Peptide conjugated, inosine-substituted antisense oligomer compound and method

• US 8,053,420 B2
• Filed: 03/31/2008
• Issued: 11/08/2011
• Est. Priority Date: 05/24/2004
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Weller, Dwight D., Hassinger, Jed N.
• Primary Examiner(s): VIVLEMORE, TRACY ANN
• Application Number:
  US12/060,135
• Publication Number:
  US 20080182973A1
• Time in Patent Office: 1,317 Days
• Field of Search: None
• US Class Current: 514/44
• CPC Class Codes: A61K 48/0008 characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3145 with the nitrogen in 3' or 5'-position
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/331 Universal or degenerate base
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

A therapeutic oligomer-peptide conjugate, and methods of using the conjugate are disclosed. The conjugate includes (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. The conjugate has greater cellular uptake than the compound alone, by virtue of the arginine-rich peptide, and substantially greater antisense activity greater activity than the conjugate in the absence of inosine for guanine substitutions.

Claims

1. A method for enhancing the cellular uptake of a substantially uncharged antisense oligonucleotide compound, wherein the compound comprises a base sequence complementary to a string of at least four contiguous cytosine bases, the base sequence comprising one or more inosine bases and no more than three contiguous guanine bases, the method comprising forming a conjugate of the compound and an arginine-rich peptide.

2. The method of claim 1, wherein the position of the one or more inosine bases in the base sequence limits the number of contiguous guanine bases in the base sequence to two or fewer.

3. The method of claim 1, wherein the base sequence comprises at least two inosine bases.

4. The method of claim 1, wherein the conjugate has enhanced water solubility during a purification step involving conjugate binding to and release from a cationic ion exchange resin, wherein the enhanced water solubility is relative to a second conjugate which is identical to the conjugate except that the second conjugate comprises four or more contiguous guanine bases.

5. The method of claim 1, wherein the compound targets a region that includes the start codon in an mRNA, and the conjugate comprises an enhanced ability to block translation of the protein encoded by the mRNA in a cell-free protein translation system, wherein the enhanced ability to block translation is relative to a second conjugate which is identical to the conjugate except that the second conjugate comprises four or more contiguous guanine bases.

6. The method of claim 1, wherein the arginine-rich peptide comprises 8 to 16 subunits including X subunits, Y subunits and optional Z subunits, wherein the arginine-rich peptide includes at least six X subunits, at least two Y subunits and at most three Z subunits, where >

50% of the subunits are X subunits, and where;

(a) each X subunit is independently arginine or an arginine analog, the analog having the structure;

R¹N═

C(NH₂)R², where R¹ is H or R;

R² is R, NH₂, NHR or NR₂, R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen and R¹ and R² may together form a ring, wherein the side chain is linked to the amino acid via R¹ or R²;

(b) each Y subunit is independently a neutral amino acid having the structure;

—

C(O)—

(CHR)_n—

NH—

, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl, wherein the substituted alkyl, alkenyl or alkynyl includes at most one heteroatom for every four carbon atoms; and

(c) each Z subunit is independently alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.

7. The method of claim 6, wherein the arginine-rich peptide has the sequence identified as SEQ ID NO:

17.

8. The method of claim 1, wherein the compound comprises a sequence of morpholino subunits, each morpholino subunit comprising a phosphorus-containing linkage connecting the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3-position of an adjacent morpholino subunit.

9. The method of claim 8, wherein each morpholino subunit comprises a phosphorodiamidate linkage, and each morpholino subunit has independently the following structure:

10. The method of claim 8, wherein the conjugate inhibits the synthesis of c-myc in a cellular system, and the compound has the sequence identified as SEQ ID NO:

1, where at least one of the four contiguous guanine (G) bases in the sequence is substituted with an inosine (I) base.

19. The method of claim 1, wherein the conjugate inhibits the synthesis of c-myc in a cellular system, and the compound has the sequence identified as SEQ ID NO:

1, where at least one of the four contiguous guanine (G) bases in SEQ ID NO;

1 is substituted with an inosine (I) base.

29. The method of claim 10, wherein the compound has the sequence identified as SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 or SEQ ID NO;

10.

30. The method of claim 19, wherein the compound has the sequence identified as SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 or SEQ ID NO;

10.

11. A method for preparing a conjugate of a substantially uncharged antisense oligonucleotide compound and an arginine-rich peptide, wherein the compound comprises a base sequence complementary to a string of at least four contiguous cytosine bases, the method comprising including at least one inosine bases in the base sequence such that the base sequence comprises no more than three contiguous guanine bases.

12. The method of claim 11, wherein including the at least one inosine base limits the number of contiguous guanine bases in the base sequence to two or fewer.

