Patent Application
20230407295
The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML file, created on Aug. 25, 2023, is named 740096_UM9-285_ST26.xml and is 438,226 bytes in size.
This disclosure relates to compositions and methods of protecting oligonucleotides for modified guide RNAs for CRISPR genome editing.
CRISPR RNA-guided genome engineering has revolutionized research into human genetic disease and many other aspects of biology. Numerous CRISPR-based in vivo or ex vivo genome editing therapies are nearing clinical trials. At the heart of this revolution are the microbial effector proteins found in class II CRISPR-Cas systems such as Cas9 (type II) and Cas12a/Cpf1 (type V) (Jinek et al. Science 337, 816-821 (2012); Gasiunas et al. PNAS 109, E2579-E2586 (2012); Zetsche et al. Cell 163, 759-771 (2015)).
The most widely used genome editing tool is the type II-A Cas9 from Streptococcus pyogenes strain SF370 (SpCas9) (Jinek et al, supra). Cas9 forms a ribonucleoprotein (RNP) complex with a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) for efficient DNA cleavage both in bacteria and eukaryotes (
For mammalian applications, Cas9 and its guide RNAs can be expressed from DNA (e.g. a viral vector), RNA (e.g. Cas9 mRNA plus guide RNAs in a lipid nanoparticle), or introduced as a ribonucleoprotein (RNP).
Unmodified single-stranded RNAs are extremely vulnerable to nuclease degradation and are rapidly degraded in cells and biological fluids such as serum. Delivering naked and unformulated crRNAs is similar to the scenario of delivering other therapeutic oligonucleotides such as antisense oligonucleotide (ASO) and short interfering RNA (siRNA), where chemical modifications are the key to in vivo efficacy. However, while complete chemical modifications significantly enhanced crRNA stability (
The present disclosure provides protecting oligonucleotides for CRISPR genome editing. In certain embodiments, the protecting oligonucleotides of the disclosure are heavily or fully chemically modified. The protecting oligonucleotides of the disclosure may confer several advantages in vivo or ex vivo, including crRNA stability, improved potency, and/or reduced off-target effects. Furthermore, in certain embodiments, the protecting oligonucleotides of the disclosure have reduced immunogenicity, e.g., a reduced ability to induce innate immune responses.
In certain aspects, the disclosure provides a protecting oligonucleotide comprising: (a) a sequence that is complementary to a crRNA; and (b) at least one chemically modified nucleotide, wherein the protecting oligonucleotide is capable of binding the crRNA, and wherein the protecting oligonucleotide confers nuclease resistance to the crRNA when bound.
In an embodiment, the modified nucleotides each independently comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
In an embodiment, each modification of the ribose group is independently selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-NH2 (2′-amino), 4′-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2′-(S)-constrained ethyl (S-cEt), a constrained MOE, and a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC).
In an embodiment, at least 80% of the ribose groups are chemically modified. In an embodiment, at least 90% of the ribose groups are chemically modified. In an embodiment, 100% of the ribose groups are chemically modified.
In an embodiment, crRNA comprises a guide sequence portion capable of hybridizing to a target polynucleotide sequence and a repeat sequence portion.
In an embodiment, the protecting oligonucleotide binds to the crRNA to forma duplex.
In an embodiment, the duplex has a melting temperature (Tm) of greater than 37° C. over the full length of the duplex.
In an embodiment, the duplex has a melting temperature (Tm) of less than 37° C. over the region of complementarity comprising the guide sequence portion.
In an embodiment, the binding of a tracrRNA to the guide sequence portion of the crRNA dissociates the protecting oligonucleotide from the crRNA.
In an embodiment, the protecting oligonucleotide further comprises at least one moiety conjugated to the protecting oligonucleotide. In an embodiment, the at least one moiety is conjugated to at least one of the 5′ end of the protecting oligonucleotide and/or the 3′ end of the protecting oligonucleotide.
In an embodiment, the at least one moiety increases cellular uptake of the guide RNA. In an embodiment, the at least one moiety promotes specific tissue distribution of the guide RNA.
In an embodiment, the at least one moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides analogs, nucleoside analogs, endocannabinoids, vitamins, receptor ligands, peptides, aptamers, and alkyl chains.
In an embodiment, the at least one moiety is selected from the group consisting of cholesterol, docosahexaenoic acid (DHA), docosanoic acid (DCA), lithocholic acid (LA), GalNAc, amphiphilic block copolymer (ABC), hydrophilic block copolymer (HBC), poloxamer, Cy5, and Cy3.
In an embodiment, the at least one moiety is conjugated to the guide RNA via a linker. In an embodiment, the linker is selected from the group consisting of an ethylene glycol chain, an alkyl chain, a polypeptide, a polysaccharide, and a block copolymer.
In an embodiment, the at least one moiety is a modified lipid. In an embodiment, the modified lipid is a branched lipid.
In an embodiment, the modified lipid is a branched lipid of Formula I, Formula I: X-MC(═Y)M-Z-[L-MC(═Y)M-R]n, where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to a chemically modified guide RNA, L is an optional linker moiety, and each R is independently a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group. In an embodiment, the modified lipid is a headgroup-modified lipid.
In an embodiment, the modified lipid is a headgroup-modified lipid of Formula II, Formula II: X-MC(═Y)M-Z-[L-MC(═Y)M-R]n-L-K-J, where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, N-alkyl, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to chemically modified guide RNA, each L is independently an optional linker moiety, and R is a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group, K is a phosphate, sulfate, or amide and J is an aminoalkane or quaternary aminoalkane group.
