ARTIFICIAL NUCLEIC ACIDS FOR RNA EDITING
20220073915 · 2022-03-10
Inventors
- Jacqueline Wettengel (Sigmaringen, DE)
- Thorsten Stafforst (Tübingen, DE)
- Philipp Reautschnig (Tübingen, DE)
- Tobias Merkle (Tübingen, DE)
Cpc classification
C12N2310/3231
CHEMISTRY; METALLURGY
C12N2310/533
CHEMISTRY; METALLURGY
C12N15/111
CHEMISTRY; METALLURGY
C12N9/78
CHEMISTRY; METALLURGY
C12N15/113
CHEMISTRY; METALLURGY
C12N2310/346
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
Abstract
The present invention concerns artificial nucleic acids for site-directed editing of a target RNA. In particular, the present invention provides artificial nucleic acids capable of site-directed editing of endogenous transcripts by harnessing an endogenous deaminase. Further, the present invention provides artificial nucleic acids for sited-directed editing of a target RNA, which are chemically modified, in particular according to a modification pattern as described herein. The invention also comprises a vector encoding said artificial nucleic acid and a composition comprising said artificial nucleic acid. Moreover, the invention provides the use of the artificial nucleic acid, the composition or the vector for site-directed editing of a target RNA or for in vitro diagnosis. In addition, the artificial nucleic acid, the composition or the vector as described herein are provided for use as a medicament or for use in diagnosis of a disease or disorder.
Claims
1. Artificial nucleic acid for site-directed editing of a target RNA, the artificial nucleic acid comprising a) a targeting sequence, which comprises a nucleic acid sequence complementary to a target sequence in the target RNA, and b) a recruiting moiety for recruiting a deaminase, wherein the targeting sequence comprises at least one nucleotide, wherein the nucleobase is chemically modified, and/or wherein the targeting sequence comprises at least one backbone modification.
2. The artificial nucleic acid according to claim 1, wherein the targeting sequence comprises at least one chemically modified nucleotide, which is chemically modified at the 2′ position.
3. The artificial nucleic acid according to claim 2, wherein the chemically modified nucleotide comprises a substituent at the 2′ carbon atom, wherein the substituent is selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro; and/or wherein the chemically modified nucleotide is selected from the group consisting of a locked nucleic acid (LNA) nucleotide, an ethylene bridged nucleic acid (ENA) nucleotide and an (S)-constrained ethyl cEt nucleotide.
4. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at least one backbone modification and wherein a nucleotide comprises a modified phosphate group, preferably selected from the group consisting of a phosphorothioate, a phosphoroselenate, a borano phosphate, a borano phosphate ester, a hydrogen phosphonate, a phosphoroamidate, an alkyl phosphonate, an aryl phosphonate and a phosphotriester.
5. The artificial nucleic acid according to any of the preceding claims, wherein at least 40% of the nucleotides of the targeting sequence are chemically modified at the 2′ position.
6. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at the position corresponding to a nucleotide to be edited, preferably an adenosine or a cytidine nucleotide to be edited, in the target sequence a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site.
7. The artificial nucleic acid according to any of the preceding claims, wherein at least one, preferably both, of the two nucleotides or variants thereof, which are positioned 5′ or 3′ of the position corresponding to a nucleotide to be edited in the target sequence, is chemically modified at the 2′ carbon atom, wherein the 2′ carbon atom is linked to a substituent selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably selected from 2′-O-methyl, 2′-O-methoxyethyl, 2′-hydrogen and 2′-fluoro; and/or wherein at least one, preferably both, of the two nucleotides or variants thereof, which are positioned 5′ or 3′ of the position corresponding to a nucleotide to be edited in the target sequence, comprises a modified phosphate group, preferably a phosphorothioate group.
8. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at the position corresponding to a nucleotide to be edited in the target sequence a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, and wherein the nucleotide, which is positioned 5′ of the position corresponding to the nucleotide to be edited, is a pyrimidine nucleotide, preferably a pyrimidine ribonucleotide or a pyrimidine deoxynucleotide, and wherein said pyrimidine nucleotide comprises a nucleobase, which is chemically modified at the 2′ position, preferably by 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl or 2′-O-fluoro.
9. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises the nucleic acid sequence
3′AcC5′, wherein A is an adenosine nucleotide or a variant thereof, preferably an adenosine ribonucleotide or a deoxyadenosine nucleotide; c is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, at the position corresponding to a nucleotide, preferably an adenosine or a cytidine, more preferably an adenosine, to be edited in the target sequence; and C is a cytidine nucleotide or a variant thereof, preferably a cytidine ribonucleotide, a modified cytidine ribonucleotide, a deoxycytidine nucleotide or a modified deoxycytidine nucleotide, more preferably a deoxycytidine nucleotide or a modified deoxycytidine nucleotide.
10. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises the nucleic acid sequence
3′As*cC*5′, wherein As is an adenosine nucleotide or a variant thereof, preferably an adenosine ribonucleotide or a deoxyadenosine nucleotide, further comprising a phosphorothioate group; c is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, at the position corresponding to a nucleotide, preferably an adenosine or a cytidine, more preferably an adenosine, to be edited in the target sequence; and C is a cytidine nucleotide or a variant thereof; wherein an asterisk (*) indicates a chemical modification of the preceding nucleotide at the 2′ carbon atom with 2′-hydrogen (2′-deoxy), 2′-O-methyl, 2′-O-methoxyethyl or 2′-fluoro.
11. The artificial nucleic acid according to any of claims 1 to 8, wherein the targeting sequence comprises the nucleic acid sequence
3′Us*cC*5′, wherein Us is an uridine nucleotide or a variant thereof, preferably an uridine ribonucleotide or a deoxyuridine nucleotide, further comprising a phosphorothioate group; c is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, at the position corresponding to a nucleotide, preferably an adenosine or a cytidine, more preferably an adenosine, to be edited in the target sequence; and C is a cytidine nucleotide or a variant thereof; wherein an asterisk (*) indicates a chemical modification of the preceding nucleotide at the 2′ carbon atom with 2′-hydrogen (2′-deoxy), 2′-O-methyl, 2′-O-methoxyethyl or 2′-fluoro;
12. The artificial nucleic acid according to any of the preceding claims, wherein at least two of the five nucleotides at the 3′ terminus of the targeting sequence comprise a modified phosphate group, preferably a phosphorothioate group.
13. The artificial nucleic acid according to any of the preceding claims, wherein at least two of the five nucleotides at the 3′ terminus of the targeting sequence are LNA nucleotides, ENA nucleotides or (S)-constrained ethyl cEt nucleotides.
14. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at least one nucleotide comprising a modified phosphate group, preferably a phosphorothioate nucleotide; at least one nucleotide selected from the group consisting of an LNA nucleotide, an ENA nucleotide and an (S)-constrained ethyl cEt nucleotide, preferably an LNA nucleotide; and at least one nucleotide comprising a substituent at the 2′ carbon atom, wherein the substituent is selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro.
15. The artificial nucleic acid according to any one of the preceding claims, wherein the targeting sequence is characterized by a modification pattern according to any one of formulae (Ia), (Ib) or (Ic):
3′N.sub.aCN.sub.b5′ (Ia) wherein N is a nucleotide or a variant thereof, preferably a ribonucleotide or a variant thereof, a deoxynucleotide or a variant thereof, more preferably a modified ribonucleotide, or a modified deoxynucleotide; C is the nucleotide at the position corresponding to the nucleotide to be edited in the target sequence and wherein C is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site; a is an integer in a range from 1 to 40, preferably from 6 to 10; b is an integer in a range from 4 to 40; and wherein a+b is in a range from 15 to 80;
3′N.sub.cNs.sub.dN.sub.aCN.sub.bNs.sub.eN.sub.f5′ (Ib) wherein N is a nucleotide or a variant thereof, preferably a ribonucleotide or a variant thereof, a deoxynucleotide or a variant thereof, more preferably a modified ribonucleotide, or a modified deoxynucleotide; C is the nucleotide at the position corresponding to the nucleotide to be edited in the target sequence and wherein C is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site; Ns is a nucleotide comprising a modified phosphate group, preferably a phosphorothioate group; c is an integer in a range from 0 to 4; d is an integer in a range from 1 to 10; a is an integer in a range from 1 to 26; b is an integer in a range from 4 to 40; e is an integer in a range from 0 to 4; f is an integer in a range from 0 to 4; wherein a+d+c is in a range from 1 to 40; wherein b+e+f is in a range from 4 to 40; and wherein a+d+c+b+e+f is in a range from 15 to 80;
3′N.sub.cNI.sub.gN.sub.hNI.sub.iN.sub.aCN.sub.bNI.sub.jN.sub.kNI.sub.lN.sub.m5′ (Ic) wherein N is a nucleotide or a variant thereof, preferably a ribonucleotide or a variant thereof, a deoxynucleotide or a variant thereof, more preferably a modified ribonucleotide, or a modified deoxynucleotide; C is the nucleotide at the position corresponding to the nucleotide to be edited in the target sequence and wherein C is a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site; NI is an LNA nucleotide or a modified LNA nucleotide; c is an integer in a range from 0 to 4, preferably from 1 to 3; g, i is an integer in a range from 1 to 5; h is an integer in a range from 1 to 30, preferably from 1 to 5; a is an integer in a range from 1 to 15; b is an integer in a range from 4 to 30; j is an integer in a range from 0 to 5, preferably from 1 to 3; k is an integer in a range from 4 to 30; l is an integer in a range from 0 to 5, preferably from 1 to 3; m is an integer in a range from 0 to 3; wherein c+g+h+i+a is in a range from 1 to 40; wherein b+j+k+I+m is in a range from 4 to 40; and wherein c+g+h+i+a+b+j+k+I+m is in a range from 15 to 80.
16. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence is characterized by a modification pattern selected from any one of the formulae II(a) to II(l):
3′Ns.sub.4N.sub.6CN.sub.7-295′; (a)
3′Ns.sub.4N.sub.6-10CN.sub.9-12Ns.sub.25′; (b)
3′Ns.sub.2N.sub.11-15CN.sub.9-12Ns.sub.25′; (c)
3′NIs.sub.2Ns.sub.2NIN.sub.6-10CN.sub.5-9NI.sub.2NNs.sub.25′; (d)
3′NIsNsNIsNsN.sub.6-10CN.sub.4-8NINNINNs.sub.25′; (e)
3′NsNIsNsNIsN.sub.6-10CN.sub.3-7NINNIN.sub.2Ns.sub.25′; (f)
3′Ns.sub.2NNINNIN.sub.6-10CN.sub.4-8NINNINNs.sub.25′, (g)
3′NsNIsNs.sub.2NIN.sub.5CN.sub.5NIN.sub.1-235′; (h)
3′NIsNsNIsNsN.sub.8CN.sub.6NIN.sub.1-235′ (i)
3′NsNIsNs.sub.2NIN.sub.5CN.sub.5NIN.sub.20NI.sub.25′; (j)
3′NIsNsNIsNsN.sub.8CN.sub.6NIN.sub.20NI.sub.25′; and (k)
3′Ns.sub.4N.sub.6CN.sub.9Ns.sub.25′, (l) wherein N is a nucleotide or a variant thereof, preferably a ribonucleotide or a variant thereof, a deoxynucleotide or a variant thereof, more preferably a modified ribonucleotide, or a modified deoxynucleotide; Ns is a nucleotide comprising a modified phosphate group, preferably a phosphorothioate group; NI is an LNA nucleotide or a modified LNA nucleotide; NIs is an LNA nucleotide or a modified LNA nucleotide, further comprising a modified phosphate group, preferably a phosphorothioate group; C is the nucleotide at the position corresponding to the nucleotide to be edited in the target sequence and wherein C is a cytidine nucleotide or a variant thereof, a deoxycytidine or a variant thereof, preferably a deoxycytidine nucleotide, or an abasic site.
17. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises a nucleic acid sequence, wherein, with the exception of the cytidine nucleotide or the variant thereof, the deoxycytidine nucleotide or the variant thereof, or the abasic site, at the position corresponding to the nucleotide to be edited in the target sequence, with the exception of LNA nucleotides, and optionally with the exception of at least one of the two nucleotides, which are positioned 5′ or 3′ to said nucleotide at the position corresponding to the nucleotide to be edited in the target sequence, all nucleotides are chemically modified at the 2′ carbon atom, which is linked to a substituent selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro.
18. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises a nucleic acid sequence selected from the group consisting of TABLE-US-00014 (SEQ ID NO: 1) 5′ U*U*C*A*C*U* UcA G*U*G*U*As*Us*Gs*Cs*C* 3′; (SEQ ID NO: 2) 5′ U*U*C*A*C*U* UcA G*U*G*U*As*Us*Gs*Cs*C* 3′; (SEQ ID NO: 3) 5′ A*C*C*U*C*C* AcU C*A*G*U*Gs*Us*Gs*As*U* 3′; (SEQ ID NO: 4) 5′ U*U*U*C*C*U* CcA C*U*G*U*Us*Gs*Cs*As*A* 3′; (SEQ ID NO: 5) 5′ U*G*U*G*U*A* UcU U*G*C*U*Gs*Us*Gs*As*G* 3′; (SEQ ID NO: 6) 5′ G*A*G*G*U*C* CcU G*G*G*G*Gs*Cs*Gs*Cs*U* 3′; (SEQ ID NO: 7) 5′ G*A*U*C*U*U* CcU G*A*U*G*Gs*Cs*Cs*As*C* 3′; (SEQ ID NO: 8) 5′ A*G*C*C*A*C* AcA C*U*C*C*Gs*Us*Cs*As*G* 3′; (SEQ ID NO: 9) 5′ G*A*U*U*U*U* CcU G*A*U*A*Gs*Cs*Us*As*C* 3′; (SEQ ID NO: 10) 5′ G*G*C*C*A*C* AcA U*U*C*U*Gs*Us*Cs*As*G* 3′; (SEQ ID NO: 11) 5′ G*A*U*C*U*U* CcU G*A*U*G*Gs*Cs*Cs*As*C* 3′; (SEQ ID NO: 12) 5′ G*G*C*C*A*C* AcA C*U*C*C*Gs*Us*Cs*As*G* 3′; (SEQ ID NO: 13) 5′ G*A*U*U*U*U* CcU G*A*U*A*Gs*Cs*As*As*C* 3′; (SEQ ID NO: 14) 5′ G*G*C*U*A*C* GcA C*U*C*U*Gs*Us*Cs*As*A* 3′; (SEQ ID NO: 15) 5′ A*G*G*C*C*G* CcG U*C*G*U*Gs*Gs*Cs*Gs*G* 3′; (SEQ ID NO: 16) 5′ C*C*G*C*U*C* CcU CcU C*A*G*C*Cs*Cs*Gs*Us* C* 3′; (SEQ ID NO: 17) 5′ A*C*G*C*C*A* CcA G*C*U*C*Cs*As*As*Cs*U* 3′; (SEQ ID NO: 18) 5′ G*U*C*U*C*A* CcA A*U*U*G*Cs*Us*Cs*Us*C* 3′; (SEQ ID NO: 19) 5′ G*A*A*A*U*A* CcA U*C*A*G*As*Us*Us*Us*G* 3′; (SEQ ID NO: 20) 5′ A*A*U*U*A*G* CcU U*C*U*G*Gs*Cs*Cs*As*U* 3′; (SEQ ID NO: 21) 5′ G*A*U*C*A*G* CcU C*C*U*G*Gs*Cs*Cs*As*U* 3′; (SEQ ID NO: 22) 5′ G*A*U*C*A*G* CcU U*C*U*G*Gs*Cs*Cs*As*U* 3′; (SEQ ID NO: 23) 5′ G*A*U*C*A*G* CcU U*C*U*G*Gs*Cs*Cs*As*U* 3′; (SEQ ID NO: 24) 5′ C*A*C*U*G*C* CcA G*G*C*A*Us*Cs*As*Gs*C* 3′; (SEQ ID NO: 25) 5′ C*A*C*U*G*C* CcG G*G*C*A*Us*Cs*As*Gs*C* 3′; (SEQ ID NO: 26) 5′ U*C*C*G*C*C* CcG A*U*C*C*As*Cs*Gs*As*U* 3′; (SEQ ID NO: 27) 5′ C*C*U*U*U*C* UcG U*C*G*A*Us*Gs*Gs*Us*C* 3′; (SEQ ID NO: 28) 5′ C*C*U*U*U*C* U*cG U*C*G*A*Us*Gs*Gs*Us*C* 3′; (SEQ ID NO: 29) 5′ C*U*U*G*A*U* AcA U*C*C*A*Gs*Us*Us*Cs*C* 3′; (SEQ ID NO: 30) 5′ U*U*U*C*A*G* GcA U*U*U*C*Cs*Us*Cs*Cs*G* 3′; (SEQ ID NO: 31) 5′ C*U*U*C*A*G* GcA U*G*G*G*Gs*Cs*As*Gs*C* 3′; (SEQ ID NO: 32) 5′ A*G*G*A*A*C* AcA A*C*C*U*Us*Us*Gs*Us*C* 3′; (SEQ ID NO: 33) 5′ U*U*U*C*A*C* AcA U*C*C*A*Us*Cs*As*As*C* 3′; (SEQ ID NO: 34) 5′ C*U*U*C*A*C* GcA U*C*C*A*Us*Cs*As*As*C* 3′; (SEQ ID NO: 35) 5′ U*G*G*G*A*C* AcA A*C*C*C*Cs*Us*Gs*Cs*C* 3′; (SEQ ID NO: 36) 5′ C*G*A*C*U*C* CcU C*U*G*G*As*Us*Gs*Us*U* 3′; (SEQ ID NO: 37) 5′ C*G*A*C*U*C* UcU C*U*G*G*As*Us*Gs*Us*U* 3′; or a fragment or variant of any of these nucleic acid sequences; wherein A is an adenosine nucleotide or a variant thereof, preferably an adenosine ribonucleotide, an adenosine deoxynucleotide, a modified adenosine ribonucleotide or a modified adenosine deoxynucleotide; C is a cytidine nucleotide or a variant thereof, preferably a cytidine ribonucleotide, a cytidine deoxynucleotide, a modified cytidine ribonucleotide or a modified cytidine deoxynucleotide; G is a guanosine nucleotide or a variant thereof, preferably a guanosine ribonucleotide, a guanosine deoxynucleotide, a modified guanosine ribonucleotide or a modified guanosine deoxynucleotide; U is an uridine nucleotide or a variant thereof, preferably an uridine ribonucleotide, an uridine deoxynucleotide, a modified uridine ribonucleotide or a modified uridine deoxynucleotide; As, Cs, Gs and Us are nucleotides or variants thereof, preferably ribonucleotides or deoxynucleotides as defined above, further comprising a phosphorothioate group; wherein an asterisk (*) indicates a chemical modification of the preceding nucleotide at the 2′ carbon atom, preferably with 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl or 2′-fluoro; and wherein a lower case letter c indicates the position corresponding to a nucleotide or a variant thereof, preferably an adenosine or cytidine, more preferably an adenosine, to be edited in the target sequence and wherein c represents a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site.
19. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at the position corresponding to a nucleotide to be edited in the target sequence a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, and wherein at least one, preferably both, of the two nucleotides or variants thereof, which are positioned 5′ or 3′ of the position corresponding to a nucleotide to be edited in the target sequence, is chemically modified at the 2′ carbon atom, wherein the 2′ carbon atom is linked to a substituent selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably selected from 2′-O-methyl, 2′-O-methoxyethyl, 2′-hydrogen and 2′-fluoro; and/or wherein at least one, preferably both, of the two nucleotides or variants thereof, which are positioned 5′ or 3′ of the position corresponding to a nucleotide to be edited in the target sequence, comprises a modified phosphate group, preferably a phosphorothioate group.
20. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises at the position corresponding to a nucleotide to be edited in the target sequence a cytidine nucleotide or a variant thereof, a deoxycytidine nucleotide or a variant thereof, or an abasic site, and wherein the nucleotide, which is positioned 5′ of the position corresponding to the nucleotide to be edited, is a pyrimidine nucleotide, preferably a pyrimidine ribonucleotide or a pyrimidine deoxynucleotide, and wherein said pyrimidine nucleotide comprises a nucleobase, which is chemically modified at the 2′ position, preferably by 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl or 2′-O-fluoro.
21. The artificial nucleic acid according to any of the preceding claims, wherein the targeting sequence comprises a nucleic acid sequence as defined in any of claims 9 to 11.
22. The artificial nucleic acid according to any of the preceding claims, wherein the recruiting moiety comprises at least one coupling agent capable of recruiting a deaminase comprising a moiety that binds to said coupling agent, wherein the coupling agent is preferably covalently linked to the 5′-terminus or to the 3′-terminus of the targeting sequence or to an internal nucleotide within the targeting sequence.
23. The artificial nucleic acid according to claim 22, wherein the coupling agent is selected from the group consisting of O6-benzylguanine, O2-benzylcytosine, chloroalkane, 1×BG, 2×BG, 4×BG, and a variant of any of these.
24. The artificial nucleic acid according to claim 22 or 23, wherein the moiety binding to said coupling agent is selected from the group consisting of a SNAP-tag, a CLIP-tag, a HaloTag, and a fragment or variant of any one of these.
25. The artificial nucleic acid according to any of the preceding claims, wherein the recruiting moiety comprises 06-benzylguanine, 1×BG, 2×BG, 4×BG or a variant of any one of these and the deaminase, preferably an adenosine deaminase, comprises a SNAP-tag or a fragment or variant thereof; the recruiting moiety comprises a chloroalkane and the deaminase, preferably an adenosine deaminase, comprises a HaloTag or a fragment or variant thereof; or the recruiting moiety comprises O2-benzylcytosine or a variant thereof and the deaminase, preferably an adenosine deaminase, comprises a Clip-tag or a fragment or variant thereof.
26. The artificial nucleic acid according to any of the preceding claims, wherein the recruiting moiety comprises a coupling agent, which is capable of recruiting more than one deaminase molecule, wherein the coupling agent is preferably selected from 2×BG, 4×BG and a variant of any one of these; and/or the recruiting moiety comprises at least two moieties of a coupling agent, wherein the at least two moieties represent the same coupling agent or a different coupling agent.
27. The artificial nucleic acid according to any one of claims 1 to 21, wherein the recruiting moiety comprises a nucleic acid sequence capable of specifically binding to the deaminase, preferably an adenosine or cytidine deaminase.
28. The artificial nucleic acid according to claim 27, wherein the recruiting moiety comprises a nucleic acid sequence capable of specifically binding to the dsRNA binding domain of the deaminase, preferably an adenosine or cytidine deaminase.
29. The artificial nucleic acid according to any one of claims 1 to 21, 27 and 28, wherein the recruiting moiety comprises a nucleic acid sequence that is capable of intramolecular base pairing, preferably capable of forming a stem-loop structure.
30. The artificial nucleic acid according to claim 29, wherein the stem-loop structure comprises a double-helical stem comprising at least two mismatches.
31. The artificial nucleic acid according to claim 29 or 30, wherein the stem loop structure comprises a loop consisting of from 3 to 8, preferably from 4 to 6, more preferably 5, nucleotides, wherein the loop preferably comprises the nucleic acid sequence GCUAA or GCUCA.
32. The artificial nucleic acid according to any of claims 1 to 21 and 27 to 31, wherein the recruiting moiety comprises a nucleic acid sequence comprising at least one nucleotide, wherein the nucleobase is chemically modified, and/or wherein the nucleic acid sequence comprises at least one backbone modification.
33. The artificial nucleic acid according to claim 32, wherein the recruiting moiety comprises a nucleic acid sequence comprising at least one chemically modified nucleotide, which is chemically modified at the 2′ position.
34. The artificial nucleic acid according to claim 33, wherein the chemically modified nucleotide comprises a substituent at the 2′ carbon atom, wherein the substituent is selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro; and/or wherein the chemically modified nucleotide is a locked nucleic acid (LNA) nucleotide, an ethylene bridged nucleic acid (ENA) nucleotide or an (S)-constrained ethyl cEt nucleotide.
35. The artificial nucleic acid according to any of claims 32 to 34, wherein the recruiting moiety comprises a nucleic acid sequence comprising at least one backbone modification and wherein the phosphate group linking the sugars of two neighbouring nucleotides is a modified phosphate group, preferably selected from the group consisting of a phosphorothioate, a phosphoroselenate, a borano phosphate, a borano phosphate ester, a hydrogen phosphonate, a phosphoroamidate, an alkyl phosphonate, an aryl phosphonate and a phosphotriester.
36. The artificial nucleic acid according to any of claims 32 to 35, wherein the recruiting moiety comprises a nucleic acid sequence, wherein at least 40% of the nucleotides are chemically modified at the 2′ position.
37. The artificial nucleic acid according to any of claims 32 to 36, wherein the recruiting moiety comprises a nucleic acid sequence, wherein at least of two of the five nucleotides at the 5′ terminus of the nucleic acid sequence comprise a phosphorothioate group.
