Compositions for enhancing transport of molecules into cells

• US 7,468,418 B2
• Filed: 04/29/2004
• Issued: 12/23/2008
• 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., Stein, David A., Nelson, Michelle H., Kroeker, Andrew D.
• Primary Examiner(s): Schnizer; Richard
• Application Number:
  US10/836,804
• Publication Number:
  US 20040265879A1
• Time in Patent Office: 1,699 Days
• Field of Search: None
• US Class Current: 530/300
• 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 conjugate, comprising

a carrier peptide attached to a nucleic acid analog having a substantially uncharged backbone and a targeting base sequence,

wherein the carrier peptide has the sequence (Arg-Y-Arg)₄, where Y is 6-aminohexanoic acid.

2. The conjugate of claim 1, wherein the peptide is effective to enhance the binding of an antisense oligomer to its target sequence, relative to the antisense oligomer in unconjugated form, as evidenced by:

(i) a decrease in expression of an encoded protein, when binding of the antisense oligomer to its target sequence is effective to block a translation start codon for the encoded protein, or

(ii) an increase in expression of an encoded protein, when binding of the antisense oligomer to its target sequence is effective to block an aberrant splice site in a pre-mRNA which encodes said protein when correctly spliced.

3. The conjugate of claim 1, wherein the peptide is effective to enhance the binding of an antisense oligomer to its target sequence, relative to the antisense oligomer in unconjugated form, as evidenced by:

(i) a decrease in expression of an encoded protein in a cell free translation system, when binding of the antisense oligomer to its target sequence is effective to block a translation start codon for the encoded protein, or

(ii) an increase in expression of an encoded protein in a cell free translation system, when binding of the antisense oligomer to its target sequence is effective to block an aberrant splice site in a pre-mRNA which encodes said protein when correctly spliced.

4. The conjugate of claim 1, wherein the peptide is effective to enhance the transport of the nucleic acid analog into a cell, relative to the analog in unconjugated form.

5. The conjugate of claim 1, wherein the nucleic acid analog is conjugated to the peptide via a linker selected from 6-aminohexanoic acid, cysteine, and an uncharged, non-natural amino acid.

6. The conjugate of claim 1, wherein the nucleic acid analog is a morpholino oligomer, comprising morpholino subunits linked by phosphorus-containing linkages between the morpholino nitrogen of one subunit and an exocyclic carbon at the morpholino 3-position of an adjacent subunit.

7. The conjugate of claim 6, wherein the morpholino subunits are joined by intersubunit linkages, in accordance with the structure:

8. The conjugate of claim 7, wherein said conjugate is effective to:

(a) inhibit expression of targeted mRNA in a protein expression system;

(b) inhibit splicing of targeted pre-mRNA;

or

(c) inhibit replication of a virus, by targeting cis-acting elements which control nucleic acid replication or mRNA transcription of the virus.

9. The conjugate of claim 8, wherein said inhibition is in a cell free system.

10. A composition for intracellular delivery of a nucleic acid analog in vivo, comprising the conjugate of claim 1, and a suspension of insoluble gas-containing microbubbles in an aqueous vehicle comprising at least one filmogenic compound selected from a protein, surfactant, lipid, polysaccharide, and combinations thereof.

11. The composition of claim 10, wherein the microbubbles are suspended in an aqueous vehicle comprising albumin, and the insoluble gas is selected from the group consisting of perfluoromethane, perfluoroethane, perfluoropropane, perfluorobutane, and periluoropentane.

12. The conjugate of claim 7, wherein each R′

is a lower alkyl.