In one aspect, the disclosure provides a double stranded oligonucleotide comprising: (a) a crRNA comprising (i) a guide sequence portion capable of hybridizing to a target polynucleotide sequence, and (ii) a repeat sequence portion; and (b) a protecting oligonucleotide that is complementary to the crRNA, wherein the crRNA comprises at least 50% modified nucleotides, and wherein the protecting oligonucleotide comprises at least one modified nucleotide.
In an embodiment, the modified nucleotide comprises a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
In an embodiment, each modification of the ribose group is independently selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-NH2 (2′-amino), 4′-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2′-(S)-constrained ethyl (S-cEt), a constrained MOE, or a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC).
In an embodiment, at least 80% of the ribose groups are chemically modified. In an embodiment, at least 90% of the ribose groups are chemically modified. In an embodiment, 100% of the ribose groups are chemically modified.
In an embodiment, the double stranded oligonucleotide comprises a melting temperature (Tm) of greater than 37° C. over the full length of the double stranded oligonucleotide.
In an embodiment, the double stranded oligonucleotide comprises a melting temperature (Tm) of less than 37° C. for the region of the double stranded oligonucleotide comprising the crRNA guide sequence portion.
In an embodiment, the binding of a tracrRNA to the repeat sequence portion of the crRNA dissociates the protecting oligonucleotide from the crRNA.
In an embodiment, the protecting oligonucleotide binds to a region of the crRNA that is not fully chemically modified.
In an embodiment, the protecting oligonucleotide binds to a region of the crRNA that comprises less than 100% modified ribose groups.
In an embodiment, the double stranded oligonucleotide further comprises at least one moiety conjugated to the protecting oligonucleotide and/or crRNA. In an embodiment, the at least one moiety is conjugated to at least one of the 5′ end and/or the 3′ end of the protecting oligonucleotide and/or crRNA.
In an embodiment, the at least one moiety increases cellular uptake of the guide RNA. In an embodiment, the at least one moiety promotes specific tissue distribution of the guide RNA.
In an embodiment, the at least one moiety is selected from the group consisting of fatty acids, steroids, secosteroids, lipids, gangliosides analogs, nucleoside analogs, endocannabinoids, vitamins, receptor ligands, peptides, aptamers, and alkyl chains.
In an embodiment, the at least one moiety is selected from the group consisting of cholesterol, docosahexaenoic acid (DHA), docosanoic acid (DCA), lithocholic acid (LA), GalNAc, amphiphilic block copolymer (ABC), hydrophilic block copolymer (HBC), poloxamer, Cy5, and Cy3.
In an embodiment, the at least one moiety is conjugated to the guide RNA via a linker. In an embodiment, the linker is selected from the group consisting of an ethylene glycol chain, an alkyl chain, a polypeptide, a polysaccharide, and a block copolymer.
In an embodiment, the at least one moiety is a modified lipid. In an embodiment, the modified lipid is a branched lipid.
In an embodiment, the modified lipid is a branched lipid of Formula I, Formula I: X-MC(═Y)M-Z-[L-MC(═Y)M-R]n, where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to a chemically modified guide RNA, L is an optional linker moiety, and each R is independently a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group. In an embodiment, the modified lipid is a headgroup-modified lipid.
In an embodiment, the modified lipid is a headgroup-modified lipid of Formula II, Formula II: X-MC(═Y)M-Z-[L-MC(═Y)M-R]n-L-K-J, where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, N-alkyl, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to chemically modified guide RNA, each L is independently an optional linker moiety, and R is a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group, K is a phosphate, sulfate, or amide and J is an aminoalkane or quaternary aminoalkane group.
In an embodiment, the guide RNA binds to a Cas9 nuclease selected from the group consisting of S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9).
In an embodiment, the Cas9 is a variant Cas9 with altered activity.
In an embodiment, the variant Cas9 is selected from the group consisting of a Cas9 nickase (nCas9), a catalytically dead Cas9 (dCas9), a hyper accurate Cas9 (HypaCas9), a high fidelity Cas9 (Cas9-HF), an enhanced specificity Cas9 (eCas9), and an expanded PAM Cas9 (xCas9).
In an embodiment, the Cas9 off-target activity is reduced relative to an unmodified guide RNA.
In an embodiment, the Cas9 on-target activity is increased relative to an unmodified guide RNA.
In one aspect, the disclosure provides a method of editing a target region of a genome in a cell altering expression of a target gene in a cell, comprising administering to said cell a genome editing system comprising: a protecting oligonucleotide of any of the embodiments recited above; one or more crRNAs of any of the embodiments recited above; one or more tracrRNAs; and an RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease.
In an embodiment, the target gene is in a cell in an organism.
In an embodiment, expression of the target gene is knocked out or knocked down.
In an embodiment, the RNA-guided nuclease or a polynucleotide encoding an RNA-guided nuclease and the one or more tracrRNAs are administered to the cell before the one or more crRNAs or the protecting oligonucleotide.
In an embodiment, the crRNA comprises a crRNA portion of any of the preceding embodiments modification pattern consisting of:
wherein rN=RNA, mN=2′-O-methyl RNA, fN=2′-fluoro RNA, N # N=phosphorothioate linkage, and N=any nucleotide.
In an embodiment, the protecting oligonucleotide of any of the preceding embodiments comprises a modification pattern consisting of:
wherein rN=RNA, mN=2′-O-methyl RNA, N # N=phosphorothioate linkage, and N=any nucleotide.