38. The artificial nucleic acid according to any of claims 32 to 37, wherein the recruiting moiety comprises a nucleic acid sequence, wherein at least of two of the five nucleotides at the 5′ terminus of the nucleic acid sequence are LNA nucleotides, ENA nucleotides or (S)-constrained ethyl cEt nucleotides.
39. The artificial nucleic acid according to any of claims 32 to 38, wherein the recruiting moiety comprises a nucleic acid sequence comprising at least one nucleotide comprising a modified phosphate group, preferably a phosphorothioate group; at least one LNA nucleotide, ENA nucleotide or (S)-constrained ethyl cEt nucleotide; and at least one nucleotide comprising a substituent at the 2′ carbon atom, wherein the substituent is selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro.
40. The artificial nucleic acid according to any of claims 1 to 21 and 27 to 39, wherein the recruiting moiety comprises a nucleic acid sequence selected from the group consisting of TABLE-US-00015 (a) (SEQ ID NO: 38) 5′ GGUGUCGAG-Na-AGA-N.sub.c-GAGAACAAUAU-G CU A/C A-AUGUUGUUCUC-N.sub.d-UCU-N.sub.b-CUCGA CACC 3′; (b) (SEQ ID NO: 39) 5′ GsGsUGUCGAG-N.sub.a-AGA-N.sub.c-GAGAACAAUAU- GCU A/C A-AUGUUGUUCUC-N.sub.d-UCU-N.sub.b-CUCGA CACC 3′; and (c) (SEQ ID NO: 40) 5′ GslGslUGUCGAG-N.sub.a-AGA-N.sub.c-GAGAACAAU AU-GCU A/C A-AUGUUGUUCUC-N.sub.d-UCU-N.sub.b-CU CGACACC 3′; or a fragment or variant of any of these nucleic acid sequences; wherein N.sub.a and N.sub.b form a mismatch, preferably wherein N.sub.a is adenosine and N.sub.b is cytidine; N.sub.c and N.sub.d form a mismatch, preferably wherein N.sub.c and N.sub.d are guanosine; Gs is a guanosine comprising a phosphorothioate group; and GsI is an LNA guanosine comprising a phosphorothioate group.
41. The artificial nucleic acid according to any of claims 1 to 21 and 27 to 39, wherein the recruiting moiety comprises a nucleic acid sequence derived from VA RNA I, or a fragment or variant thereof.
42. The artificial nucleic acid according to any of claims 1 to 21, 27 to 39 and 41, wherein the recruiting moiety comprises the nucleic acid sequence TABLE-US-00016 (SEQ ID NO: 41) GCACACCTGGGTTCGACACGCGGGCGGTAACCGCATG GATCACGGCGGACGGCCGGATTCGGGGTTCGAACCCC GGTCGTCCGCCATGATACCCTTGC, or a fragment or variant thereof.
43. The artificial nucleic acid according to any of claims 40 to 42, wherein the recruiting moiety comprises a nucleic acid sequence as defined in said claims, wherein at least one nucleotide, preferably at least 40%,50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% of the nucleotides, comprises a substituent at the 2′ carbon atom, wherein the substituent is selected from the group consisting of a halogen, an alkoxy group, a hydrogen, an aryloxy group, an amino group and an aminoalkoxy group, preferably from 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl and 2′-fluoro.
44. The artificial nucleic acid according to any of the preceding claims, wherein the recruiting moiety comprises a nucleic acid sequence selected from the group consisting of TABLE-US-00017 (a) (SEQ ID NO: 42) 5′ G*G*U*GU*C*GAG-N.sub.a-AGA-N.sub.c-GAGAAC*AA U*AU*-GC*U* A/C A- AU*GU*U*GU*U*C*U* C*-N.sub.d-U*C*U*-N.sub.b*-C*U*C*GAC*AC*C* 3′; (b) (SEQ ID NO: 43) 5′ Gs*Gs*U*GU*C*GAG-N.sub.a-AGA-N.sub.c-GAGAAC* AAU*AU*-GC*U* A/C A-AU*GU*U*GU*U*C*U* C*-N.sub.d-U*C*U*-N.sub.b*-C*U*C*GAC*AC*C* 3′; and (c) (SEQ ID NO: 44) 5′ Gsl*Gsl*U*GU*C*GAG-N.sub.a-AGA-N.sub.c-GAGA AC*AAU*AU*-GC*U* A/C A-AU*GU*U*GU*U*C *U*C*-N.sub.d-U*CU*-N.sub.b*-C*U*C*GAC*AC*C* 3′; or a fragment or variant of any of these sequences; wherein N.sub.a and N.sub.b form a mismatch, preferably wherein N.sub.a is adenosine and N.sub.b is cytidine; N.sub.c and N.sub.d form a mismatch, preferably wherein N.sub.c and N.sub.d are guanosine; Gs is a guanosine comprising a phosphorothioate group; GsI is an LNA guanosine comprising a phosphorothioate group; and wherein an asterisk (*) indicates a modification of the nucleotide at the 2′ carbon atom, preferably with 2′-hydrogen, 2′-O-methyl, 2′-O-methoxyethyl or 2′-fluoro.
45. Artificial nucleic acid for site-directed editing of a target RNA, the artificial nucleic acid comprising a) a targeting sequence, which comprises or consists of a nucleic acid sequence complementary or partially complementary to a target sequence in the target RNA, and b) a recruiting moiety for recruiting a deaminase, wherein the recruiting moiety comprises a nucleic acid sequence capable of specifically binding to the deaminase, preferably an adenosine or cytidine deaminase, wherein the recruiting moiety is characterized by any one of the features defined in claims 27 to 44.
46. The artificial nucleic acid according to any of the preceding claims, which further comprises a moiety, which enhances cellular uptake of the artificial nucleic acid.
47. The artificial nucleic acid according to claim 46, wherein the moiety enhancing cellular uptake is a triantennary N-acetyl galactosamine (GalNAc3), which is preferably conjugated with the 3′ terminus or with the 5′ terminus of the artificial nucleic acid.
48. The artificial nucleic acid according to any of the preceding claims, comprising in 5′ to 3′ direction the recruiting moiety and the targeting sequence defined in the preceding claims.
49. The artificial nucleic acid according to any of the preceding claims, which is an RNA.
50. The artificial nucleic acid according to any of the preceding claims, wherein the deaminase is an adenosine deaminase or a fragment or variant thereof, preferably selected from the group consisting of ADAR1, ADAR2 and a fragment or variant thereof, more preferably a peptide or protein comprising an adenosine deaminase domain; or a cytidine deaminase or a fragment or variant thereof, preferably Apobec1 or a fragment or variant thereof, more preferably a peptide or protein comprising a cytidine deaminase domain.
51. The artificial nucleic acid according to any of the preceding claims, wherein the deaminase is an adenosine deaminase, preferably a eukaryotic adenosine deaminase, more preferably a vertebrate adenosine deaminase, even more preferably a mammalian adenosine deaminase, most preferably a human adenosine deaminase, or a cytidine deaminase, preferably a eukaryotic cytidine deaminase, more preferably a vertebrate cytidine deaminase, even more preferably a mammalian cytidine deaminase, even more preferably a murine or a human cytidine deaminase, most preferably mApobec1.
52. The artificial nucleic acid according to any of the preceding claims, wherein the site-directed editing comprises the deamination of adenosine or cytidine in the target sequence.
53. Vector encoding the artificial nucleic acid according to any of the preceding claims.
54. Cell comprising the artificial nucleic acid according to any of claims 1 to 52 or the vector according to claim 53.
55. Composition comprising the artificial nucleic acid according to any one of claims 1 to 52, the vector according to claim 53 or the cell according to claim 54, and an additional excipient, preferably a pharmaceutically acceptable excipient.
56. The composition according to claim 55 comprising the artificial nucleic acid or the vector in the form of a nanoparticle, preferably a lipid nanoparticle or a liposome.
57. The composition according to claim 55 or 56, wherein the artificial nucleic acid or the vector is complexed by a cationic compound.
58. The composition according to claim 57, wherein the cationic compound is a cationic lipid.
59. Kit comprising the artificial nucleic acid according to any one of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, or the composition according to any of claims 55 to 58.
60. Use of the artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for site-directed editing of a target RNA.
61. Use of the artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for in vitro diagnosis of a disease or disorder.
62. Method for site-directed editing of a target RNA, which comprises contacting a target RNA with the artificial nucleic acid according to any of claims 1 to 52.
63. The artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for use as a medicament.
64. The artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for use in the treatment or prophylaxis of a disease or disorder selected from the group consisting of infectious diseases, tumour diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders.
