxycarbonyl (Boc).
  • Peptides containing various C-terminal hydrophobic linkages were prepared as follows. Peptides were prepared for direct condensation with an amine or hydroxy group of the PMO by including combinations of natural and/or non-natural amino acids at the C-terminal end of the peptide during SPPS. Specifically, the linkages were comprised of the amino acids glycine, beta-alanine, and/or 6-aminohexanoic acid, used in different combinations of one or two residues. Peptide synthesis was otherwise identical to the synthesis of other peptide acids.
  • Peptides with masked amine groups for direct condensation with an amine or hydroxy group of a PMO were prepared as follows. Free peptide amino groups interfere with direct condensation of the carboxy terminus of the peptide with an amine or hydroxy group of a PMO, and must therefore be masked. Peptide sequences, such as rTat and pTat (Table 1), that contain amine side-chains were prepared by using the 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde) amine side chain protecting group. Peptide synthesis was otherwise identical to the synthesis of other peptide acids.
  • Lysine Dde groups survived the resin cleavage and deprotection of other amino acid side chain protecting groups.
  • the side chain amines remain masked by Dde through conjugation and are subsequently deprotected by treatment with 2% hydrazine in DMF.
  • a C-terminally reactive peptide-benzotriazolyl ester was prepared by dissolving the peptide-acid (15 ⁇ mol), HBTU (14.25 ⁇ mol), and HOBt (15 ⁇ mol) in 200 ⁇ l NMP and adding DIEA (22.5 ⁇ mol). Immediately after addition of DIEA, the peptide solution was added to 1 ml of a 12 mM solution of 5′-piperazine-functionalized, 3′-acetyl-PMO in DMSO. After 180 minutes at 30° C., the reaction was diluted with a four-fold excess of water.
  • the crude conjugate was purified first through a CM-Sepharose weak cation exchange column (Sigma, St. Louis, Mo.) to remove unconjugated PMO, and then through a reversed phase column (HLB column, Waters, Milford, Mass.).
  • the conjugate was lyophilized and analyzed by MALDI-TOF MS, SCX HPLC, and CE.
  • G-Rich Oligomers form G-Tetraplex Aggregates when Conjugated to Arginine-Rich Peptides
  • This example details the development of an arginine-rich peptide-PMO conjugate that targets the c-myc gene for a coronary artery bypass graft (CABG) clinical application.
  • the goal of the project described in this example was to develop processes for the synthesis and purification of the conjugate with acceptable yield and purity.
  • methods for analysis of the conjugate must characterize it and identify impurities throughout the synthetic process and in any formulations used in the clinic. Quantitation of the amount of free peptide remaining in the product is probably the most critical analytical capability as free peptide is potentially more toxic than conjugate or free PMO.
  • the aggregation state of the molecule can therefore be manipulated by changing the loading environment. This provided additional evidence that the later-eluting peaks correspond to aggregate forms of the conjugate, the traditional disaggregants urea and guanidinium are apparently effective in disaggregating the conjugate as it is loaded onto the resin.
  • conjugate's aggregate form has a much longer retention time suggested that, however the molecules are interacting, the charges on the arginine side-chains are still available for interaction with the ion-exchange resin. This would be the case if the PMO-portions of conjugate molecules were interacting with each other to form multimeric aggregates where the peptide portions did't involved in the interaction.
  • the aggregation problem seems to be unique to the c-myc PMO sequence, or at least to guranine-rich oligomers (GROs).
  • GROs guranine-rich oligomers
  • the conjugation of similar peptides to sequences that have low G-content present no aggregation problems.
  • Conjugates of other PMOs with consecutive G-runs exhibit the same type of aggregation on SCX HPLC.
  • the linker between the PMO and the peptide doesn't seem to have any affect on aggregation.
  • the N-(4-maleimidobutyryloxy) succinimide ester (GMBS) thioether linkage gives a very similar SCX elution pattern to Gly2-Ahx, Ahx2, or Ahx amide linkages.
  • the observed aggregation observed with conjugated GRO is consistent with PMO to PMO interactions that form the G-tetraplex structures seen in DNA and RNA with runs of consecutive guanines.
  • the PMO sequence of the cmyc 20-mer PMO described above (SEQ ID NO:1) is 5′-ACG TTG AGG GGC ATC GTC GC-3′ it's four-G run spanning positions 8-11.
  • Evidence for the formation of G-tetraplex structures is provided when the effects of different cations on the degree of aggregation is measured by SCX HPLC. It is well-known that cationic interactions are important for coordinating DNA and RNA tetraplexes.
  • guanine-rich nucleic acid sequences can adopt intermolecular or intramolecular quadruplex structures that are stabilized by the presence of G-quartets (see FIG. 5 ).
  • G-quartets are stacked planar hydrogen-bonded guanine tetramers and cause guanine-rich nucleic acids to form quadruplex structures.
  • the potential roles of quadruplex formation in vivo have been investigated because some biologically important G-rich sequences are capable of forming G-quartets under physiological conditions in vitro.
  • the number of reports describing specific G-quartet-binding proteins is now considerable (Shafer and Smirnov 2000).
  • G-rich oligomers can form a variety of possible quadruplex structures, depending on both thermodynamic and kinetic considerations.
  • the structures formed can be influenced by oligonucleotide base sequence and concentration, as well as the conditions (temperature and buffer) used for annealing, especially the presence of monovalent cations such as K + and Na + .
  • Quadruplexes can be formed by one, two or four molecules of oligonucleotide, which are referred to as monomer, dimer and tetramer structures, respectively.
  • Monomer and dimer quadruplexes have been classified further based on the positioning of their loop regions into chair (lateral loop) or basket (diagonal loop) forms.
  • the relative strand orientation (5′ to 3′ polarity) of the four strands of the quadruplex may be parallel, antiparallel, or mixed (Dapic, Abdomerovic et al. 2003).
  • Conjugates were eluted with 4 ml of 2M guanidinium chloride.
  • the salty conjugate solutions eluted from the ion-exchange columns were desalted by loading on 2 ml Waters HLB reversed-phase columns, washing with 4 ml H 2 O, and eluting with 4 ml 50% ACN. Finally, the solutions were lyophilized.
  • fLUC firefly luciferase
  • RNA polymerase promoter upstream of the c-myc:fLUC sequences and allowed RNA to be produced from the plasmid, after linearization with NotI, using the T7 polymerase-based Megascript kit and protocol (Ambion).
  • In vitro translations were carried out using transcribed RNA at a final concentration in each reaction of 1 nM, with 12 ⁇ l nuclease-treated rabbit reticulocyte lysate (Promega) in addition to PMO, (RAhxR) 4 -PMO, or water.
  • the improved translational inhibition of the peptide conjugated, inosine substituted PMO is in marked contrast to the decreased inhibition observed with the unconjugated PMOs described in Example 3 and shown in FIG. 6 .
  • the enhanced steric blocking inhibition that arginine-rich peptides confer to antisense PMO completely overcomes the decreased steric blocking properties observed with unconjugated inosine substituted PMO.
  • PMO with inosine substitutions are unexpectedly enhanced to a greater degree than non-inosine substituted PMO.
  • the mechanism for the enhanced steric blocking properties that arginine-rich peptides confer to inosine substituted PMO is currently unknown.

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