Compositions and Methods for Modifying a Predetermined Target Nucleic Acid Sequence

• US 20150211023A1
• Filed: 12/16/2012
• Published: 07/30/2015
• Est. Priority Date: 12/16/2011
• Status: Active Grant

• Current Assignee: Targetgene Biotechnologies Ltd.
• Original Assignee: Targetgene Biotechnologies Ltd.
• Inventors: Shiboleth, Yoel Moshe, Weinthal, Dan Michael
• Granted Patent: US 11,458,157 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/711 Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/7115 Nucleic acids or oligonucleotides having modified bases, i.e. other than adenine, guanine, cytosine, uracil or thymine
• CPC Class Codes: A61K 31/712 Nucleic acids or oligonucleotides having modified sugars, i.e. other than ribose or 2'-deoxyribose
• CPC Class Codes: A61K 31/7125 Nucleic acids or oligonucleotides having modified internucleoside linkage, i.e. other than 3'-5' phosphodiesters
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C07K 2319/01 containing a localisation/targetting motif
• CPC Class Codes: C07K 2319/09 containing a nuclear localisation signal
• CPC Class Codes: C12N 15/00 Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor mutants or genetically engineered microorganisms, per se C12N1/00, C12N5/00, C12N7/00; new plants per se A01H; plant reproduction by tissue culture techniques A01H4/00; new animals per se A01K67/00; use of medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases, gene therapy A61K48/00
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/8213 Targeted insertion of genes into the plant genome by homologous recombination
• CPC Class Codes: C12N 15/825 involving pigment biosynthesis
• CPC Class Codes: C12N 15/8251 Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
• CPC Class Codes: C12N 15/902 using homologous recombination
• CPC Class Codes: C12N 15/907 in mammalian cells
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

Provided herein are compositions and methods for modifying a predetermined nucleic acid sequence. A programmable nucleoprotein molecular complex containing a polypeptide moiety and a specificity conferring nucleic acid (SCNA) which assembles in-vivo, in a target cell, and is capable of interacting with the predetermined target nucleic acid sequence is provided. The programmable nucleoprotein molecular complex is capable of specifically modifying and/or editing a target site within the target nucleic acid sequence and/or modifying the function of the target nucleic acid sequence.

Claims

1. A nucleoprotein composition for modifying a predetermined target site in a target nucleic acid sequence in a target cell, the composition comprising:

(a) a polynucleotide molecule encoding a chimeric polypeptide, said polypeptide comprising;

(i) a functional domain capable of modifying said target site, the functional domain being devoid of a specific nucleic acid binding site; and

(ii) a linking domain that is capable of interacting with a specificity conferring nucleic acid, the linking domain being devoid of a specific target nucleic acid binding site;

and;

(b) a specificity conferring nucleic acid (SCNA) comprising;

(i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site; and

(ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide;

whereby assembly of the polypeptide and the SCNA within the target cell forms a functional nucleoprotein complex, capable of specifically modifying said target nucleic acid at the target site.

2. The composition of claim 1, wherein said functional domain comprises a catalytic domain.

3. The composition of claim 1, wherein said modifying the target nucleic acid is selected from:

mutation, deletion, insertion, replacement, binding, digestion, double-strand-break creation, nicking, methylation, acetylation, ligation, recombination, helix unwinding, chemical modification, labelling, activation and inactivation.

4. The composition of claim 1, wherein said chimeric polypeptide further comprises a subcellular localization domain.

5. The composition of claim 1, wherein the SCNA comprises a nucleic acid selected from the group consisting of a single-strand DNA, a single strand RNA, a double strand RNA, a modified DNA, a modified RNA, a locked-nucleic acid (LNA) and a peptide-nucleic acid (PNA) or combinations thereof.

6. The composition of claim 1, wherein said target nucleic acid is DNA.

7. The composition of claim 1, wherein the recognition region of the SCNA comprises a chemical modification selected from the group consisting of 5′

-end modification, 3′

-end modification, and internal modification;

wherein the chemical modification is selected from the group consisting of a nucleotide modification, and addition of a non nucleotide moiety.

12. The composition of claim 1, wherein said recognition region of the SCNA comprises a polynucleotide motif capable of specifically attaching to the linking domain of the chimeric protein.

36. A method of treating a genetic disease in an organism, the method comprising expressing in a cell of the organism the nucleo-protein programmable molecular complex of claim 1.

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15. A method for modifying a predetermined target site within a target nucleic acid sequence by a programmable nucleo-protein molecular complex, the method comprising the steps of:

a. delivering a nucleic acid sequence encoding a programmable chimeric polypeptide to a host cell, said chimeric polypeptide comprising;

(i) a functional domain capable of modifying said target site, the functional domain being devoid of a aspecific nucleic acid binding site; and

(ii) a linking domain that is capable of interacting with a specificity conferring nucleic acid, the linking domain being devoid of a specific target-nucleic acid binding site;

b. delivering a specificity-conferring nucleic acid (SCNA) molecule, or a nucleic acid encoding the SCNA to said host cell, said SCNA molecule comprising;

(i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site; and

(ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide with high binding affinity;

wherein expression of the polypeptide in the cell harboring the SCNA enables attachment of said chimeric polypeptide to the SCNA, forming an active programmed nucleoprotein complex, thereby targeting the chimeric polypeptide to the predetermined target nucleic acid sequence within the host cell, enabling the modification of the predetermined target site of the target nucleic acid sequence by said active programmed nucleo-protein molecular complex.

