Compositions and Methods for Genome Editing
Described herein are compositions for targeting and editing genomes. Also described herein are methods for targeting and editing genomes utilizing the compositions in the instant disclosure.
1 . An engineered guide RNA that comprises:
(a) at least one chemical modification, and
(b) a targeting domain, wherein the targeting domain hybridizes to a target RNA when administered to a subject, thereby forming a complex that recruits an RNA editing entity present in a cell of the subject;
wherein the RNA editing entity, when associated with the engineered guide RNA and the target RNA, performs a targeted editing of a base of a nucleotide of the target RNA.
2 . The engineered guide RNA of claim 1 , wherein the engineered guide RNA comprises a mismatch relative to the target RNA.
3 . The engineered guide RNA of claim 2 , wherein the mismatch comprises a base in the engineered guide RNA opposite to and unpaired with a base in the target RNA molecule.
4 . The engineered guide RNA of claim 2 , wherein the mismatch comprises an A/C mismatch and wherein the A is in the target RNA molecule and the C is in the engineered guide RNA.
5 . The engineered guide RNA of claim 4 , wherein the A in the A/C mismatch comprises the base of the nucleotide in the target RNA molecule chemically modified by the RNA editing entity.
6 . The engineered guide RNA of claim 5 , wherein the engineered guide RNA comprises a C opposite the base of the nucleotide in the target RNA chemically modified by the RNA editing entity.
7 . The engineered guide RNA of claim 5 , wherein the target RNA molecule comprises a G adjacent to and 5′ of the base of the nucleotide in the target RNA chemically modified by the RNA editing entity.
8 . The engineered guide RNA of claim 6 , wherein the engineered guide further comprises a G adjacent to and 5′ of the C opposite to and unpaired with the A in the target RNA molecule chemically modified by the RNA editing entity.
9 . The engineered guide RNA of any of claims 5 - 8 , wherein the engineered guide RNA comprises an unmodified nucleotide on either side of the mismatch.
10 . The engineered guide RNA of any one of claims 5 - 9 , wherein the engineered guide RNA does not comprise a second mismatch within 2 nucleotides of the mismatch.
11 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification is positioned:
(a) proximal to a 5′ end of the engineered guide RNA;
(b) proximal to a 5′ end of a region of the engineered guide RNA; or
(c) both (a) and (b).
12 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification is positioned:
(a) proximal to a 3′ end of the engineered guide RNA;
(b) proximal to a 3′ end of a region of the engineered guide RNA; or
(c) both (a) and (b).
13 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification is positioned proximal to a 5′ end of the engineered guide RNA, proximal to a 5′ end of a region of the engineered guide RNA, proximal to a 3′ end of the engineered guide RNA, proximal to a 3′ end of a region of the engineered guide RNA, or any combination thereof.
14 . The engineered guide RNA of any one of the preceding claims, wherein the engineered guide RNA, when present in an aqueous solution and not bound to the target RNA, does not bind to the RNA editing entity with a dissociation constant less than about 100 nM.
15 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a substitution of one or both of non-linking phosphate oxygen atoms in a phosphodiester backbone linkage of the engineered guide RNA as provided in Table 2.
16 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a substitution of one or more of linking phosphate oxygen atoms in a phosphodiester backbone linkage of the engineered guide RNA as provided in Table 2.
17 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a modification to a sugar of a nucleotide of the engineered guide RNA as provided in Table 2.
18 . The engineered guide RNA of claim 17 , wherein the modification to the sugar of the nucleotide of the engineered guide RNA comprises at least one locked nucleic acid (LNA).
19 . The engineered guide RNA of claim 17 , wherein the modification to the sugar of the nucleotide of the engineered guide RNA comprises at least one unlocked nucleic acid (UNA).
20 . The engineered guide RNA of claim 17 , wherein the modification to the sugar comprises a modification of a constituent of the sugar, wherein the sugar comprises a ribose sugar.
21 . The engineered guide RNA of claim 20 , wherein the modification to the constituent of the ribose sugar of the nucleotide of the engineered guide RNA comprises a 2′-O-methyl group.
22 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises replacement of a phosphate moiety of the engineered guide RNA with a dephospho linker as provided in Table 2.
23 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a modification of a phosphate backbone of the engineered guide RNA as provided in Table 2.
24 . The engineered guide RNA of claim 23 , wherein the engineered guide RNA comprises a phosphothioate group.
25 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a modification to a base of a nucleotide of the engineered guide RNA.
26 . The engineered guide RNA of claim 25 , wherein the at least one chemical modification comprises an unnatural base of a nucleotide as provided in Table 2.
27 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises a morpholino group, a cyclobutyl group, pyrrolidine group, or peptide nucleic acid (PNA) nucleoside surrogate.
28 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification comprises at least one stereopure nucleic acid as provided in Table 2.
29 . The engineered guide RNA of any one of the preceding claims, wherein the engineered guide RNA comprises from 1 to 100 chemical modifications, each of which can be independently the same or different.