In certain embodiments, the protecting oligonucleotide of any one of the preceding embodiments is a protecting oligonucleotide modification pattern selected from any of RC01-10 of Table 1.
In certain embodiments, the crRNA of any one of the preceding embodiments is a crRNA modification pattern selected from any of crRNA 1-134 of Table 2.
In certain embodiments, protecting oligonucleotide and/or crRNA of any one of the preceding embodiments further comprises one or more additional modified nucleotides, each independently selected from a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
In certain embodiments, each modification of the ribose group is independently selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 4′-thio, a bicyclic nucleotide, a locked nucleic acid (LNA), a 2′-(S)-constrained ethyl (S-cEt), a constrained MOE, and a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC).
In certain embodiments, each modification of the phosphate group is independently selected from the group consisting of a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.
In certain embodiments, each modification of the nucleobase group is independently selected from the group consisting of 2-thiouridine, 4-thiouridine, N6 -methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, and halogenated aromatic groups.
The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments taken in conjunction with the accompanying drawings. The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Provided herewith are protecting oligonucleotides, including heavily or fully chemically protecting oligonucleotides. In certain embodiments, protecting oligonucleotides and crRNAs with 5′ and/or 3′ conjugated moieties are provided. Methods of using the protecting oligonucleotides and crRNAs of the disclosure for genome editing with a CRISPR nuclease and kits for performing the same are also provided.
Unless otherwise defined herein, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques provided herein are usually performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications unless otherwise specified, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art, unless otherwise specified. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
Unless otherwise defined herein, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.
So that the disclosure may be more readily understood, certain terms are first defined.
As used herein, the term “protecting oligonucleotide” refers to an oligonucleotide that comprises complementarity to a crRNA and confers increased nuclease resistance to the crRNA when bound relative to a crRNA that is not bound by a protecting oligonucleotide.
As used herein, the term “guide RNA” or “gRNA” refer to any nucleic acid that promotes the specific association (or “targeting”) of an RNA-guided nuclease such as a Cas9 to a target sequence (e.g., a genomic or episomal sequence) in a cell.
As used herein, a “modular” or “dual RNA” guide comprises more than one, and typically two, separate RNA molecules, such as a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), which are usually associated with one another, for example by duplexing. gRNAs and their component parts are described throughout the literature (see, e.g., Briner et al. Mol. Cell, 56(2), 333-339 (2014), which is incorporated by reference).
As used herein, a “unimolecular gRNA,” “chimeric gRNA,” or “single guide RNA (sgRNA)” comprises a single RNA molecule. The sgRNA may be a crRNA and tracrRNA linked together. For example, the 3′ end of the crRNA may be linked to the 5′ end of the tracrRNA. A crRNA and a tracrRNA may be joined into a single unimolecular or chimeric gRNA, for example, by means of a four nucleotide (e.g., GAAA) “tetraloop” or “linker” sequence bridging complementary regions of the crRNA (at its 3′ end) and the tracrRNA (at its 5′ end).
As used herein, a “repeat” sequence or region is a nucleotide sequence at or near the 3′ end of the crRNA which is complementary to an anti-repeat sequence of a tracrRNA.
As used herein, an “anti-repeat” sequence or region is a nucleotide sequence at or near the 5′ end of the tracrRNA which is complementary to the repeat sequence of a crRNA.
Additional details regarding guide RNA structure and function, including the gRNA/Cas9 complex for genome editing may be found in, at least, Mali et al. Science, 339(6121), 823-826 (2013); Jiang et al. Nat. Biotechnol. 31(3). 233-239 (2013); and Jinek et al. Science, 337(6096), 816-821 (2012); which are incorporated by reference herein.
As used herein, a “guide sequence” or “targeting sequence” refers to the nucleotide sequence of a gRNA, whether unimolecular or modular, that is fully or partially complementary to a target domain or target polynucleotide within a DNA sequence in the genome of a cell where editing is desired. Guide sequences are typically 10-30 nucleotides in length, preferably 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and are at or near the 5′ terminus of a Cas9 gRNA.
As used herein, a “target domain” or “target polynucleotide sequence” is the DNA sequence in a genome of a cell that is complementary to the guide sequence of the gRNA.
In addition to the targeting domains, gRNAs typically include a plurality of domains that influence the formation or activity of gRNA/Cas9 complexes. For example, as mentioned above, the duplexed structure formed by first and secondary complementarity domains of a gRNA (also referred to as a repeat: anti-repeat duplex) interacts with the recognition (REC) lobe of Cas9 and may mediate the formation of Cas9/gRNA complexes (Nishimasu et al. Cell 156: 935-949 (2014); Nishimasu et al. Cell 162(2), 1113-1126 (2015), both incorporated by reference herein). It should be noted that the first and/or second complementarity domains can contain one or more poly-A tracts, which can be recognized by RNA polymerases as a termination signal. The sequence of the first and second complementarity domains are, therefore, optionally modified to eliminate these tracts and promote the complete in vitro transcription of gRNAs, for example through the use of A-G swaps as described in Briner 2014, or A-U swaps. These and other similar modifications to the first and second complementarity domains are within the scope of the present disclosure.