16. The method of claim 15, wherein said target nucleic acid is DNA.

17. The method of claim 16, wherein said target DNA is genomic DNA, wherein the target genomic DNA is of a eukaryotic origin.

19. The method of claim 15, wherein said target nucleic acid sequence is an extra-chromosomal nucleic acid sequence, selected from the group consisting of mitochondria, chloroplast, amyloplast and chromoplast.

20. The method of claim 15, wherein said target nucleic acid sequence is selected from a viral nucleic acid sequence, a prokaryotic nucleic acid sequence and a synthetic nucleic acid sequence.

21. The method of claim 15, wherein said modification is selected from the group consisting of mutation, deletion, insertion, replacement, binding, digestion, double-strand-break creation, nicking, methylation, acetylation, ligation, recombination, helix unwinding, chemical modification, labelling, activation and inactivation.

22. The method of claim 15, wherein the chimeric protein comprises a protein moiety having a nucleic acid functional modifier, wherein the functional modification is selected from the group consisting of transcriptional activation, transcriptional inactivation, RNA transcript silencing, alternative RNA splicing, chromatin rearrangement, cellular parasite and virus inactivation and change in cellular localization or compartmentalization of said target nucleic acid sequence.

23. The method of claim 15, wherein said SCNA comprises a nucleic acid molecule selected from the group consisting of a single-strand DNA, a single strand RNA, a double strand RNA, a modified DNA, a modified RNA, a locked-nucleic acid (LNA) and a peptide-nucleic acid (PNA) or combinations thereof.

24. The method of claim 15, wherein the interaction between the SCNA and the target nucleic acid is through base pairing, selected from the group consisting of a full double helix base pairing, a partial double helix base pairing, a full triple helix base pairing, a partial triple helix base pairing, and D-loops or branched forms, formed by said pairing.

25. The method of claim 15, wherein the recognition region of the SCNA comprises a modification selected from the group consisting of 5′

-end modification, 3′

-end modification, and internal modification, wherein said modification is selected from the group consisting of a nucleotide modification, Biotin, Fluorescein, Amine-linkers, oligo-peptides, Aminoallyl, a dye molecule, fluorophores, Digoxigenin, Acrydite, Adenylation, Azide, NHS-Ester, Cholesteryl-TEG, Alkynes, Photocleavable Biotin, Thiol, Dithiol, Modified bases, phosphate, 2-Aminopurine, Trimer-20, 2,6-Diaminopurine, 5-Bromo-deoxiUridine, DeoxiUridine, Inverted dT, dideoxi-nucleotides, 5-methyl deoxyCytidine, deoxylnosine, 5-nitroindole, 2-O-methyl RNA bases, Iso-dC, Iso-dG, Flourine modified bases and Phosphorothioate bonds.

28. The method of claim 15, wherein the recognition region of the SCNA comprises a nucleotide motif capable of interacting with the linking domain of the chimeric protein.

31. A nucleo-protein complex formed by the method of claim 15, wherein the physical association between the linking domain of the protein moiety and the recognition region of the specificity conferring nucleic acid moiety form a programmed functional complex within the target cell.

33. A host cell having a predetermined genetic modification in a predetermined target site, created by the method of claim 15, said host cell is selected from the group consisting of vertebrate cell, mammalian cell, human cell, animal cell, plant cell, invertebrate cell, nematodal cell, insect cell, and stem cell.

35. A transgenic organism or knock out organism having a predetermined genetic modification formed by the method of claim 15.

38. The host cell of claim 24, said host cell is selected from the group consisting of vertebrate cell, mammalian cell, human cell, animal cell, plant cell, invertebrate cell, nematodal cell, insect cell, and stem cell.

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37. A host cell comprising:

a) a polypeptide comprising;

(i) a functional domain capable of modifying a target site in a target nucleic acid sequence in the cell, the functional domain being devoid of a specific nucleic acid binding site; and

(ii) a linking domain that is capable of interacting with a specificity conferring nucleic acid, the linking domain being devoid of a specific target-nucleic acid binding site;

and;

(b) a specificity conferring nucleic acid (SCNA) comprising;

(i) a nucleotide sequence complementary to a region of the target nucleic acid flanking the target site; and

(ii) a recognition region capable of specifically attaching to the linking domain of the polypeptide;

whereby assembly of the polypeptide and the SCNA within the host cell forms a functional nucleoprotein complex, capable of specifically modifying the target nucleic acid at the target site.