Peptide-Based In Vivo siRNA Delivery System

• US 20120165393A1
• Filed: 12/15/2011
• Published: 06/28/2012
• Est. Priority Date: 12/17/2010
• Status: Active Grant

• Current Assignee: Arrowhead Pharmaceuticals, Inc.
• Original Assignee: Arrowhead Madison Inc (Arrowhead Pharmaceuticals, Inc.)
• Inventors: Rozema, David B., Wakefield, Darren H., Lewis, David L., Wolff, Jon A., Blokhin, Andrei V., Hadwiger, Philipp, Kitas, Eric A., Hoffman, Torsten, Jahn-Hofmann, Kerstin, Mohr, Peter, Roehl, Ingo, Benson, Jonathan D., Carlson, Jeffrey C., Martin Mueller, Hans, Ott, Guenther
• Granted Patent: US 8,501,930 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61K 47/549 Sugars, nucleosides, nucleotides or nucleic acids
• 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 47/6415 Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/20 for DNA viruses
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3515 Lipophilic moiety, e.g. cholesterol
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific

Abstract

The present invention is directed compositions for targeted delivery of RNA interference (RNAi) polynucleotides to hepatocytes in vivo. Targeted RNAi polynucleotides are administered together with co-targeted melittin delivery peptides. Delivery peptides provide membrane penetration function for movement of the RNAi polynucleotides from outside the cell to inside the cell. Reversible modification provides physiological responsiveness to the delivery peptides.

Claims

1. A conjugate delivery system composition for delivering an RNA interference polynucleotide to a liver cell in vivo comprising:

RNAi-A+Melittin−

(L−

Gal)_x

wherein,

Melittin is a melittin peptide

L is physiologically labile linkage,

Gal is an Asialoglycoprotein Receptor (ASGPr) ligand,

x is an integer having a value greater than 80% of primary amines of Melittin, RNAi is an RNA interference polynucleotide, and

A is either a hydrophobic group having at least 20 carbon atoms or a galactose cluster.

2. The composition of claim 1 wherein the RNA interference polynucleotide is selected from the groups consisting of:

DNA, RNA, dsRNA, siRNA, and microRNA.

3. The composition of claim 1 wherein the liver cell consists of a hepatocyte.

4. The composition of claim 1 wherein the ASGPr ligands are reversibly linked to at least 90% of the amines on a plurality of melittin peptides.

5. The composition of claim 1 wherein the Melittin peptide is selected from the list consisting of:

Seq. ID 1, Seq. ID 7, Seq. ID 11, Seq. ID 51, Seq. ID 57, Seq. ID 58, Seq. ID 92, and Seq. ID 96.

6. The composition of claim 1 wherein the Melittin peptide consists of D-form amino acids.

7. The composition of claim 1 wherein L is a disubstituted maleamate.

8. The composition of claim 1 wherein L is a amidobenzyl carbamate.

9. The composition of claim 7 further comprising a polyethyleneglycol (PEG) covalently linked to the amino terminus of the melittin peptide.

10. The composition of claim 7 further comprising an ASGPr ligand-PEG conjugate covalently linked to the amino terminus of the melittin peptide.

11. The composition of claim 8 further comprising a polyethyleneglycol (PEG) covalently linked to the amino terminus of the melittin peptide.

12. The composition of claim 8 further comprising an ASGPr ligand-PEG conjugate covalently linked to the amino terminus of the melittin peptide.

13. The composition of claim 1 wherein the ASGPr ligand is selected from the group consisting of lactose, galactose, N-acetylgalactosamine (GalNAc), galactosamine, N-formylgalactosamine, N-acetyl-galactosamine, N-propionylgalactosamine, N-n-butanoylgalactosamine, and N-iso-butanoyl-galactosamine.

16. The composition of claim 1 wherein the galactose cluster consists of an N-acetylgalactosamine trimer.

17. The composition of claim 1 wherein the hydrophobic group consists of cholesterol.

18. The composition of claim 1 wherein the composition is provided in a pharmaceutically acceptable carrier or diluent.

19. A method for inhibiting expression of a gene expressed in a liver cell of a mammal comprising:

a) forming an RNAi polynucleotide having sequence identical or nearly identical to a portion of sequence of said gene; and

,

a) injecting into a vessel in said mammal a composition of claim 1.

14. The composition of 1 wherein RNAi is linked to A via a physiologically labile linkage L².

15. The composition of 14 wherein L² is orthogonal to L.

20. A method of manufacturing an RNA interference polynucleotide delivery composition comprising:

a) forming a melittin peptide;

b) forming a plurality of uncharged masking agents each comprising an ASGPr ligand covalently linked to a disbustituted maleic anhydride or a dipeptide amindobenzyl amine reactive carbonate;

c) modifying greater than 80% of primary amines on a population of melittin peptides with the masking agents of step b,

d) linking the RNA interference polynucleotide to a hydrophobic group having at least 20 carbon atoms or a galactose trimer;

f) providing the RNA interference polynucleotide and the modified melittin peptide in solution suitable for administration in vivo.