65. The artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for use in the treatment or prophylaxis of a disease or disorder, wherein the treatment or prophylaxis comprises a step of site-directed editing of a target RNA.
66. The artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, the composition according to any of claims 55 to 58 or the kit according to claim 59 for use in the diagnosis of a disease or disorder, which is preferably selected from the group consisting of infectious diseases, tumour diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders.
67. Method for treating a subject with a disease or a disorder, the method comprising administering an effective amount of the artificial nucleic acid according to any of claims 1 to 52, the vector according to claim 53, the cell according to claim 54, or the composition according to any of claims 55 to 58 to the subject.
68. The method according to claim 67, wherein the disease or the disorder is selected from the group consisting of infectious diseases, tumour diseases, cardiovascular diseases, autoimmune diseases, allergies and neurological diseases or disorders.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0147] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.
[0148]
[0149]
[0151]
[0153]
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[0157]
[0159]
[0161]
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[0166]
EXAMPLES
[0168] The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.
Example 1
[0169] Unmodified RNA oligonucleotides were produced by in vitro transcription from linear synthetic DNA templates (purchased from Sigma-Aldrich, Germany) with T7 RNA polymerase (Thermo Scientific, USA) at 37° C. overnight. The resulting RNA was precipitated in ethanol and purified via urea (7M) polyacrylamide (15%) gel electrophoresis (PAGE), extracted into water, precipitated with ethanol and resuspended and stored in nuclease-free water. All chemically modified RNA oligonucleotides purchased from Biospring (Germany), Eurogentec (Belgium) or Dharmacon (USA). Long sequences were assembled from two pieces by ligation. As a first step, a plasmid-borne approach was applied in order to screen for suitable guideRNA sequences. A reporter editing assay (
[0170] In the reporter editing assay, Firefly luciferase was expressed under control of a CMV promotor from a pShuttle-CMV plasmid. The W417X amber mutation was introduced via overlap PCR. Sequences of the cloned products were determined by Sanger sequencing. The RIG-guideRNAs were expressed under control of the U6 promotor from a modified pSilencer backbone similar as described in Wettengel et al. (Wettengel, J., Reautschnig, J., Geisler, S., Kahle, P. J., Stafforst, T. Harnessing human ADAR2 for RNA repair—Recoding a PINK1 mutation rescues mitophagy. Nucl. Acids Res. 45, 2797-2808 (2017). Sequences of the cloned products were determined by Sanger sequencing. Sequences of the applied R/G-guideRNAs are provided in Table 1.
TABLE-US-00012 TABLE 1 R/G guideRNAs R/G guide RNAs expressed from plasmid 5′-3′ sequence Luciferase R/G-v1 (GUGGAAUAGUAUAACAAUAU GCUAAAUGUUGUUAUAGUAUC CCACGUGCAGCCAGCCGUCCU CUAGAGGGCCCUGAAGAGGGC CC) (SEQ ID NO: 61) Luciferase R/G-v4 (GUGGAAGAGGAGAACAAUAU GCUAAAUGUUGUUCUCGUCUC CCACGUGCAGCCAGCCGUCCU CUAGAGGGCCCUGAAGAGGGC CC) (SEQ ID NO: 62) Luciferase R/G-v9.4 (GUGGUCGAGAAGAGGAGAAC AAUAUGCUAAAUGUUGUUCUC GUCUCCUCGACCACGUGCAGC CAGCCGUCCUCUAGAGGGCCC UGAAGAGGGCCC) (SEQ ID NO: 63) Chemically synthesized ASOs 5′-3′ sequence ACTB 3′UTR 18 nt [GC AAU G](CCA) [UC AC] [C*][U*][C*][C*][C] (SEQ ID NO: 64) ACTB 3′UTR ASO (GGUGA AUAGUAUAAC AAUAU v1 GCUAA AUGUUGUUAUAGUAUCC ACC) [GC AAU G](CCA) [UC AC][C*][U*][C*] [C*][C](SEQ ID NO: 65) ACTB 3′UTR ASO (GGUGAAG AGGAGAACAA v4 UAUGCUAAAU GUUGUUCUC GUCUCCACC)[GC AAU G] (CCA) [UC AC][C*][U*] [C*][C*][C] (SEQ ID NO: 66) ACTB 3′UTR ASO (GGU GUC GAG AAG AGG v9.4 AGA AC AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CC) [GC AAU G] (CCA) [UC AC][C*][U*] [C*][C*][C] (SEQ ID NO: 67) GAPDH 3′UTR 18 nt [AG GGG U](CCA) [CA UG][G*][C*] [A*][A*][C] (SEQ ID NO: 68) GAPDH 3′UTR (GGUGA AUAGUAUAAC ASO v1 AAUAUGCUAA AUGUUGU UAUAGUAUCCACC) [AG GGG U](CCA) [CA UG][G*][C*] [A*][A*][C] (SEQ ID NO: 69) GAPDH 3′UTR (GGUGAAG AGGAGAACAA UAUGCUAAAU ASO v4 GUUGUUCUCGUCUCCACC)[AG GGG U] (CCA)[CA UG][G*][C*][A*][A*] [C](SEQID NO: 70) GAPDH 3′UTR (GGU GUC GAG AAG AGG AGA AC ASO v9.4 AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CC) [AG GGG U](CCA) [CA UG] [G*][C*][A*][A*][C] (SEQ ID NO: 71) GAPDH 3′UTR [G*][G*][U](G)[U][C] ASO v9.5 (GAG AAG AGG AGA A)[C] (AA)[U](A)[U](G) [C][U] (A AA)[U](G)[UU](G) [UUCUC](G)[UCUCCUC](GA) [C](A) [CCAGGGGU](CCA) [CAUG][G*][C*][A*][A*] [C](SEQ ID NO: 72) GAPDH ORF1 ASO [GGG GUG](CCA) [AG CA] 18 nt [G*][U*][U*][G*][G] (SEQ ID NO: 73) GAPDH ORF1 ASO (GGU GUC GAG AAG AGG AGA v9.4 AC AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CC) [GGG GUG](CCA)[AG CA][G*] [U*][U*][G*][G] (SEQ ID NO: 74) GAPDH ORF2 ASO [GGG GUG](CCA)[AG CA] 18 nt [G*][U*][U*][G*][G] (SEQ ID NO: 75) GAPDH ORF2 ASO (GGU GUC GAG AAG AGG AGA v9.4 AC AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CC) [GU UUU U](CCA) [GA CG] [G*][C*][A*][G*][G] (SEQ ID NO: 76) GAPDH ORF1 ASO [G]*[G]*[U](G)[U][C] v25 (GAG AAG AGG AGA A)[C] (AA) [U](A)[U](G) [C] [U](A AA)[U](G)[U][U] (G)[U][U][C][U][C](G) [U][C][U][C][C][U][C] (G A)[C](A) [C][C] (UUGUCAUGGAUGACCUUGGCCA) [G]{G}[GG UG](CCA) [AGCA]{G*}[U*][U*]{G*} [G](SEQ ID NO: 77) GAPDH ORF1 ASO [G]*[G]*[U](GUCGAG AAG R/G unmod v25 AGG AGA ACAAUAUGCUAAAUG UUGUUCUCGUCUCCUCG ACACC UUGUCAUGGAUGACCUU GGCCA) [G][G][GG UG](CCA) [AGCA][G*][U*][U*]{G*} [G](SEQ ID NO: 78) SERPINA ASO v9.4 (GGU GUC GAG AAG AGG AGA AC AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CC) [CCU UUC](UCG) [UCG A] [U*][G*][G*][U*][C] (SEQ ID NO: 79) SERPINA ASO 40 nt (CAUGGCCCCAGCAGCUUCAGUC) [C]{C}[UUUC](UCG)[UCGA] {T*}[G*][G*]{T*}[C] (SEQ ID NO: 80) SERPINA ASO v25 [G*][G*][U](G)[U][C] (GAG AAG AGG AGA A)[C] (AA) [U](A)[U](G) [C] [U](A AA)[U](G)[U][U] (G)[U][U][C][U][C](G) [U][C][U][C][C][U][C] (G ACACC CAUGGCCCCAGCA GCUUCAGUC)[C]{C}[UUUC] (UCG) [UCGA]{T*}[G*] [G*]{T*}[C] (SEQ ID NO: 81) SERPINA ASO v25 [G*][G*][U](G)[U][C] (GAG AAG AGG AGA A)[C] (AA) [U](A)[U](G) [C] [U](A AA)[U](G)[U][U] (G)[U][U][C][U][C](G) [U][C][U][C][C][U][C] (G ACACC CAUGGCCCCAGCA GCUUCAGUC)[C]{C}[UUUCU] (CG) [UCGA][T*][G*] [G*]{T*}[C] (SEQ ID NO: 82) STAT1 ASO v25 [G*][G*][U](G)[U][C] (GAG AAG AGG AGA A)[C] (AA) [U](A)[U](G) [C] [U](A AA)[U](G)[U][U] (G)[U][U][C][U][C] (G)[U][C][U][C] [C][U][C](GACACCCAG ACACAGAAAUCAACUCAGU) [C][T][UGAU](ACA) [UCCA]{G*}[U*][U*] {C*}[C] (SEQ ID NO: 83) GAPDH 3′UTR (GGUGA AUAGUAUAAC unmod ASO v1 AAUAUGCUAA AUGUUGUUA UAGUAUCCACC AG GGG UCCACA UG GCAAC) (SEQ ID NO: 84) GAPDH 3′UTR (GGUGAAG AGGAGAACAA UAU unmod ASO v4 GCUAAAU GUUGUUCUCGUCUCC ACCAG GGG UCCACA UGGCAAC) (SEQ ID NO: 85) GAPDH 3′UTR (GGU GUC GAG AAG AGG AGA unmod ASO v9.4 AC AAU AUG CUA AAU GUUGUU CUCGUC UCC UCG ACA CCAG GGG U CCACAU GGCAAC) (SEQ ID NO: 86) Sense guideRNAs for RT PCR 5′-3′ sequence GAPDH sense (GGACCAACUGCUUGGCACCCCUG GCCAAGGUCAUCCAUGACAACUUU GGUAUCGUGGAAGGACC) (SEQ ID NO: 87) STAT1 sense (GGGAACUGGAUCUAUCAAGACUG AGUUGAUUUCUGUGUCUGAAGUGU AAGUGAACACAGAA) (SEQ ID NO: 88) SERPINA1 sense (GGACCATCGACGAGAAAGGGACT GAAGCTGCTGGGGCCATGTTTTTA GAGGCCATACCCAT) (SEQ ID NO: 89) Legend to Table 1:(N) = RNA base, [N] = 2′-OMe RNA base, *= Phosphorothioate linkage, {N} = LNA base
