Compositions for enhancing transport of molecules into cells

• US 10,300,149 B2
• Filed: 01/10/2017
• Issued: 05/28/2019
• Est. Priority Date: 04/29/2003
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Iversen, Patrick L., Moulton, Hong M., Nelson, Michelle H., Stein, David A., Kroeker, Andrew D.
• Primary Examiner(s): Schnizer, Richard A
• Application Number:
  US15/402,449
• Publication Number:
  US 20170368203A1
• Time in Patent Office: 868 Days
• Field of Search: None
• 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-oligomer analog conjugate comprising a carrier peptide covalently linked to a nucleic acid analog, the nucleic acid analog comprising a substantially uncharged backbone and a targeting base sequence, and the carrier peptide consisting of ten to fifteen subunits selected from X and Y subunits, including eight to thirteen X subunits, two to four Y subunits, at least one linker subunit linking the nucleic acid analogue to the peptide carboxy terminus, 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 each R is independently 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; and

(b) each Y subunit is independently a hydrophobic amino acid, and

wherein each linker subunit comprises 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 each Y subunit comprises 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.

3. 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.

4. The peptide-nucleic acid analog conjugate of claim 1, wherein each Y subunit is selected from the group consisting of phenylalanine, tyrosine, tryptophan, leucine, isoleucine, and valine.

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

6. The peptide-nucleic acid analog conjugate of claim 1, wherein the carrier peptide has the formula (ArgYArg)₄.

7. The peptide-nucleic acid analog conjugate of claim 6, wherein each Y subunit is selected from the group consisting of phenylalanine, tyrosine, tryptophan, leucine, isoleucine, and valine.

8. The peptide-nucleic acid analog conjugate of claim 6, wherein each Y subunit is phenylalanine.

9. The peptide-nucleic acid analog conjugate of claim 8, wherein the linker comprises a β

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

10. The peptide-nucleic acid analog conjugate of claim 6, wherein each Y subunit is tyrosine.

11. The peptide-nucleic acid analog conjugate of claim 10, wherein the linker comprises a β

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

12. The peptide-nucleic acid analog conjugate of claim 6, 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.

13. The peptide-nucleic acid analog conjugate of claim 12, wherein the morpholino subunits are linked by phosphorous-containing linkages in accordance with Formula (I):

14. The peptide-nucleic acid analog conjugate of claim 13, wherein X in Formula (I) is dimethylamino.

15. The peptide-nucleic acid analog conjugate of claim 6, wherein the carrier peptide is attached to the 5′

terminus or the 3′

terminus of the nucleic acid analog.

16. The peptide-nucleic acid analog conjugate of claim 15, 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;

18. The peptide-nucleic acid analog conjugate of claim 6, 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.