Peptide oligonucleotide conjugates

• US 9,862,946 B2
• Filed: 09/11/2015
• Issued: 01/09/2018
• Est. Priority Date: 05/05/2011
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Hanson, Gunnar J.
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US14/851,434
• Publication Number:
  US 20160237426A1
• Time in Patent Office: 851 Days
• Field of Search: 435 61, 435 911, 435 9131, 435455, 435458, 435375, 514 1, 514 2, 514 55, 514 11, 514 44, 530300, 536 231, 536 245, 424 91
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 47/64 Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent peptidic linkers A61K47/65
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• 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 15/1136 against growth factors, growth regulators, cytokines, lymphokines or hormones
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 15/87 Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/33 of the base
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/33 Alteration of splicing

Abstract

Oligonucleotide analogs conjugated to carrier peptides are provided. The disclosed compounds are useful for the treatment of various diseases, for example diseases where inhibition of protein expression or correction of aberrant mRNA splice products produces beneficial therapeutic effects.

Claims

1. A conjugate comprising:

(a) a carrier peptide comprising amino acid subunits, the carrier peptide comprising a proline amino acid at the carboxy terminus of the carrier peptide; and

(b) a phosphorodiamidate morpholino oligonucleotide comprising a substantially uncharged backbone and a targeting base sequence for sequence-specific binding to a target nucleic acid; and

(c) a covalent attachment between the nucleic acid analog and the carrier peptide, the covalent attachment comprising the carboxy-terminal proline and an optional linker group;

wherein;

two or more of the amino acid subunits are positively charged amino acids, no more than seven contiguous amino acid subunits are arginine, and the carrier peptide is covalently attached to the nucleic acid analogue, the covalent attachment between the nucleic acid analog and the carrier peptide is not 6-aminohexanoic acid or β

-alanine.

2. The conjugate of claim 1, wherein at least one of the positively charged amino acids is arginine.

3. The conjugate of claim 1, wherein each of the positively charged amino acids is arginine.

4. The conjugate of claim 1, wherein all of the amino acid subunits, except the carboxy terminal proline are positively charged amino acids.

5. The conjugate of claim 1, wherein the carrier peptide comprises the sequence (R^d)_m, wherein R^d is independently, at each occurrence, a positively charged amino acid and m is an integer ranging from 2 to 12.

6. The conjugate of claim 5, wherein each R^d is arginine.

7. The conjugate of claim 1, wherein each amino acid subunit, except the carboxy terminal proline, is arginine.

8. The conjugate of claim 1, wherein the carrier peptide comprises at least one hydrophobic amino acid, the hydrophobic amino acid comprising a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain wherein the alkyl, alkenyl and alkynyl side chain includes at most one heteroatom for every six carbon atoms.

9. The conjugate of claim 1, wherein the carrier peptide comprises at least one neutral amino acid having the following structure (Y^b):

—

C(O)—

(CHR^e)_n—

NH—

(Y^b)

wherein n is 2 to 7 and each R^e is independently, at each occurrence, hydrogen or methyl.

10. The conjugate of claim 1, wherein each of the amino acid subunits are natural amino acids.

11. The conjugate of claim 1, wherein the uncharged backbone comprises a sequence of morpholino ring structures joined by intersubunit linkages, the intersubunit linkages joining a 3′

-end of one morpholino ring structure to a 5′

-end of an adjacent morpholino ring structure, wherein each morpholino ring structure is bound to a base-pairing moiety, such that the oligonucleotide can bind in a sequence-specific manner to the target nucleic acid, wherein the intersubunit linkages have the following general structure (I);

12. The conjugate of claim 11, wherein at least one of the morpholino ring structures has the following structure (i):

13. The conjugate of claim 11, wherein each linkage is linkage (A) and X is —

N(CH₃)₂.

14. The conjugate of claim 11, wherein W and Y are each O at each occurrence.

15. The conjugate of claim 11, wherein the nucleic acid analogue has the following structure (XVII):

16. The conjugate of claim 15, wherein R¹⁸ is the carrier peptide.

17. The conjugate of claim 15, wherein R¹⁹ is the carrier peptide.

18. The conjugate of claim 15, wherein R¹⁹ is

19. The conjugate of claim 15, wherein L¹ has the following structure (XXIX):

20. The conjugate of claim 15, wherein R¹⁸ is the carrier peptide, L² is a direct bond, and the carrier peptide forms a bond between the carboxy terminus of the carrier peptide and the 3′

terminal nitrogen of the nucleic acid analogue.