In Vivo Delivery of Oligonucleotides

• US 20160040161A1
• Filed: 12/12/2012
• Published: 02/11/2016
• Est. Priority Date: 12/12/2011
• Status: Active Grant

• Current Assignee: OncoImmunin, Inc.
• Original Assignee: OncoImmunin, Inc.
• Inventors: PACKARD, Beverly, KOMORIYA, Akira
• Granted Patent: US 10,557,136 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/7084 Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
• CPC Class Codes: A61K 48/0025 wherein the non-active part clearly interacts with the delivered nucleic acid
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/3517 Marker; Tag
• CPC Class Codes: C12N 2320/32 Special delivery means, e.g. tissue-specific
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

This invention provides a method for the in vivo delivery of oligonucleotides. The invention utilizes the presence of one or plurality of HES linked to an oligonucleotide to deliver a nucleic acid sequence of interest into the cytoplasm of cells and tissues of live organisms. The delivery vehicle is nontoxic to cells and organisms. Since delivery is sequence-independent and crosses membranes in a receptor-independent manner, the delivered oligonucleotide can target complementary sequences in the cytoplasm as well as in the nucleus of live cells. Sequences of bacterial or viral origin can also be targeted. The method can be used for delivery of genes coding for expression of specific proteins, antisense oligonucleotides, siRNAs, shRNAs, Dicer substrates, miRNAs, anti-miRNAs or any nucleic acid sequence in a living organism. The latter include mammals, plants, and microorganisms such as bacteria, protozoa, and viruses.

Claims

1. A composition for delivering a therapeutic oligonucleotide to a subject, said composition comprising a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to a nucleic acid sequence in vivo and modulates the level of a protein encoded or regulated by the nucleic acid.

2. The composition of claim 1, wherein the therapeutic oligonucleotide is from about 8 nucleotides to about 750 nucleotides.

3. The composition of claim 1, wherein the therapeutic oligonucleotide is single stranded or double stranded.

5. The composition of claim 1, wherein the HES-oligonucleotide comprises 3 or more fluorophores capable of forming one or more HES.

6. The composition of claim 1, wherein the therapeutic oligonucleotide is a member selected from:

siRNA, shRNA, miRNA, a Dicer substrate, an aptamer, a decoy and an antisense.

7. The composition of claim 6, wherein the therapeutic oligonucleotide is an antisense oligonucleotide that specifically hybridizes to an RNA.

8. The composition of claim 7, wherein the therapeutic antisense oligonucleotide:

(a) is a substrate for RNAse H when hybridized to the RNA;

(b) is not a substrate for RNAse H when hybridized to the RNA;

(c) is DNA or a DNA mimic;

or

(d) is specifically hybridizable to a target region of the RNA selected from the group consisting of

(i) a sequence within 30 nucleotides of the AUG start codon of an mRNA;

(ii) nucleotides 1-10 of a miRNA;

(iii) a sequence in the 5′

untranslated region of an mRNA;

(iv) a sequence in the 3′

untranslated region of an mRNA;

(v) an intron/exon junction of an mRNA;

(vi) a sequence in a precursor-miRNA (pre-miRNA) or primary-miRNA (pri-miRNA) that when bound by the oligonucleotide blocks miRNA processing; and

(vii) an intron/exon junction and a region 1 to 50 nucleobases 5′

of an intron/exon junction of an RNA.

14. The composition of claim 1, wherein the therapeutic oligonucleotide can induce RNA interference (RNAi).

18. The composition according to claim 1 that contains one or more modified nucleoside motifs selected from:

2′

OME, locked nucleic acid (LNA), alpha LNA, 2′

-Fluoro (2′

F), 2′

-O(CH2)₂OCH₃(2′

-MOE), 2′

-OCH₃(2′

-O-methyl), PNA and morpholino.

21. The composition according to claim 1 that contains one or more modified internucleoside linkages selected from:

phosphorothioate, phosphorodithioate, phosphoramide, 3′

-methylene phosphonate, O-methylphosphoroamidiate, PNA, morpholino, C-5 propyne and 5-methyl C.

26. The method of claim 29 wherein the disease or disorder is selected from the group consisting of:

an infectious disease, cancer, a proliferative disease or disorder, a neurological disease or disorder, and inflammatory disease or disorder, a disease or disorder of the immune system, a disease or disorder of the cardiovascular system, a metabolic disease or disorder, a disease or disorder of the skeletal system, and a disease or disorder of the skin or eyes.

29. A method for treating a disease or disorder in a subject, said method comprising administering to a subject in need thereof, a therapeutically effective amount of the composition of claim 1;

wherein the disease or disorder is characterized by

(a) overexpression or underexpression of the target a nucleic acid in the subject, or

(b) overexpression or underexpression of a protein encoded by the target nucleic in the subject.

30. The composition of claim 8, wherein the therapeutic oligonucleotide is siRNA, shRNA or a Dicer substrate.

31. The composition of claim 8, wherein the therapeutic oligonucleotide is a Dicer substrate 18-35 nucleotides in length.

4. (canceled)

9-13. -13. (canceled)

15-16. -16. (canceled)

19-20. -20. (canceled)

22-24. -24. (canceled)

25. A method for modulating a target nucleic acid or protein in a subject, said method comprising administering to the subject a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to a target nucleic acid sequence in vivo and thereby modulates the level of a protein encoded or regulated by the nucleic acid.

27. A method for modulating a target nucleic acid or protein in a cell ex vivo, said method comprising administering to the cell a therapeutically effective amount of an H-type excitonic structure (HES)-oligonucleotide containing a therapeutic oligonucleotide that specifically hybridizes to the target nucleic acid sequence in the cell and thereby modulates the level of protein encoded or regulated by the nucleic acid.

28. (canceled)