Attorney Docket No.199235.769601 MECP2-TARGETING ENGINEERED GUIDE RNAS AND POLYNUCLEOTIDES CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority under 35 U.S.C. § 119 from U.S. Provisional Application No.63/567,457 filed on March 20, 2024, U.S. Provisional Application No.63/639,003 filed on April 26, 2024, U.S. Provisional Application No.63/660,145 filed on June 14, 2024, U.S. Provisional Application No.63/691,590 filed on September 6, 2024, and U.S. Provisional Application No.63/752,489 filed on January 31, 2025, the disclosures of which are incorporated herein by reference in their entirety. BACKGROUND [0002] Payloads that mediate RNA editing can be viable therapies for genetic diseases. However, highly efficacious payloads that can maximize on-target RNA editing while minimizing off- target RNA editing are needed. Moreover, payloads that are capable of facilitating protein knockdown are also needed. Additionally, payloads that are capable of restoring translation in pathogenic nonsense mutations are also needed. SUMMARY [0003] Described herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA is capable of hybridizing to a target MECP2 RNA, wherein the target MECP2 RNA is encoded by a mutant allele, wherein: the engineered guide RNA upon hybridization to target MECP2 RNA forms a guide-target RNA scaffold with the target RNA; two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to the target MECP2 RNA; upon hybridization of the engineered guide RNA to the target RNA, one or more target adenosines in the target MECP2 RNA are edited by an RNA editing entity; and the engineered guide RNA facilitates an increased level of editing of the target MECP2 RNA encoded by the mutant allele, as compared to a level of editing of an otherwise comparable target RNA encoded by a wildtype allele, wherein the mutant allele encodes a R168X mutation in the target MECP2 RNA. In some embodiments, the engineered guide facilitates editing of the target MECP2 RNA encoded by the mutant allele at a higher specificity relative to the RNA encoded by the wildtype allele. In some embodiments, the engineered guide RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the engineered guide RNA comprises a sequence of SEQ ID NO: 34. In some embodiments, the engineered guide RNA is capable of recruiting ADAR1, ADAR2, or a combination thereof. In some embodiments, the engineered guide RNA facilitates allele-specific
Attorney Docket No.199235.769601 editing by ADAR1, ADAR2, or a combination thereof of the mutant allele. In some embodiments, the two or more structural features independently comprise a bulge and further comprise a structural feature selected from the group consisting of a mismatch, a bulge, an internal loop, and a combination thereof. [0004] Also disclosed herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA is capable of hybridizing to one or more target RNAs derived from one or more species, wherein the one or more target RNAs is encoded by one or more mutant alleles, wherein: the engineered guide RNA independently hybridizes to each target RNA in the one or more target RNAs independently forming a guide- target RNA scaffold with each target RNA; two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to each target RNA; upon hybridization of the engineered guide RNA to the target RNA, one or more target adenosines in the target RNA are edited by an RNA editing entity; and the engineered guide RNA facilitates editing of each target RNA in the one or more target RNAs derived from one or more species at comparable editing levels. In some embodiments, the one or more species independently comprise a human, a primate, a non-human primate, or a mouse. In some embodiments, the non-human primate is a cynomolgus macaque. In some embodiments, the comparable editing level is an RNA editing level of clinical relevance. In some embodiments, the two or more structural features independently comprise a bulge and further comprise a structural feature selected from the group consisting of a mismatch, a bulge, an internal loop, and a combination thereof. In some embodiments, the engineered guide RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55 or SEQ ID NO: 56. In some embodiments, the engineered guide RNA comprises a sequence of any one of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 41, SEQ ID NO: 47, SEQ ID NO: 55 or SEQ ID NO: 56. [0005] Also disclosed herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein the engineered guide RNA is capable of hybridizing to a target MECP2 RNA that comprises a premature stop codon, wherein: the engineered guide RNA, upon hybridization to the target MECP2 RNA forms a guide-target RNA scaffold with the target MECP2 RNA; two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to the target MECP2 RNA, wherein the two or more structural features independently comprise a mismatch, a bulge, an internal loop, or a combination thereof; and wherein the engineered guide RNA comprises a sequence with at