Along with the first and second complementarity domains, Cas9 gRNAs typically include two or more additional duplexed regions that are necessary for nuclease activity in vivo but not necessarily in vitro (Nishimasu 2015, supra). A first stem-loop near the 3′ portion of the second complementarity domain is referred to variously as the “proximal domain,” “stem loop 1” (Nishimasu 2014, supra; Nishimasu 2015, supra) and the “nexus” (Briner 2014, supra). One or more additional stem loop structures are generally present near the 3′ end of the gRNA, with the number varying by species: S. pyogenes gRNAs typically include two 3′ stem loops (for a total of four stem loop structures including the repeat: anti-repeat duplex), while S. aureus and other species have only one (for a total of three). A description of conserved stem loop structures (and gRNA structures more generally) organized by species is provided in Briner 2014, which is incorporated herein by reference. Additional details regarding guide RNAs generally may be found in WO2018026976A1, which is incorporated herein by reference.
The inventors have surprisingly discovered that the use of protecting oligonucleotides confers increased nuclease resistance to crRNAs. The protecting oligonucleotides described herein comprise complementarity to a crRNA. The protecting oligonucleotides confer nuclease resistance to the crRNAs when bound. In preferred embodiments, the protecting oligonucleotides described herein bind to a region of the crRNA the contains at least one unmodified ribose group. In certain embodiments, the protecting oligonucleotide binds to a region of the crRNA the contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 unmodified ribose groups.
In certain embodiments, the protecting oligonucleotide is about 5 to about 20 nucleotides in length (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length). In certain embodiments, the protecting oligonucleotide is 11, 12, 13, 14, 15, or 16 nucleotides in length. The length of the protecting oligonucleotide is shorter than the crRNA to which the protecting oligonucleotide comprises complementarity to (i.e., the protecting oligonucleotide comprises complementarity to a region of the crRNA, wherein said region is not the full length of the crRNA).
In certain embodiments, the protecting oligonucleotide binds to the crRNA to form a duplex, optionally wherein the protecting oligonucleotide binds to a region of the crRNA that is not fully chemically modified.
In certain embodiments, the duplex has a melting temperature (Tm) of greater than 37° C. over the full length of the duplex.
In certain embodiments, the duplex has a melting temperature (Tm) of less than 37° C. over the region of complementarity comprising the guide sequence portion.
In certain embodiments, the binding of a tracrRNA to the guide sequence portion of the crRNA dissociates the protecting oligonucleotide from the crRNA.
Exemplary protecting oligonucleotide sequences are recited below, wherein “N” corresponds to any nucleotide:
The protecting oligonucleotides and guide RNAs (i.e., crRNAs and tracrRNAs) of the disclosure possess improved in vivo stability, improved genome editing efficacy, and/or reduced immunotoxicity relative to unmodified or minimally modified protecting oligonucleotides and guide RNAs.
The protecting oligonucleotides and guide RNAs of the disclosure contain one or more modified nucleotides comprising a modification in a ribose group, a phosphate group, a nucleobase, or a combination thereof.
Chemical modifications to the ribose group may include, but are not limited to, 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxy ethyl) (MOE), 2′-NH2(2′-amino), 4′-thio, 2′-O-Allyl, 2′-O-Ethylamine, 2′-O-Cyanoethyl, 2′-O-Acetalester, or a bicyclic nucleotide, such as locked nucleic acid (LNA), 2′-(S)-constrained ethyl (S-cEt), constrained MOE, or 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNANC).
The term “4′-thio” as used herein corresponds to a ribose group modification where the sugar ring oxygen of the ribose is replaced with a sulfur.
Chemical modifications to the phosphate group may include, but are not limited to, a phosphorothioate, phosphonoacetate (PACE), thiophosphonoacetate (thioPACE), amide, triazole, phosphonate, or phosphotriester modification.
In an embodiment, the crRNA portion of the chemically modified guide RNA comprises between 1 and 20 phosphorothioate modifications (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 phosphorothioate modifications). In an embodiment, the crRNA portion of the chemically modified guide RNA comprises between 1 and 20 phosphorothioate modifications (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 phosphorothioate modifications) and comprises at least about 50% activity relative to a guide RNA that does not comprise phosphorothioate modifications (e.g., 50% activity, 60% activity, 70% activity, 80% activity, 90% activity, 95% activity, or 100% activity, relative to a guide RNA that does not comprise phosphorothioate modifications).
Chemical modifications to the nucleobase may include, but are not limited to, 2-thiouridine, 4-thiouridine, N6-methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, or halogenated aromatic groups.
The chemically modified guide RNAs may have one or more chemical modifications in the crRNA portion and/or the tracrRNA portion for a modular or dual RNA guide. The chemically modified guide RNAs may also have one or more chemical modifications in the single guide RNA for the unimolecular guide RNA.
The protecting oligonucleotides and guide RNAs may comprise at least about 50% to at least about 100% chemically modified nucleotides, at least about 60% to at least about 100% chemically modified nucleotides, at least about 70% to at least about 100% chemically modified nucleotides, at least about 80% to at least about 100% chemically modified nucleotides, at least about 90% to at least about 100% chemically modified nucleotides, and at least about 95% to at least about 100% chemically modified nucleotides.
The protecting oligonucleotides and guide RNAs may comprise at least about 50% chemically modified nucleotides, at least about 60% chemically modified nucleotides, at least about 70% chemically modified nucleotides, at least about 80% chemically modified nucleotides, at least about 90% chemically modified nucleotides, at least about 95% chemically modified nucleotides, at least about 99% chemically modified, or 100% (fully) chemically modified nucleotides.
The protecting oligonucleotides and guide RNAs may comprise at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% chemically modified nucleotides.
Protecting oligonucleotides and guide RNAs that comprise at least about 80% chemically modified nucleotides to at least about 99% chemically modified nucleotides are considered “heavily” modified, as used herein.