30 . The engineered guide RNA of any one of any one of the preceding claims, wherein the at least one chemical modification does not comprise a naturally occurring chemical modification to a nucleic acid in a eukaryotic cell.
31 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification increases specificity of the engineered guide RNA binding to the target RNA compared to a specificity of an otherwise identical reference polynucleotide without the at least one chemical modification.
32 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification increases resistance to nuclease digestion of the engineered guide RNA compared to resistance to nuclease digestion of an otherwise identical reference polynucleotide without the at least one chemical modification as measured in an in vitro assay.
33 . The engineered guide RNA of any one of the preceding claims, wherein the at least one chemical modification decreases immunogenicity of the engineered guide RNA compared to immunogenicity of an otherwise identical reference polynucleotide without the at least one chemical modification as measured in an in vitro assay.
34 . The engineered guide RNA of any one of preceding claims, wherein the target RNA comprises RAB7A, ABCA4, SERPINA1, SERPINA1 E342K, HEXA, LRRK2, SNCA, APP, Tau, CFTR, ALAS1, ATP7B, ATP7B G1226R, HFE C282Y, LIPA c.894 G>A, PCSK9 start site, or SCNN1A start site, a fragment any of these, or any combination thereof.
35 . The engineered guide RNA of claim 34 , wherein the target RNA comprises SERPINA1 E342K.
36 . The engineered guide RNA of claim 34 , wherein the engineered guide RNA has at least 70%, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% sequence identity with any one of SEQ ID NOs: 1-2.
37 . The engineered guide RNA of claim 34 , wherein the target RNA encodes ABCA4.
38 . The engineered guide RNA of any one of the preceding claims, wherein the RNA editing entity is:
(a) ADAR or APOBEC;
(b) a catalytically active fragment of (a);
(c) fusion polypeptide comprising (a) or (b); or
(d) any combination of (a)-(c).
39 . The engineered guide RNA of claim 38 , wherein the RNA editing entity comprises ADAR, and wherein the ADAR comprises ADAR1, ADAR2, ADAR3, or a combination thereof.
40 . The engineered guide RNA of any one of the preceding claims, wherein the RNA editing entity is endogenous to the cell of the subject.
41 . The engineered guide RNA of any one of the preceding claims, wherein the RNA editing entity is exogenously provided.
42 . The engineered guide RNA of any one of the preceding claims, further comprising a structural loop stabilized scaffold.
43 . The engineered guide RNA of claim 42 , wherein the structural loop stabilized scaffold comprises a stem loop, a junction, a T junction, a clover leaf, a pseudoknot, or any combination thereof.
44 . The engineered guide RNA of claim 42 or 43 , wherein the structural loop stabilized scaffold comprises at least 1, least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 stem loop structures.
45 . The engineered guide RNA of any one of claims 42 - 44 , wherein the structural loop stabilized scaffold comprises a tRNA scaffold.
46 . The engineered guide RNA of any one of the preceding claims, further comprising an RNA editing entity recruiting domain.
47 . The engineered guide RNA of any one of the preceding claims, wherein the engineered guide RNA is conjugated to a targeting moiety.
48 . The engineered guide RNA of claim 47 , wherein the targeting moiety targets a neuronal cell.
49 . The engineered guide RNA of claim 47 , wherein the targeting moiety targets a liver cell.
50 . The engineered guide RNA of claim 47 , wherein the targeting moiety targets a macular cell.
51 . The engineered guide RNA of any one of the preceding claims, wherein the engineered guide RNA is encapsulated in particles.
52 . The engineered guide RNA of claim 51 , wherein the particles comprise nanoparticles.
53 . The engineered guide RNA of claim 51 , wherein the particles comprise liposomes.
54 . A pharmaceutical composition in unit dose form comprising:
(a) the engineered guide RNA of any one of claims 1 - 53 ; and
(b) a pharmaceutically acceptable: excipient, carrier, or diluent.
55 . A method of treating or preventing a disease or a condition in a subject in need thereof, the method comprising: administering to the subject the engineered guide RNA of any one of claims 1 - 53 .
56 . The method of claim 55 , wherein the administering is intrathecally, intraocularly, intravitreally, retinally, intravenously, intramuscularly, intraventricularly, intracerebrally, intracerebellarly, intracerebroventricularly, intraperenchymally, subcutaneously, or a combination thereof.
57 . The method of claim 55 or 56 , wherein the disease or the condition comprises a neurological disease or condition.
58 . The method of claim 57 , wherein the neurological or neurodevelopmental disease or condition comprises Parkinson's disease, Alzheimer's disease, or dementia.
59 . The method of claim 55 or 56 , wherein the disease or the condition comprises a liver disease or condition.
60 . The method of claim 59 , wherein the liver disease or condition comprises liver cirrhosis.
61 . The method of claim 59 , wherein the liver disease or condition comprises alpha-1 antitrypsin deficiency (AAT deficiency).
62 . The method of claim 55 or 56 , wherein the disease or the condition comprises macular degeneration.
63 . The method of claim 62 , wherein the macular degeneration comprises Stargardt's disease.