[0171] Flp-In 293 T-REx cells (R78007, Thermo Fisher scientific) containing the respective gnomically integrated ADAR version were generated as described in Wettengel et al. and in Heep et al. (Heep, M., Mach, P., Reautschnig, P., Wettengel, J., Stafforst, T. Applying Human ADAR1p110 and ADAR1p150 for Site-Directed RNA Editing—G/C Substitution Stabilizes GuideRNAs Against Editing. Genes 8, 34 (2017)). Cells were cultured in DMEM+10% FBS+100 μg/ml hygromycin B+15 μg/ml blasticidin S. For editing, 2.5×10.sup.5 cells/well (ADAR1p110, ADAR1p150) or 3×10.sup.5 cells/well (ADAR2) were seeded into poly-D-lysine-coated 24-well plates in 500 μl DMEM+10% FBS+10 ng/ml doxycycline. Twenty-four hours later, transfection was performed with the luciferase reporter plasmid (300 ng) and the R/G-guideRNA (1300 ng) using a Lipofectamine-2000 to plasmid ratio of 3:1. The medium was changed every 24 h until harvest. RNA was isolated and sequenced 72 h post transfection, as described above.
[0172] Even though being less effective in recruiting ADAR2 (35% reduced editing yield), sequence variant 9.4 turned out to improve editing yield with ADAR1p110 by almost twofold.
[0173] In a next step, the plasmid-borne expression of the guideRNA was replaced by the administration of chemically stabilized antisense oligonucleotides (ASO). In the first round, three chemically stabilized ASO designs (v1, v9, v9.4) were tested for the editing of a respective 5′-UAG site in the 3′-UTR of GAPDH and ACTB. While the ADAR recruiting domain comprised of natural ribonucleotides, the 17 nt antisense part of the ASO was designed as an Antagomir-like modified gapmer10 (global 2′-Omethylation, 3′-terminal phosporthioate linkages,
[0174] To assess the individual ADAR preference of such ASOs, we lipofected them into engineered 293 Flp-In T-REx cells expressing a specific ADAR isoform (ADAR2, ADAR1p110 or ADAR1p150)11 under control of a CMV tet-on promotor. 48 Hours before ASO transfection, 2×10.sup.5 of the respective ADAR-Flp-In 293 T-REx cells per well were seeded in 24 well plates in DMEM+10% FBS containing 10 ng/mL doxycycline for induction of ADAR gene expression. After 48 hours cells were detached and reverse-transfected in 96 well plates. To this end, the respective ASO (5 pmol/well unless stated otherwise) and Lipofectamine 2000 (0.75 μL/well) were each diluted with OptiMEM to a volume of 10 μL in separate tubes, respectively. After 5 minutes, both solutions were mixed and 100 μL cell suspension (5×10.sup.4 cells) in DMEM+10% FBS+10 ng/mL doxycycline was added to the transfection mixture inside 96 wells. 24 hours later, cells were harvested for RNA isolation and sequencing, as described above.
[0175] Notably, particularly high editing yields (75-85%) were detected for both targets in ADAR1p150-expressing cells (
Example 2
[0176] In a further series of experiments, endogenously expressed ADAR was harnessed for the editing of a 5′-UAG codon in the 3′-UTR of the two housekeeping genes GAPDH and ACTB in HeLa cells by simple lipofection of the respective ASOs.
[0177] To this end, HeLa cells (Cat. No.: ATCC CCL-2) were cultured in DMEM+10% FBS+P/S (100U/mL penicillin and 100 μg/mL streptomycin. 5×10.sup.4 cells in 100 μL DMEM+10% FBS 600 units IFN-α, Merck, catalog number IF007, lot number 2937858) were added to a transfection mix of 0.5 μL Lipofectamine 2000 and 5 pmol guideRNA/well in a 96-well format. For concurrent editing with two different ASOs, 2.5 pmol of each respective ASO were co-transfected. After 24 hours cells were harvested for RNA isolation and sequencing.
[0178] A control ASO comprising only of the specificity domain but lacking the ADAR recruiting domain did not elicit any editing (
[0179] Also in this series of experiments, editing of both transcripts was further analysed after simultaneous co-transfection of two guideRNAs. Also in this setting, the editing yields remained unchanged at high levels (
[0180] In a next step, the chemical modification was extended to the ADAR recruiting domain. Specifically, the 5′-terminus was stabilized by 2′-O-methylation and phosphorthioate linkages and all pyrimidines were substituted with their 2′-O-methylated analogs. Even though heavily modified, this ASO design v9.5 was equal or even better in recruiting endogenous ADAR in HeLa cells (
[0181] In order to assess, which ADAR isoform was recruited by ASO v9.5 in HeLa cells, the expression of ADARs was determined in Western Blot experiments.
[0182] For western blotting, cells were harvested and lysed in urea-lysis buffer (8 M urea, 100 mM NaH.sub.2PO.sub.4, 10 mM Tris, pH 8.0) 72 h after reverse transfection of the siRNA. Shear force was applied using a 23-gauge syringe, and the cell debris were removed by centrifugation at 30.000 g for 15 min at 4° C. Then a Bradford assay was used to normalize total protein amounts, and appropriate amounts of protein lysate in 1× Laemmli-buffer were loaded onto an SDS-PAGE (4% stacking, 12% separating gel). Proteins were transferred on a PVDF membrane using a tank-blotting-system at 30 V overnight. The membrane was blocked in 5% nonfat dry milk TBST+50 μg/ml avidin for 2h at room temperature, and was afterwards incubated with the primary antibodies (5% nonfat dry milk TBST+1:1000 α-ADAR1, Santa Cruz, sc-73408 or α-ADAR2, Santa Cruz, sc-73409+1:40.000 α-beta-actin, Sigma Aldrich, A5441) at 4° C. overnight. The secondary antibodies (5% nonfat dry milk TBST+1:10.000 α-Mouse-HRP+1:50.000 Precision Protein™ StrepTactin-HRP Conjugate, Bio-Rad, #1610381) were incubated for 1.5h at room-temperature. After each antibody incubation, the membrane was washed 3×5 min with TBST. Detection was performed using 1 ml of Clarity Western ECL Substrate (Biorad) and a Fusion SL Vilber Lourmat (Vilber).