Protecting oligonucleotides and guide RNAs that comprise 100% chemically modified nucleotides are considered “fully” modified, as used herein.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified ribose group at about 50% of the nucleotides to about 100% of the nucleotides, at about 60% of the nucleotides to about 100% of the nucleotides, at about 70% of the nucleotides to about 100% of the nucleotides, at about 80% of the nucleotides to about 100% of the nucleotides, at about 90% of the nucleotides to about 100% of the nucleotides, and at about 95% of the nucleotides to about 100% of the nucleotides
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified ribose group at about 50% of the nucleotides, at about 60% of the nucleotides, at about 70% of the nucleotides, at about 80% of the nucleotides, at about 90% of the nucleotides, at about 95% of the nucleotides, at about 99% of the nucleotides, or at 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified ribose group at about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides.
Protecting oligonucleotides and guide RNAs that have at least about 80% of the ribose groups chemically modified to at least about 99% of the ribose groups chemically modified are considered “heavily” modified, as used herein.
Protecting oligonucleotides and guide RNAs that have 100% of the ribose groups chemically modified are considered “fully” modified, as used herein.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified phosphate group at about 50% of the nucleotides to about 100% of the nucleotides, at about 60% of the nucleotides to about 100% of the nucleotides, at about 70% of the nucleotides to about 100% of the nucleotides, at about 80% of the nucleotides to about 100% of the nucleotides, at about 90% of the nucleotides to about 100% of the nucleotides, and at about 95% of the nucleotides to about 100% of the nucleotides
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified phosphate group at about 50% of the nucleotides, at about 60% of the nucleotides, at about 70% of the nucleotides, at about 80% of the nucleotides, at about 90% of the nucleotides, at about 95% of the nucleotides, at about 99% of the nucleotides, or at 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified phosphate group at about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of nucleotides.
Protecting oligonucleotides and guide RNAs that have at least about 80% of the phosphate groups chemically modified to at least about 99% of the phosphate groups chemically modified are considered “heavily” modified, as used herein.
Protecting oligonucleotides and guide RNAs that have 100% of the phosphate groups chemically modified are considered “fully” modified, as used herein.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified nucleobase at about 50% of the nucleotides to about 100% of the nucleotides, at about 60% of the nucleotides to about 100% of the nucleotides, at about 70% of the nucleotides to about 100% of the nucleotides, at about 80% of the nucleotides to about 100% of the nucleotides, at about 90% of the nucleotides to about 100% of the nucleotides, and at about 95% of the nucleotides to about 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified nucleobase at about 50% of the nucleotides, at about 60% of the nucleotides, at about 70% of the nucleotides, at about 80% of the nucleotides, at about 90% of the nucleotides, at about 95% of the nucleotides, at about 99% of the nucleotides, or at 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise a chemically modified nucleobase at about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides.
Protecting oligonucleotides and guide RNAs that have at least about 80% of the nucleobases chemically modified to at least about 99% of the nucleobases chemically modified are considered “heavily” modified, as used herein.
Protecting oligonucleotides and guide RNAs that have 100% of the nucleobases chemically modified are considered “fully” modified, as used herein.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise any combination of chemically modified ribose groups, chemically modified phosphate groups, and chemically modified nucleobases at about 50% of the nucleotides to about 100% of the nucleotides, at about 60% of the nucleotides to about 100% of the nucleotides, at about 70% of the nucleotides to about 100% of the nucleotides, at about 80% of the nucleotides to about 100% of the nucleotides, at about 90% of the nucleotides to about 100% of the nucleotides, and at about 95% of the nucleotides to about 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise any combination of chemically modified ribose groups, chemically modified phosphate groups, and chemically modified nucleobases at about 50% of the nucleotides, at about 60% of the nucleotides, at about 70% of the nucleotides, at about 80% of the nucleotides, at about 90% of the nucleotides, at about 95% of the nucleotides, at about 99% of the nucleotides, or at 100% of the nucleotides.
In certain exemplary embodiments, the protecting oligonucleotides and guide RNAs may comprise any combination of chemically modified ribose groups, chemically modified phosphate groups, and chemically modified nucleobases at about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the nucleotides.
Protecting oligonucleotides and guide RNAs that have at least about 80% of any combination of the ribose groups, the phosphate groups, and the nucleobases chemically modified to at least about 99% of the nucleobases chemically modified are considered “heavily” modified, as used herein.
Protecting oligonucleotides and guide RNAs that have 100% of any combination of the ribose groups, the phosphate groups, and the nucleobases chemically modified are considered “fully” modified, as used herein.
The heavily and fully chemically modified protecting oligonucleotides and guide RNAs of the disclosure possess several advantages over the minimally modified protecting oligonucleotides and guide RNAs in the art. Heavily and fully chemically modified protecting oligonucleotides and guide RNAs are expected to ease chemical synthesis, further enhance in vivo stability, and provide a scaffold for terminally appended chemical functionalities that facilitate delivery and efficacy during clinical applications to genome editing.
The chemical modification pattern used in the guide RNA is such that activity of the guide RNA is maintained when paired with an RNA-guided DNA endonuclease, e.g., Cas9.
In an embodiment, the chemically modified guide RNAs of the disclosure comprise at least about 50% activity relative to an unmodified guide RNA (e.g., 50% activity, 60% activity, 70% activity, 80% activity, 90% activity, 95% activity, or 100% activity, relative to an unmodified guide RNA).
The activity of a guide RNA can be readily determined by any means known in the art. In an embodiment, % activity is measured with the traffic light reporter (TLR) Multi-Cas Variant 1 system (TLR-MCV1), described below. The TLR-MCV1 system will provide a % fluorescent cells which is a measure of % activity.