13. The method of claim 11, wherein at least two inosine bases are included.

14. The method of claim 11, wherein the compound targets a region that includes the start codon in an mRNA, and including the one or more inosine base enhances the ability of the conjugate to block translation of a protein encoded by the mRNA in a cell-free protein translation system, wherein the enhanced ability to block translation is relative to a second conjugate which is identical to the conjugate, except that the second conjugate comprises four or more contiguous guanine bases.

15. The method of claim 11, wherein the arginine-rich peptide comprises 8 to 16 subunits including X subunits, Y subunits, and optional Z subunits, wherein the arginine-rich peptide includes at least six X subunits, at least two Y subunits, and at most three Z subunits, where >

50% of the subunits are X subunits, and where;

(a) each X subunit is independently arginine or an arginine analog, the analog having the structure;

R¹N═

C(NH₂)R², where R¹ is H or R;

R² is R, NH₂, NHR, or NR₂, R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen and R¹ and R² may together form a ring, wherein the side chain is linked to the amino acid via R¹ or R²;

(b) each Y subunit is independently a neutral amino acid having the structure;

—

C(O)—

(CHR)—

NH—

, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl, wherein the substituted alkyl, alkenyl or alkynyl includes at most one heteroatom for every four carbon atoms; and

(c) each Z subunit is independently alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.

16. The method of claim 15, wherein the arginine-rich peptide has the sequence identified as SEQ ID NO:

17.

17. The method of claim 11, wherein the compound comprises a sequence of morpholino subunits, each morpholino subunit comprising a phosphorus-containing linkage connecting the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3-position of an adjacent morpholino subunit.

18. The method of claim 17, wherein each morpholino subunit comprises phosphorodiamidate linkages, and each morpholino subunit has independently the following structure:

32. The method of claim 15, wherein the arginine-rich peptide has the sequence identified as SEQ ID NO:

16.

20. An oligomer-peptide conjugate comprising:

(a) an arginine-rich peptide conjugated to a substantially uncharged antisense oligonucleotide compound, the compound comprising a base sequence complementary to a string of at least four contiguous cytosine bases and wherein the base sequence comprises at least one inosine bases and no more than three contiguous guanine bases.

21. The conjugate of claim 20, wherein the position of the at least one inosine bases in the base sequence limits the number of contiguous guanine bases in the base sequence to two or fewer.

22. The conjugate of claim 21, wherein the base sequence comprises at least two inosine bases.

23. The conjugate of claim 20, wherein the compound targets a region that includes the start codon in an mRNA, and the conjugate comprises an enhanced ability to block translation of a protein encoded by the mRNA in a cell-free protein translation system, wherein the enhanced ability to block translation is relative to a second conjugate which is identical to the conjugate, except that the second conjugate comprises four or more contiguous guanine bases.

24. The conjugate of claim 20, wherein the arginine-rich peptide comprises 8 to 16 subunits including X subunits, Y subunits, and optional Z subunits, wherein the arginine-rich peptide includes at least six X subunits, at least two Y subunits, and at most three Z subunits, where >

50% of the subunits are X subunits, and where;

(a) each X subunit is independently arginine or an arginine analog, the analog having the structure;

R¹N═

C(NH₂)R², where R¹ is H or R;

R² is R, NH₂, NHR, or NR₂, R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen and R¹ and R² may together form a ring, wherein the side chain is linked to the amino acid via R¹ or R²;

(b) each Y subunit is independently a neutral amino acid having the structure;

—

C(O)—

(CHR)_n—

NH—

, where (i) n is 2 to 7 and each R is independently H or methyl, or (ii) n is 1 and R is substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl, wherein the substituted alkyl, alkenyl or alkynyl includes at most one heteroatom for every four carbon atoms; and

(c) each Z subunit is independently alanine, asparagine, cysteine, glutamine, glycine, histidine, lysine, methionine, serine or threonine.

25. The conjugate of claim 24, wherein the arginine-rich peptide has the sequence identified as SEQ ID NO:

17.

26. The conjugate of claim 20, wherein the compound comprises a sequence of morpholino subunits, each morpholino subunit comprising a phosphorus-containing linkage connecting the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3-position of an adjacent morpholino subunit.

27. The conjugate of claim 26, wherein each morpholino subunit comprises a phosphorodiamidate linkage, and each morpholino subunit has independently the following structure:

28. The conjugate of claim 20, wherein the conjugate inhibits the synthesis of c-myc in a cellular system, and the compound has the sequence identified as SEQ ID NO:

1, where at least one of the four contiguous guanine (G) bases in SEQ ID NO;

1 is substituted with an inosine (I) base.

31. The conjugate of claim 28, wherein the compound has the sequence identified as SEQ ID NO:

2, SEQ ID NO;

3, SEQ ID NO;

4, SEQ ID NO;

5, SEQ ID NO;

6, SEQ ID NO;

7, SEQ ID NO;

8, SEQ ID NO;

9 or SEQ ID NO;

10.

33. The conjugate of claim 24, wherein the arginine-rich peptide has the sequence identified as SEQ ID NO:

16.