[0183] In Western Blot, only ADAR1p110 was found to be well expressed, whereas ADAR1p150 was only faintly visible but clearly inducible by IFN-α (
[0184] When transfecting an siRNA against ADAR2 or mock, respectively, the editing yield remained unaffected at 35% and 70%, depending on IFN-α, respectively (
[0185] When varying the amount of ASO v9.5 between 20 pmol and 40 fmol/96 well (
[0186] The time profile of the editing yield was further assayed over five days after transfection of 5 pmol/well into quickly dividing HeLa cells (10% FBS). For that purpose, HeLa cells were transfected as described above. Prior to transfection, cells were treated with IFN-α for 24 hours (where indicated). Cells were harvested for RNA isolation at the respective time points indicated. For time points later than 24 hours post transfection, cells were detached after 24 hours and transferred into 24-well plates in order to avoid overgrowth of the cells. Medium (containing IFN-α where indicated) was changed every 24 hours. The maximum editing yield was typically observed in a time window of 12-48 hours after transfection and dropped down slowly (
[0187] In order to assess the scope of cell lines, in which the recruitment of endogenous ADAR works efficiently, ASO v9.5 was applied to a panel of 10 immortalized human standard (cancer) cell lines (
[0188] In order to better assess the potential therapeutic scope of ADAR-recruiting ASOs, a panel of seven primary cells from different tissues was tested, including fibroblasts (from a Parkinson patient), and commercially acquired astrocytes, hepatocytes (several donors), epithelial cells from the retina and the bronchia, and endothelial cells from arterial and venous vessels (
Example 3
[0189] Following the characterization of ASO design 9.4 for the editing of 5′-UAG triplets in the 3′-UTR, the editing of a 5′-UAG triplet in the ORF of GAPDH in ADAR-expressing 293 cell lines was tested with an ASO based on v9.4 (see also Example 1). Comparison of the editing yields obtained with the three ADARs showed that the editing yields in the ORF followed the same trend as in the 3′-UTR before (ADAR1p150>ADAR1p110 ADAR2), albeit with generally lower editing yields (11%-55%, see
[0190] The ASO architecture was further optimized in order to improve the on-target binding kinetics by increasing the length of specificity domain and by including LNA modifications. We identified ASO design v25, which comprises of the unaltered ADAR-recruiting domain, but contained a 40 nt specificity domain, which was partly modified by 2′-O-methylation, phosphorothioate linkage and contained three LNA modifications (
Example 4
[0191] In order to evaluate the therapeutic potential of such ASOs, the editing of two therapeutically relevant deamination sites was tested. First, the phosphorylation site in endogenous STAT1 (Tyr701) was targeted, deamination of which switches function of the protein as a transcription factor. After editing, the respective 5′-UIU codon encodes for Cys, an amino acid that is unable to mimic phosphorylated Tyr. An ASO based on the v25 design described above was used in these experiments. Editing yields of 21.0±6.2% were achieved in primary fibroblasts and up to 7% in RPE cells prior to IFN-α treatment (
[0192] As a second site, the editing of the PiZZ mutation (E342K) in the SERPINA1 transcript, the most common cause of α1-antitrypsin deficiency (A1AD), was tested. Loss of antitrypsin, which regulates neutrophil elastase activity, causes severe damage of the lungs. Furthermore, mutated antitrypsin accumulates in the liver and leads to severe liver damage. First, the editing of the E342K mutation (5′-CAA triplet) was tested upon overexpression of the mutated SERPINA1 cDNA in ADAR1p150-expressing 293 cells applying an ASO build on the v9.4 design. In order to obtain SERPINA1 cDNA for cloning, total RNA was isolated from HepG2 cells and reverse transcribed. The E342K mutation was inserted into the cDNA by PCR and both SERPINA1 wild-type and the E342K mutant were each cloned on a pcDNA3.1 vector under control of the CMV promotor using HindIII and ApaI restriction. For genomic integration of SERPINA1 using the piggyBac transposon system, the wild-type and mutant cDNA was cloned on a PB-CA vector using the same restriction sites as above. 1×10.sup.6 HeLa cells were seeded in a six-well plate 24 hours before transfection. 1 μg of the piggyBac transposase vector (Transposagen Biopharmaceuticals) and 2.5 μg of the SERPINA1 PB-CA vector were co-transfected using 10.5 μL FuGENE6 (Promega) according to the manufacturer's protocol. After 24 hours, cells were selected for 2 weeks in DMEM+10% FBS medium containing 10 μg/mL puromycin. For editing, stably transfected or plasmid transfected (300 ng plasmid/0.9 μL FuGENE6 for Hela and 100 ng plasmid/0.3 μL Lipofectamine2000 for Flp-ADAR1p150 cells) cells were reverse transfected with the respective ASO as described above. After 24 hours, cell culture supernatant was collected for the A1AT-ELISA and cells were harvested for RNA isolation and sequencing. The A1AT-ELISA was performed with a commercial kit (cat. no.: ab108799, Abcam) according to the manufacturer's protocol. Samples from three biological replicates were measured in technical duplicates. The MAT protein amount was calculated from a standard curve using linear regression.
[0193] Only in presence of the ASO, an editing yield of 29±2% was determined at the targeted site (
Example 5
[0194] In order to test the guideRNA stabilities, guideRNAs have been incubated for a defined amount of time (0 min, 5 min, 10 min, 1 h, 3 h, 6 h, 12 h or 24 h) in PBS buffer containing 10% FBS. After incubation, the guideRNAs were separated on a 15% Urea (7M)-PAGE, stained with SYBR Gold and were photographed and quantified with a Typhoon FLA biomolecular imager. The guideRNAs with the unmodified 3 nt anticodon typically had very short half-lifes in serum (minutes). The guideRNA with a 3′-UCU anticodon targeting the 5′-AAA codon, e.g.
Example 6
[0195] In a parallel approach, guideRNAs conjugated with a coupling agent were employed for editing endogenous transcripts with tagged ADARs. For example, BG-conjugated guideRNAs were used in combination with SNAP-tagged ADARs (see