Exemplary chemical modification patterns are described in Table 1 and Table 2 below.
It will be understood to those of skill in the art that the base sequence of the first 20 nucleotides of the exemplary crRNAs recited in Table 2 above are directed to a specific target. This 20-nucleotide base sequence may be changed based on the target nucleic acid, however the chemical modifications remain the same. An exemplary unmodified crRNA sequence, from 5′ to 3′, is
where “N” corresponds to any nucleotide (e.g., A, U, G, or C). An exemplary unmodified tracrRNA sequence, from 5′ to 3′, is
It will be further understood to those of skill in the art that the guide sequence may be 10-30 nucleotides in length, preferably 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20, 21, 22, 23 or 24 nucleotides in length), and is at or near the 5′ terminus of a Cas9 gRNA.
Chemically modified guide RNAs, including chemically modified crRNAs and tracrRNAs, are described in more detail in US20210363518 and US20210388348, each of which is incorporated herein by reference.
The protecting oligonucleotides described herein may be designed to bind to any region of the crRNA modification patterns that contain at least one unmodified ribose group, thereby conferring nuclease resistance to the crRNA.
A crRNA and a tracrRNA hybridize together by forming a duplex between the repeat region of the crRNA and the anti-repeat region of the tracrRNA. In certain embodiments, modular, or dual RNA, guide RNAs are provided with modifications in the repeat region and the anti-repeat region to enhance the affinity between the two regions and form a stronger duplex.
The high-affinity interaction may be enhanced by increasing the GC nucleotide content in the duplex formed by the repeat regions and the anti-repeat region. Nucleotide modifications, such as 2′-Fluoro and 2′-O-Methyl modifications, may also be introduced, which increase the melting temperature (Tm) of the duplex. Further modifications include the use of orthogonal and non-naturally occurring nucleotides. The various repeat region/anti-repeat region modifications described herein enhance the stability of the duplex, helping to prevent the crRNA and tracrRNA from folding into sub-optimal structures and therefore promoting higher genome editing efficacy.
The protecting oligonucleotides and/or crRNAs of the disclosure may be modified with terminally conjugated moieties. As used herein, a “terminally conjugated moiety” or “moiety” refers to a compound which may be linked or attached to the 5′ and/or 3′ end of the crRNA and/protecting oligonucleotide. Terminally conjugated moieties can provide increased stability, increased ability to penetrate cell membranes, increase cellular uptake, increase circulation time in vivo, act as a cell-specific directing reagent, and/or provide a means to monitor cellular or tissue-specific uptake.
In certain embodiments, the terminally conjugated moiety is conjugated to the 5′ end of the crRNA. In certain embodiments, the terminally conjugated moiety is conjugated to the 3′ end of the crRNA. In certain embodiments, the terminally conjugated moiety is conjugated to the 5′ end of the protecting oligonucleotide. In certain embodiments, the terminally conjugated moiety is conjugated to the 3′ end of the protecting oligonucleotide.
In certain exemplary embodiments, a terminally conjugated moiety includes, but is not limited to, fatty acid, steroid, secosteroid, lipid, ganglioside analog, nucleoside analogs, endocannabinoid, vitamin, receptor ligand, peptide, aptamer, alkyl chain, fluorophore, antibody, nuclear localization signal, and the like.
In certain exemplary embodiments, a terminally conjugated moiety includes, but is not limited to, cholesterol, cholesterol-triethylene glycol (TEGChol), docosahexaenoic acid (DHA), docosanoic acid (DCA), lithocholic acid (LA), GalNAc, amphiphilic block copolymer (ABC), hydrophilic block copolymer (HBC), poloxamer, Cy5, Cy3, and the like.
In certain exemplary embodiments, the at least one terminally conjugated moiety is a modified lipid, including a branched lipid (such as the structure shown in Formula I) or a headgroup-modified lipid (such as the structure shown in Formula II).
X-MC(═Y)M-Z-[L-MC(═Y)M-R]n Formula I
where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to the rest of the structure, L is an optional linker moiety, and each R is independently a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group.
X-MC(═Y)M-Z-[L-MC(═Y)M-R]n-L-K-J Formula II
where X is a moiety that links the lipid to the guide RNA, each Y is independently oxygen or sulfur, each M is independently CH2, NH, N-alkyl, O or S, Z is a branching group which allows two or three (“n”) chains to be joined to the rest of the structure, each L is independently an optional linker moiety, and R is a saturated, monounsaturated or polyunsaturated linear or branched moiety from 2 to 30 atoms in length, a sterol, or other hydrophobic group, K is a phosphate, sulfate, or amide and J is an aminoalkane or quaternary aminoalkane group.
The moieties may be attached to the terminal nucleotides of the guide RNA via a linker. Exemplary linkers include, but are not limited to, an ethylene glycol chain, an alkyl chain, a polypeptide, a polysaccharide, a block copolymer, and the like.
In certain embodiments, the moiety is conjugated to the 5′ end and/or 3′ end of any one of RC01 to RC10 (i.e., RC01, RC02, RC03, RC04, RC05, RC06, RC07, RC08, RC09, or RC10).
As described herein, the chemically modified guide RNAs of the disclosure may be constructed as single guide RNAs (sgRNAs) by linking the 3′ end of a crRNA to the 5′ end of a tracrRNA. The linker may be an oligonucleotide loop, including a chemically modified oligonucleotide loop. In certain embodiments, the oligonucleotide loop comprises a GAAA tetraloop. The linker may be a non-nucleotide chemical linker, including, but not limited to, ethylene glycol oligomers (see, e.g., Pils et al. Nucleic Acids Res. 28(9): 1859-1863 (2000)).
