Compositions for enhancing transport of molecules into cells

• US 9,572,899 B2
• Filed: 11/05/2008
• Issued: 02/21/2017
• Est. Priority Date: 04/29/2003
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Moulton, Hong M., Nelson, Michelle H., Kroeker, Andrew D., Stein, David A.
• Primary Examiner(s): SCHNIZER, RICHARD A
• Application Number:
  US12/265,499
• Publication Number:
  US 20090082547A1
• Time in Patent Office: 3,030 Days
• Field of Search: None
• US Class Current: 1/1
• 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 49/0043 Fluorescein, used in vivo
• CPC Class Codes: A61K 49/0054 Macromolecular compounds, i.e. oligomers, polymers, dendrimers
• CPC Class Codes: A61K 49/0056 Peptides, proteins, polyamino 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 15/87 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 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

Compositions and methods for enhancing delivery of molecules, e.g. biological agents, into cells are described. The composition is a conjugate of the biological agent, preferably a nucleic acid analog having a substantially uncharged backbone, covalently linked to a peptide transporter moiety as described. Conjugation of the peptide transporter to a substantially uncharged nucleic acid analog, such as a morpholino oligomer, is also shown to enhance binding of the oligomer to its target sequence and enhance antisense activity.

Claims

1. A peptide-nucleic acid analog conjugate comprising:

i) a carrier peptide consisting of from ten to fifteen subunits connected by amide bonds, wherein each subunit is selected from X and Y subunits, including from eight to thirteen X subunits and from two to four contiguous Y subunits;

ii) a nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence; and

iii) a linker consisting of one or more linker subunits connected by amide bonds, and optionally a piperazine moiety; and

iv) a covalent attachment between the nucleic acid analog and the carrier peptide, the covalent attachment consisting of amide bonds to the linker,

wherein;

(a) each X subunit is independently an amino acid subunit comprising a side chain of the structure R¹N═

C(NH₂)R², where R¹ is H or R;

R² is R, NH₂, NHR, or NR₂, where R is lower alkyl or lower alkenyl and optionally comprises oxygen or nitrogen or R¹ and R² may together form a ring; and

wherein the side chain is linked to the amino acid via R¹ or R2;

(b) each Y subunit is independently a hydrophobic amino acid comprising a substituted or unsubstituted alkyl, alkenyl, alkynyl, aryl or aralkyl side chain, wherein the alkyl, alkenyl or alkynyl side chain includes at most one heteroatom for every six carbon atoms; and

(c) each linker subunit is of the formula —

C(O)—

(CH₂)_n—

NH—

, wherein n is an integer from 2 to 7.

2. The peptide-nucleic acid analog conjugate of claim 1, wherein the linker comprises a β

-alanine subunit, a 6-aminohexanoic acid subunit or combinations thereof.

3. The peptide-nucleic acid analog conjugate of claim 1, wherein each Y subunit is phenylalanine.

4. The peptide-nucleic acid analog conjugate of claim 1, wherein each X subunit is arginine.

5. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide comprises the sequence identified as SEQ ID NO:

13.

6. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide comprises the sequence identified as SEQ ID NO:

20.

7. The peptide-nucleic acid analog conjugate of claim 1, wherein the nucleic acid analog comprises a morpholino nucleic acid analog, the morpholino nucleic acid analog comprising morpholino subunits linked by phosphorus-containing linkages, the phosphorus-containing linkages linking the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3-position of an adjacent morpholino subunit.

8. The peptide-nucleic acid analog conjugate of claim 7, wherein the morpholino subunits are linked by phosphorous-containing linkages in accordance with the structure:

9. The peptide-nucleic acid analog conjugate of claim 8, wherein X is dimethylamino.

10. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide is attached to the 5′

terminus or the 3′

terminus of the nucleic acid analog.

11. The peptide-nucleic acid analog conjugate of claim 1, wherein the targeting base sequence of the nucleic acid analog is targeted to an antisense target in a polynucleotide, wherein the antisense target is a splice site in a pre-mRNA, a translation start site in an mRNA or a cis-acting element in a viral genome.

12. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide comprises two Y subunits.

13. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide comprises the sequence identified as SEQ ID NO:

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29 or SEQ ID NO;

30.

14. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide consists of the sequence identified as SEQ ID NO:

13, SEQ ID NO;

14, SEQ ID NO;

15, SEQ ID NO;

16, SEQ ID NO;

17, SEQ ID NO;

18, SEQ ID NO;

20, SEQ ID NO;

21, SEQ ID NO;

22, SEQ ID NO;

23, SEQ ID NO;

24, SEQ ID NO;

25, SEQ ID NO;

26, SEQ ID NO;

27, SEQ ID NO;

28, SEQ ID NO;

29 or SEQ ID NO;

30.

15. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide comprises the sequence identified as SEQ ID NO:

20.

16. The peptide-nucleic acid analog conjugate of claim 10, wherein the carrier peptide is attached to the 5′

terminus of the nucleic acid analog.

17. The peptide nucleic-acid analog conjugate of claim 16, wherein the carrier peptide is attached to the 5′

terminus of the nucleic acid analog via a piperazinyl moiety having the following structure;

22. The peptide-nucleic acid analog conjugate of claim 17, wherein the nucleic acid analog comprises a morpholino nucleic acid analog, the morpholino nucleic acid analog comprising morpholino subunits linked by phosphorus-containing linkages, the phosphorus-containing linkages linking the morpholino nitrogen of one morpholino subunit to an exocyclic carbon at the morpholino 3′

-position of an adjacent morpholino subunit.

18. A peptide moiety comprising an amino acid sequence selected from one of SEQ ID NOS:

13-41, and a linker covalently bound to the carboxy terminus of the peptide moiety consisting of one or more linker subunits of the formula —

C(O)(CH₂)_nNH—

, wherein n is an integer from 2 to 7.

19. The peptide moiety of claim 18, wherein the amino acid sequence is as SEQ ID NO:

13.

20. A peptide moiety consisting of an amino acid sequence selected from one of SEQ ID NOS:

13-41, and a linker covalently bound to the carboxy terminus of the peptide moiety consisting of one or more linker subunits of the formula —

C(O)(CH₂)_nNH—

, wherein n is an integer from 2 to 7.

21. The peptide moiety of claim 20, wherein the amino acid is SEQ ID NO:

13.