Attorney Docket No.199235.769601 least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 31 – SEQ ID NO: 37, SEQ ID NO: 47, SEQ ID NO: 51, or SEQ ID NO: 55 – SEQ ID NO: 60. In some embodiments, the target MECP2 RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 68. In some embodiments, the engineered guide RNA is from about 60 nucleotides to about 200 nucleotides in length. In some embodiments, upon hybridization of the engineered guide RNA to the target MECP2 RNA, one or more target adenosines in the target MECP2 RNA are edited by an RNA editing entity. In some embodiments, the RNA editing entity is an endogenous RNA editing entity. In some embodiments, the RNA editing entity is a human endogenous RNA editing entity. In some embodiments, the endogenous RNA editing entity is a human ADAR1, a human ADAR2, or both. In some embodiments, the premature stop codon results in a R168X mutation in the polypeptide encoded by the target RNA. In some embodiments, the target MECP2 RNA encodes a MECP2 polypeptide comprising a R168X mutation. In some embodiments, the RNA editing entity edits one or more adenosines in the premature stop codon. In some embodiments, at least one of the two or more structural features independently comprises the bulge. In some embodiments, the bulge is an asymmetrical bulge. In some embodiments, the bulge is a symmetrical bulge. In some embodiments, at least one of the two or more structural features independently comprises the internal loop. In some embodiments, the internal loop is a symmetrical internal loop. In some embodiments, the internal loop is an asymmetrical internal loop. In some embodiments, the polynucleotide encoding the engineered guide RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52 or SEQ ID NO: 61 – SEQ ID NO: 66. In some embodiments, the guide-target RNA scaffold comprises at least one 6/6 symmetric internal loop. In some embodiments, the guide-target RNA scaffold further comprises a symmetric bulge. In some embodiments, the guide-target RNA scaffold comprises a 12/12 symmetric internal loop or a 10/10 symmetric internal loop. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 35 or SEQ ID NO: 37. In some embodiments, the guide-target RNA scaffold comprises two 6/6 symmetric internal loops. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 47, or SEQ ID NO: 51. In some embodiments, the guide-target RNA scaffold comprises at least nine wobble base pairs. In some embodiments, the engineered guide RNA comprises a polynucleotide sequence of SEQ ID NO: 32, SEQ ID NO: 35, or SEQ
Attorney Docket No.199235.769601 ID NO: 37. In some embodiments, the target MECP2 RNA is encoded by a mutant allele. In some embodiments, the engineered guide RNA facilitates an increased level of editing of the target MECP2 RNA encoded by the mutant allele, as compared to a level of editing of an otherwise comparable target RNA encoded by a wildtype allele. [0006] Also disclosed herein is a viral vector comprising a polynucleotide encoding an engineered guide RNA as described herein. In some embodiments, the viral vector is an AAV vector, a lentiviral vector, or a retroviral vector. [0007] Also disclosed herein is a recombinant AAV encapsidating a vector, wherein the vector comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO: 962 – SEQ ID NO: 964, or SEQ ID NO: 965, and an AAV inverted terminal repeat. In some embodiments, the sequence is SEQ ID NO: 962. In some embodiments, the sequence is SEQ ID NO: 963. In some embodiments, the sequence is SEQ ID NO: 964. In some embodiments, the sequence is SEQ ID NO: 965. In some embodiments, the vector comprises a sequence of SEQ ID NO: 69. In some embodiments, the vector comprises a sequence of SEQ ID NO: 70. In some embodiments, the vector comprises a sequence of SEQ ID NO: 959. In some embodiments, the vector comprises a sequence of SEQ ID NO: 960. In some embodiments, the vector encodes an engineered guide RNA, wherein the engineered guide RNA, upon hybridization to a target MECP2 RNA, forms a guide-target RNA scaffold that independently comprises two or more structural features. In some embodiments, upon hybridization of the engineered guide RNA to the target MECP2 RNA, one or more target adenosines in the target MECP2 RNA are edited by an RNA editing entity. In some embodiments, the target MECP2 RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 68. In some embodiments, the two or more structural features comprises a mismatch, a bulge, an internal loop, or a combination thereof. In some embodiments, at least one of the two or more structural features comprise the bulge. In some embodiments, the bulge is an asymmetrical bulge. In some embodiments, the bulge is a symmetrical bulge. In some embodiments, at least one of the two or more structural features comprise the internal loop. In some embodiments, the internal loop is an asymmetrical internal loop. In some embodiments, the internal loop is a symmetrical internal loop. In some embodiments, the RNA editing entity comprises a human ADAR1, or a human ADAR2. In some embodiments, thevector further comprises a sequence encoding a SmOPT and U7 hairpin sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO: 76. In some embodiments, the vector further comprises a sequence encoding a SmOPT and