RNA-guided nucleases according to the present disclosure include, without limitation, naturally-occurring Type II CRISPR nucleases such as Cas9, as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present disclosure include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9). In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (e.g., complex with) a gRNA; and (b) together with the gRNA, associate with, and optionally cleave or modify, a target region of a DNA that includes (i) a sequence complementary to the targeting domain of the gRNA and, optionally, (ii) an additional sequence referred to as a “protospacer adjacent motif,” or “PAM,” which is described in greater detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in broad terms, by their PAM specificity and cleavage activity, even though variations may exist between individual RNA-guided nucleases that share the same PAM specificity or cleavage activity. Skilled artisans will appreciate that some aspects of the present disclosure relate to systems, methods and compositions that can be implemented using any suitable RNA-guided nuclease having a certain PAM specificity and/or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (e.g., Cas9 vs. Cpf1), species (e.g., S. pyogenes vs. S. aureu ) or variation (e.g., full-length vs. truncated or split; naturally-occurring PAM specificity vs. engineered PAM specificity).
Various RNA-guided nucleases may require different sequential relationships between PAMs and protospacers. In general, Cas9s recognize PAM sequences that are 5′ of the protospacer as visualized relative to the top or complementary strand.
In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases generally recognize specific PAM sequences. S. aureus Cas9, for example, recognizes a PAM sequence of NNGRRT, wherein the N sequences are immediately 3′ of the region recognized by the gRNA targeting domain. S. pyogenes Cas9 recognizes NGG PAM sequences. It should also be noted that engineered RNA-guided nucleases can have PAM specificities that differ from the PAM specificities of similar nucleases (such as the naturally occurring variant from which an RNA-guided nuclease is derived, or the naturally occurring variant having the greatest amino acid sequence homology to an engineered RNA-guided nuclease). Modified Cas9s that recognize alternate PAM sequences are described below.
RNA-guided nucleases are also characterized by their DNA cleavage activity: naturally-occurring RNA-guided nucleases typically form DSBs in target nucleic acids, but engineered variants have been produced that generate only SSBs (discussed above; see also Ran 2013, incorporated by reference herein), or that do not cut at all.
The RNA-guided nuclease Cas9 may be a variant of Cas9 with altered activity. Exemplary variant Cas9 nucleases include, but are not limited to, a Cas9 nickase (nCas9), a catalytically dead Cas9 (dCas9), a hyper accurate Cas9 (HypaCas9) (Chen et al. Nature, 550(7676), 407-410 (2017)), a high fidelity Cas9 (Cas9-HF) (Kleinstiver et al. Nature 529(7587), 490-495 (2016)), an enhanced specificity Cas9 (eCas9) (Slaymaker et al. Science 351(6268), 84-88 (2016)), and an expanded PAM Cas9 (xCas9) (Hu et al. Nature doi: 10.1038/nature26155 (2018)).
The RNA-guided nucleases may be combined with the chemically modified guide RNAs of the present disclosure to form a genome-editing system. The RNA-guided nucleases may be combined with the chemically modified guide RNAs to form an RNP complex that may be delivered to a cell where genome-editing is desired. The RNA-guided nucleases may be expressed in a cell where genome-editing is desired with the chemically modified guide RNAs delivered separately. For example, the RNA-guided nucleases may be expressed from a polynucleotide such as a vector or a synthetic mRNA. The vector may be a viral vector, including, be not limited to, an adeno-associated virus (AAV) vector or a lentivirus (LV) vector.
It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.
To test the compatibility between a certain chemical modification of the crRNA and SpyCas9/base editor (BE) activity, we established a scenario in which cell lines stably express either SpyCas9 nuclease or base editor, tracrRNA, and a fluorescent reporter gene (
The protecting oligonucleotides used in the Examples are recited below in Table 3. Table 4 below recites crRNAs used in the Examples.
Inspired by the design of short DNA/RNA heteroduplex oligonucleotide (HDO) (Nishina et al., Nature Communications, 2015) and by the well-established observation that double-stranded RNAs are more resistant to nuclease degradation than single-stranded RNAs (Lam et al., Molecular Therapy Nucleic Acids, 2015), we sought to enhance the crRNA stability by designing short, fully chemically stabilized oligos that can protect the unmodified regions of C20 (
Protecting Oligos Enhance the Potency of crRNA
To test whether protecting oligos that enhanced C20 stability can also increase its potency, we electroporated the C20-protecting oligo complexes at non-saturating dosages. We found that protecting oligos significantly enhanced the potency of C20 (
Addition of conjugations such as N-acetylgalactosamine (GalNac) and Cholesterol (TegChol) to therapeutic oligonucleotide is critical for achieving potent and targeted in vivo delivery. To test whether conjugations interfere with the crRNA activity, we synthesized both crRNA and protecting oligos with either GalNac or Cholesterol conjugation and tested the activity via electroporation in the stable cell line. We found that while directly conjugating of Cholesterol to C20 significantly compromised the activity, C20 with Cholesterol-conjugated protecting oligos did not show reduced activity (
To directly visualize enhanced cellular uptake of C20 by conjugated protecting oligos in vivo, we delivered Cy3-labeled C20 with and without cholesterol- or DHA-conjugated protecting oligos in adult mouse brains by intrastriatal (IS) injection. We found that compared to unconjugated protecting oligos, both cholesterol- and DHA-conjugated protecting oligos significantly enhanced the in vivo distribution and cellular uptake of C20 (
To test if the C20-protecting oligo complex can be taken up into cells passively (simply by adding it to the media, without electroporation or transfection), escape from endosomes, and support editing, we incubated the stable cell line shown in
We tested whether the C20-protecting oligo complex can support genome editing by AAV co-delivery in vivo. We chose to test in a double transgenic reporter mouse model, Cas9/mTmG+/+. This mouse model was generated by crossing the homozygous Cas9+/+ mice68 with the homozygous mTmG+/+ reporter mice (Muzumdar et al. Genesis. 45: 593-605. 2007). The Cas9+/+ mouse model constitutively expresses SpyCas9, and the only transgene delivered from an AAV vector is the tracrRNA, which simplified the experimental process. The mTmG+/+ reporter consists of loxP sites on either side of a membrane-targeted tdTomato (mT) cassette, which constitutively expresses red fluorescent, and a downstream membrane-targeted EGFP (mG) that cannot be expressed until the mT cassette was deleted (
First, we injected scAAV9 that expresses tracrRNA into adult Cas9/mTmG+/+ mouse brain by IS injection. Two weeks later, we performed IS injection again to deliver naked C20, which targets the two loxP sites to generate block deletion of the mT cassette and allow the mG expression. We then detected GFP expression by performing Immunohistochemistry (IHC) on formalin-fixed mouse brain sections using anti-EGFP antibodies.