TABLE-US-00013 TABLE 2 guideRNAs for use with tagged ADARs target gRNA sequence editing of various endogenous transcripts: 5′-UTR 5′-UsCsAUUAAACC CCA GAGUCsCsGsGsA-3′ SNAP-ADAR (SEQ ID NO: 92) 5′-UTR 5′-UsCsUGAAUAAU CCA GGAAAsAsGsCsA-3′ GAPDH (SEQ ID NO: 93) isoform 2 ORF 5′-UsAsUAGGGGUG CCA AGCACsUsUsGsG-3′ #1 (SEQ ID GAPDH NO: 94) ORF 5′-UsAsUGGUUUUU CCA CACGGsCsAsGsG-3′ #2 (SEQ ID GAPDH.sup.b) NO: 95) ORF 5′-GsGsUGCAGAUU CCA GGUGGsGsAsCsG-3′ #1 (SEQ ID GUSB NO: 96) ORF 5′-AsCsAGACUUGG CCA CUGAGsGsGsGsG-3′ #2 (SEQ ID GUSB NO: 97) 3′-UTR 5′-UsAsUGUGUCGG CCA CGGAAsCsAsGsG-3′ SNAP- (SEQ ID ADAR NO: 98) 3′-UTR 5′-AsAsUAAGGGGU CCA CAUGGsCsAsAsC-3′ GAPDH.sup.C) (SEQ ID NO: 99) 3′-UTR 5′-UsCsGAGCAAUG CCA UCACCsUsCsCsC-3′ ACTB (SEQ ID NO: 100) 3′-UTR 5′-UsAsUUUCCCUG CCA GAAUAsGsAsUsG-3′ GUSB (SEQ ID NO: 101) KRAS 5′-GsAsUGCUCCAA CCA CCACAsAsGsUsU-3′ target (SEQ ID NO: 102) A/1 KRAS 5′-CsCsUCUCUUGC CCA CGCCAsCsCsAsG-3′ target (SEQ ID NO: 103) 2 STAT1 5′-CsUsCUCUUGAU ACA UCCAGsUsUsCsC-3′ Y701 (SEQ ID NO: 104) editing of all 16 adenosine- containing triplets in GAPDH isoform 1: 5′-GAA 5′-CsAsCAUGCGAU UCC CAUUGsAsUsGsA-3′ (SEQ ID NO: 105) 5′-GAU 5′ - UsAsUCGACCAA ACC CGUUGsAsCsUsC-3′ (SEQ ID NO: 106) 5′-GAC 5′-CsAsCGUCAUGA GCC CUUCCsAsCsCsA-3′ (SEQ ID NO: 107) 5′-GAG 5′- AsAsCGAGGGAU CCC GCUCCsUsGsGsA-3′ (SEQ ID NO: 108) 5′-CAA 5′-GsAsAGAGGCUG UCG UCAUAsCsUsUsC-3′ (SEQ ID NO: 109) 5′-CAU 5′-CsAsACACGUCA ACG AAGGGsGsUsCsA-3′ (SEQ ID NO: 110) 5′-CAC 5′-AsAsCGCCAGGG GCG CUAAGsCsAsGsU-3′ (SEQ ID NO: 111) 5′-CAG 5′-UsAsCGCAUCGA CCG UCCUCsAsUsCsA-3′ (SEQ ID NO: 112) 5′-AAA 5′-UsAsCAUGACCC UCU UGGCUsCsCsCsC-3′ (SEQ ID NO: 113) 5′-AAU 5′-GsAsCUACCCAA ACU CGUUGsUsCsAsU-3′ (SEQ ID NO: 114) 5′-AAC 5′-AsGsUCGCCACA GCU UCCCGsGsAsGsG-3′ (SEQ ID NO: 115) 5′-AAG 5′-UsCsUAUAUCCA CCU UACCAsGsAsGsU-3′ (SEQ ID NO: 116) 5′-UAA 5′-AsGsGAGGGGUC UCA CUCCUsUsGsGsA-3′ (SEQ ID NO: 117) 5′-UAU 5′-CsUsACGCAACA ACA UCCACsUsUsUsA-3′ (SEQ ID NO: 118) 5′-UAC 5′-CsCsGAGCGCCA GCA GAGGCsAsGsGsG-3′ (SEQ ID NO: 119) 5′-UAG 5′-UsAsUGGUUUUU CCA GACGCsCsAsCsG-3′ (SEQ ID NO: 120) avoiding off-target editing of neighbouring adenosine: 5′-CAA 5′-GsAsAGAGGCUCU CG UCAUAsCsUsUsC-3′ methoxy (SEQ ID NO: 121) 5′-CAA 5′-GsAsAGAGGCUG CG UCAUAsCsUsUsC-3′ fluoro (SEQ ID NO: 122) 5′-AAA 5′-UsAsCAUGACCCU CU UGGCUsCsCsCsC-3′ methoxy (SEQ ID NO: 123) 5′-AAA 5′-UsAsCAUGACC
CU UGGCUsCsCsCsC-3′ fluoro (SEQ ID NO: 124) 5′-AAC 5′-AsGsUCGCCACA GC UUCCCGsCsAsGsG-3′ methoxy (SEQ ID NO: 125) 5′-AAC 5′-AsGsUCGCCACA GC
UCCCGsCsAsGsG-3′ fluoro (SEQ ID NO: 126) 5′-UAA 5′-AsGsGAGGGGUCU CA CUCCUsUsGsGsA-3′ methoxy (SEQ ID NO: 127) 5′-UAA 5′-AsGsGAGCGCUC
CA CUCCUsUsGsGsA-3′ fluoro (SEQ ID NO: 128) Legend to Table 2: Nucleotides highlighted in bold are unmodified and are placed opposite the triplet with the target adenosine in the middle. Nucleotides highlighted in italic are modified with 2′-O-methylation, 2′-fluorinated nucleotides are grayed out. The backbone contains terminal phosphorothioate linkages as indicated by “s”. The first three nucleotides at the 5′-end are not complementary to the mRNA substrate, but serve as linker sequence between gRNA and SNAP-tag.
[0196] For this study, all NH.sub.2-guideRNAs were purchased from Biospring (Germany) as HPLC-purified ssRNAs with a 5′-C6 amino linker. As an alternative to commercial BG derivatives, our protocol can be used to introduce the BG moiety. Benzylguanine connected to a carboxylic acid linker2,3 (12 μl, 60 mM in DMSO) was in-situ activated as an OSu-ester by incubation with EDCl.HCl (12 μl, 17.4 mg/ml in DMSO) and NHS (12 μl, 17.8 mg/ml in DMSO) for 1 h at 30° C. Then, the NH.sub.2-guideRNA (25 μl, 6 μg/μl) and DIPEA (12 μl, 1:20 in DMSO) were added to the pre-activation mix and incubated (90 min, 30° C.).20 19 The crude BG-guideRNA was purified from unreacted NH.sub.2-guideRNA by 20% urea PAGE and then extracted with H.sub.2O (700 μl, overnight at 4° C.). RNA precipitation was done with sodium acetate (0.1 volumes, 3.0 M) and ethanol (3 volumes, 100%, overnight at −80° C.). The BG-guideRNA was washed with ethanol (75%) and dissolved in water (60 μl).
[0197] Cell lines were generated that stably express SNAP-ADAR1 (SA1), SNAP-ADAR2 (SA2), 2 and their hyper-active E Q variants 10 SA1Q and SA2Q. Each respective enzyme (SA1 (wt & Q) and SA2 (wt and Q)) was integrated as a single copy under control of the dox-inducible CMV promotor at the FRT site into the genome of 293 Flip-In cells (R78007, Thermo Fisher scientific) as described before (see Wettengel, J., Reautschnig, J., Geisler, S., Kahle, P. J., Stafforst, T. Harnessing human ADAR2 for RNA repair—Recoding a PINK1 mutation rescues mitophagy. Nucl. Acids Res. 45, 2797-2808 (2017); or Cox, D. B. T., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., Zhang, F. RNA editing with CRISPR-Cas13, Science, 10.1126/science.aaq0180 (2017). Enzyme expression of all four enzymes was inducible by doxycycline (10 ng/ml) to roughly comparable levels as validated by Western blot and fluorescence microscopy (data not shown). Also at the RNA level, the expression levels of SA1 (wt & Q) and SA2 (wt and Q) were roughly comparable with average FPKM values of 679 and 814 for SA1(Q) and SA2(Q), respectively. The E Q mutation did not change the protein localization. SA1(Q) is localized to cytoplasm and nucleoplasm; SA2(Q) is mainly localized to cytoplasm. In order to determine the location of the different SNAP-ADAR proteins, 1×10.sup.5 cells were seeded in 500 μl selection media with or without doxycline (10 ng/ml) on poly-D-lysine-coated cover slips in a 24-well format. After one day, BG-FITC labeling of the SNAP-tag and nuclear staining was done. To validate SNAP-ADAR protein amounts, Western blot analysis was used. For this, 3×10.sup.5 cells were seeded in 500 μl selection media with or without doxycline (10 ng/ml) in a 24-well format for one day. Then, cells were lysed with urea buffer (8 M urea in 10 mM Tris, 100 mM NaH.sub.2PO.sub.4, pH 8.0). Protein lysate (5 μg) was separated by SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories, USA) for immunoblotting with primary antibodies against the SNAP-tag (1:1000, P9310S, New England Biolabs, USA) and g-actin (1:40000, A5441, Sigma Aldrich, USA). Afterwards, the blot was incubated with HRP-conjugated secondary antibodies against rabbit (1:10000, 111-035-003, Jackson Immuno Research Laboratories, USA) and mouse (1:10000, 115-035-003, Jackson Immuno Research Laboratories, USA) and visualized by enhanced chemiluminescence.
[0198] Editing was initiated by transfection of the short, chemically stabilized BG-guideRNA, and was analyzed for formal A-to-G conversion in cDNA at specific 5′-UAG triplets in the 3′-UTRs of the four targeted endogenous mRNAs: ACTB, GAPDH, GUSB, and SA1/2. For both wildtype enzymes (SA1/2), editing yields of 40-80% were achieved (
Example 7
[0199] A major objective in RNA editing is the suppression of off-site editing (see
Example 8
[0200] Branched linkers and multiple copies of the BG-derived recruiting moieties were tested with regard to their effect on RNA editing. To this end, various guideRNAs were tested side-by-side against the Tyr701 codon in the endogenous STAT1 transcript in 293-Flp-In cells expressing SNAP-ADAR1Q (24 h induction with 10 ng/ml doxycycline prior to guideRNA transfection, editing analysis was done 24 h post guideRNA transfection). Specifically, guideRNAs were applied that contained either a 5′-amino linker or both, a 5′- and a 3′-amino linker and coupled to one or two of the recruiting moieties, respectively. The resulting guideRNAs can potentially recruit from one to eight SNAP-ADAR1Q deaminases, as illustrated by