Attorney Docket No.199235.769601 U7 hairpin sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO: 77. In some embodiments, the vector further comprises a sequence encoding a SmOPT and U5 hairpin sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO: 79. [0008] Also disclosed herein is a pharmaceutical composition comprising: (a) an engineered guide RNA as described herein or a polynucleotide encoding an engineered guide RNA as described herein, or a recombinant AAV as described herein; and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. [0009] Also disclosed herein is a method of increasing a level of full-length MECP2 in a cell, the method comprising administering to the cell an engineered guide RNA as described herein or a polynucleotide encoding an engineered guide RNA as described herein, a recombinant AAV as described herein, or a pharmaceutical composition as described herein. In some embodiments, the level of full-length MECP2 increases by at least: 10%, 30%, 40%, or 50% relative to an otherwise comparable cell that was not administered the engineered guide RNA, the polynucleotide encoding the engineered guide RNA, the recombinant AAV or the pharmaceutical composition. In some embodiments, the level of full-length MECP2 is restored to functional levels in the cell. [0010] Also disclosed herein is a method of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition as described herein. In some embodiments, the disease or condition comprises a Rett Syndrome. In some embodiments, the Rett Syndrome arises from a MECP2 polypeptide comprising a R168X mutation. In some embodiments, the subject is human or a non-human animal. [0011] Also disclosed herein is an engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein: (a) the engineered guide RNA, upon hybridization to a sequence of a target MECP2 RNA that comprises a premature stop codon, forms a guide-target RNA scaffold with the sequence of the target MECP2 RNA, wherein the target MECP2 RNA comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO: 50, SEQ ID NO: 54, or SEQ ID NO: 68.; (b) two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to the sequence of the target MECP2 RNA, wherein the two or more structural features independently comprise a mismatch, a bulge, an internal loop, or a combination thereof; (c) the structural feature is not present within the engineered guide
Attorney Docket No.199235.769601 RNA or the target MECP2 RNA prior to the hybridization of the engineered guide RNA to the target MECP2 RNA; and (d) the engineered guide RNA is from about 60 nucleotides to about 200 nucleotides in length. [0012] Also disclosed herein is an AAV virion encapsidating a DNA vector genome comprising a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, and SEQ ID NO: 52. In some embodiments, the AAV virion is an AAV1 virion, AAV2 virion, AAV3 virion, AAV4 virion, AAV5 virion, AAV6 virion, AAV7 virion, AAV8 virion, AAV9 virion, AAV10 virion, AAV11 virion, or a derivative, a chimera, or a variant thereof. In some embodiments, the AAV virion is a recombinant AAV (rAAV) virion, a hybrid AAV virion, a chimeric AAV virion, a self-complementary AAV (scAAV) virion, or any combination thereof. [0013] Also disclosed herein is a pharmaceutical composition comprising: (a) an AAV virion encapsidating a DNA vector genome as described herein, and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. [0014] Also disclosed herein is a method of administering to a subject an effective amount of an AAV virion encapsidating a DNA vector genome as described herein, or a pharmaceutical composition as described herein. In some embodiments, the subject is a mouse, a non-human primate, or a human. In some embodiments, the pharmaceutical composition is in unit dose form. [0015] Also disclosed herein is a method of treating a Rett syndrome in a subject comprising administering to a subject an effective amount of an AAV virion encapsidating a DNA vector genome as described herein, or a pharmaceutical composition as described herein. In some embodiments, the subject is a mouse, a non-human primate, or a human. In some embodiments, the pharmaceutical composition is in unit dose form. [0016] Also disclosed herein is a method of editing an MECP2 RNA transcript in a subject comprising administering to a subject an effective amount of an AAV virion encapsidating a DNA vector genome as described herein, or a pharmaceutical composition as described herein. In some embodiments, the subject is a mouse, a non-human primate, or a human. In some embodiments, the pharmaceutical composition is in unit dose form. In some embodiments, the editing of the MECP2 RNA transcript comprises editing of a MECP2 RNA transcript that comprises a R168X mutation. [0017] Also disclosed herein is a kit comprising an AAV virion encapsidating a DNA vector genome as described herein, or a pharmaceutical composition as described herein. INCORPORATION BY REFERENCE
Attorney Docket No.199235.769601 [0018] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS [0019] Novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which exemplary principles of the present disclosure are utilized, and the accompanying drawings of which: [0020] FIG.1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side). [0021] FIG.2 shows a schematic of editing of a MECP2 transcript. The wild type transcript contains a CGA codon (arginine). The mutant transcript contains a TGA (UGA for RNA) stop codon, which is implicated in Rett Syndrome. The results from an RNA edited by ADAR transcript show the edited mutant transcript with the TGI (UGI for RNA) codon (tryptophan) and the edited WT transcript with the CGI codon (arginine) that results in a synonymous edit. [0022] FIG.3 shows an alignment of exon 4 of MECP2 in human wild type (WT), cynomolgus (cyno) WT, human mutant, cyno mutant, mouse WT, and mouse mutant cells. [0023] FIG.4 shows percent RNA editing of the MECP2 R168X mutation in engineered HEK cells for a number of exemplary engineered guide RNAs of the present disclosure. [0024] FIG.5 shows flow cytometry readthrough of mouse Mecp2 and human MECP2 showing the restoration of full-length mouse Mecp2 and human MECP2 protein as assessed by flow cytometry in engineered HEK cells that express MECP2 R168X-Flag reporter transcripts and are transfected with the engineered guide RNAs of TABLE 3.