We chose to test both cholesterol and DHA conjugates, and both cleavable and stable linkers for the protecting oligos. We also included C20 with protecting oligos without any conjugation and C20 without protecting oligos as controls.
By IHC staining using an anti-EGFP antibody, we found that C20 without protecting oligos did not generate any editing in the brain. At the same time, we only observed a few EGFP-positive cells around the injection site with C20 and protecting oligos with no conjugation. In contrast, C20 with either DHA or cholesterol conjugation and with either cleavable or stable linker generated more efficient editing with broader distribution (
Using the adult wild-type B6 mice, we tested C20 targeting the mouse Pcsk9 gene with either 14 nucleotides or 16 nucleotides protecting oligo having a trivalent GalNac conjugation. We also included a non-targeting control C20, and PBS as controls. First, we injected adult mice with AAV to express either SpyCas9-ABE8e and tracrRNA (
Next, we tested whether our co-delivery approach can generate therapeutic-relevant editing by testing in the mouse model of HT1. The HT1 mouse model used in this study is the FahPM/PM mouse, which possesses a G-to-A point mutation in the last nucleotide of exon 8 of the Fah gene, which encodes fumarylacetoacetate hydrolase. This point mutation generates exon 8 skipping and causes FAH deficiency (
We first injected AAV to express SpyCas9-ABE8e and tracrRNA by RO injection. After five weeks, we performed RO injections of C20 with a previously validated spacer sequence targeting the point mutation in the FahPM/PM mouse, with either 14 nucleotides or 16 nucleotides trivalent GalNac-conjugated protecting oligos, at a single 80 mg/kg dose. The mice were kept on NTBC the whole time to maintain their body weight and allow time for the genome editing to occur. We then sacrificed three mice from each group and extracted liver genomic DNA to measure the editing efficiency by targeted amplicon deep sequencing, as well as detecting FAH-positive hepatocytes via IHC staining using anti-FAH antibodies. For the remaining mouse from each group, we cycled NTBC off and on (7-10 days off followed by 2 days on) for three months to allow FAH-positive hepatocytes to expand and monitor the mouse body weight to test if genome editing by our co-delivery approach can correct the phenotypes of this mouse model (
The editing efficiency we achieved in the liver, though significant, was low. We then sought to improve the efficiency by optimizing the dosing regimen. The asialoglycoprotein receptor (ASGPR), which is expressed on the surface of the hepatocytes and responsible for the uptake of GalNac-conjugated oligos, has a recycle time of around 10-15 minutes. Data from previous studies also showed that due to ASGPR saturation, the hepatocytes can only uptake limited amount of the GalNac-conjugated molecules after bolus intravenous injection.77 Our initial study injected a single dose of C20 with GalNac-conjugated protecting oligo at 80 mg/kg, far beyond the capacity of available ASGPR. We reasoned that instead of a single large dose, dividing the oligos into three consecutive daily RO injections with one-third of the previous dose (26 mg/kg) may be able to deliver more oligos into the hepatocyte, thus boosting the editing efficiency (
To test this idea, we first RO injected AAV to express SpyCas9-ABE8e and tracrRNA in the B6 mice. Then, after five weeks, we performed three consecutive daily RO injections of C20 targeting the Pcsk9 gene complexed with a 14 nt GalNac-conjugated protecting oligos at 26 mg/kg. We then harvested the mouse liver genomic DNA and performed targeted amplicon deep sequencing to measure the editing efficiency. We found that compared to a single dose of 80 mg/kg oligo, three consecutive daily doses at 26 mg/kg significantly improved the editing efficiency (
We then further explored the mechanisms of protecting oligo activity. We compared the activities of C20 with protecting oligonucleotides of different lengths or that anneal to different regions of C20 (
This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/336,340, filed Apr. 29, 2022, and U.S. Provisional Patent Application Ser. No. 63/438,842, filed Jan. 13, 2023. The entire contents of the above-referenced patent applications are incorporated by reference in their entirety herein.
This invention was made with government support under Grant No. TR002668 awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63438842 | Jan 2023 | US | |
| 63336340 | Apr 2022 | US |
