Antisense oligonucleotide compositions
The present invention relates to antisense oligonucleotide (ASO) compositions and particularly to compositions and methods for the cytosolic delivery of antisense oligonucleotides (ASOs). Hybrid ASOs, part single-stranded and part double-stranded, are provided, hybridizing to form a double-stranded region that can non-covalently bond to nucleic-acid-binding protein regions. In this way, ASO::protein complexes may be produced that facilitate delivery of antisense DNA into target cells. Such complexes may be used to down-regulate gene expression in cells.
1. A pharmaceutically acceptable antisense oligonucleotide composition useful for delivering antisense oligonucleotide into the cytosol of a cell, comprising a complex formed from:
(i) a pair of partially complementary, single-stranded oligonucleotides hybridized to give at least one single-stranded antisense sequence and at least one double-stranded protein-binding sequence; and
(ii) a shuttle protein capable of conveying genetic material through a pore in a cell membrane, the shuttle protein comprising a nucleic-acid-binding domain that recognizes the double-stranded protein-binding sequence and is non-covalently bound thereto;
wherein the shuttle protein is an attenuated toxin protein or a protein homologous thereto, in which said attenuated toxin protein or homologous protein at least one toxin domain has been replaced by a said nucleic acid binding domain;
and wherein the composition further comprises:
(iii) a pore-forming protein which is capable of interaction with the said complex to facilitate translocation of the complex into the cytosol of the cell.
2. A composition of claim 1 , comprising an oligonucleotide that is modified by sulfonation to increase stability.
3. An antisense oligonucleotide composition as claimed in claim 1 , wherein the protein-binding sequence is CGG-N 11 -CCG, where “N” is any purine or pyrimidine base.
4. An antisense oligonucleotide composition as claimed in claim 1 , wherein the protein-binding sequence is:
5′-CGGCTGCTCTGATGCCG-3′ (SEQ ID NO: 19) or 5′-CGGCATCAGAGCAGCCG-3′ (SEQ ID NO: 20).
5. An antisense oligonucleotide composition as claimed in claim 1 , wherein the shuttle protein is homologous with an attenuated toxin protein.
6. An antisense oligonucleotide composition as claimed in claim 5 , wherein the shuttle protein contains B. anthracis lethal factor (LF) domain I (LFn).
7. An antisense oligonucleotide composition as claimed in claim 6 , wherein at least one of domains (II-IV) of LF is replaced by the nucleic-acid-binding domain.
8. An antisense oligonucleotide composition as claimed in claim 1 , wherein the nucleic-acid-binding domain of the shuttle protein is Saccharomyces cerevisiae GAL4.
9. An antisense oligonucleotide composition as claimed in claim 1 in which the pore-forming protein is B. anthracis virulence factor Protective Antigen PA83.
10. An antisense oligonucleotide composition as claimed in claim 1 , in which the antisense sequence is single-stranded DNA designed to hybridize to messenger RNA derived from a target gene.
11. An antisense oligonucleotide composition as claimed in claim 10 in which the target gene is expressed by a virus.
12. An antisense oligonucleotide composition as claimed in claim 11 in which the target gene is expressed by Human Papilloma Virus (HPV).
13. A method of transporting an antisense oligonucleotide across a membrane of a cell, the method comprising: providing an antisense oligonucleotide composition as claimed in claim 1 , and applying the antisense oligonucleotide composition to the cell.
14. A method of treating a subject infected with Human Papilloma Virus or having cervical cancer, comprising applying to the subject a therapeutically effective amount of a composition as claimed in claim 12 , whereby the composition is pharmaceutically acceptable.