Attorney Docket No.199235.769601 [0025] FIG.6 shows a comparison of percent RNA editing (x-axis) versus % MECP2FLAG+ cells (top) and percent RNA editing (x-axis) versus gMFI of MECP2-Flag transfected cells (bottom) for the engineered guide RNAs of TABLE 3. No gMFI was measured in controls. The figure shows percent editing of mouse Mecp2. [0026] FIG.7 shows restoration of MECP2 (by point mutation recoding) in mouse primary neurons by RNA editing as assessed by flow cytometry. The Y-axis shows the percent MECP2 protein positive cells and the X-axis shows the treatment groups - no therapy (no tx), low dose and high dose of an AAV administered guide RNA targeting Mecp2 RNA along with a wild type (WT) cell control. [0027] FIG.8 shows percent on-target RNA editing efficiency of human MECP2 and mouse Mecp2 R168X mutant transcripts relative to human MECP2 and mouse WT Mecp2, in engineered HEK293 cells, for a number of exemplary engineered guide RNAs of the present disclosure. [0028] FIG.9 shows percent on-target RNA editing of human and mouse MECP2 R168X mutant transcripts in engineered HEK293 cells relative to human and mouse WT MECP2 transcripts for a guide RNA in the present disclosure. [0029] FIG.10 shows percent on-target editing of a target adenosine (top) and percent MECP2 protein expression (bottom) in human iPSC-derived neurons expressing human MECP2 mutant (R168X) or wild type (WT) transcripts following treatment with an engineered guide RNA of the present disclosure. [0030] FIGs.11A and FIG.11B shows RNA editing of MECP2 mutant and WT transcripts. FIG.11A shows percent on-target RNA editing of human and mouse MECP2 R168X mutant transcripts in engineered HEK293 cells relative to human and mouse WT MECP2 transcripts for a guide RNA the present disclosure. FIG.11B shows secondary structures of the guide RNA targeting different MECP2 alleles. [0031] FIGs.12A and FIG.12B shows RNA editing of MECP2 transcripts in disease-relevant in vitro models. FIG.12A shows percent on-target editing of a target adenosine in Mecp2 (mouse) or MECP2 (human) mutant (R168X) or wild type (WT) transcripts in ex vivo mouse neurons and human iPSC-derived neurons following treatment with an engineered guide RNA of the present disclosure. Y axis shows percent editing (target A), and X axis shows treatment group, the data is superimposed on each other (not stacked) and the blue box in the box plots represent Mecp2 (mouse) or MECP2 (human) R168X mutant transcript editing, and grey box represents wild type Mecp2 (mouse) or MECP2 (human) transcript editing. FIG.12B shows percent MECP2 protein restoration after treatment with exemplary engineered guide RNA (SEQ
Attorney Docket No.199235.769601 ID NO: 34) in ex vivo mouse neurons and human iPSC-derived neurons, as assessed by flow cytometry. Y axis shows percent positive MECP2+ nuclei of live cells, and X axis shows treatment group. The data is superimposed on each other (not stacked) and the blue box in the box plots represents MECP2 protein levels in cells expressing the wild type Mecp2 (mouse) or MECP2 (human) transcript, and grey box represents Mecp2 (mouse) or MECP2 (human) protein levels in cells expressing the MECP2 R168X mutant transcript. [0032] FIG.13 provides immunofluorescence images of human iPSC-derived neurons transduced with scAAV PHP.eB encoding a guide RNA (SEQ ID NO: 34) (bottom) as compared to untreated WT (top), and untreated mutant (middle). Images show localization of MECP2 (left), DAPI staining of cell nuclei (middle), and MAP2 (right). [0033] FIGs.14A - FIG.14E shows the results of targeted editing of Mecp2 transcripts and MECP2 protein quantification from in vivo mouse experiments. FIG.14A shows the editing rate in the brainstem, cerebellum, and remaining brain (ROB) after treatment with a guide RNA (SEQ ID NO: 34), and vehicle. Y axis shows percent editing (target A), and X axis shows treatment group, the data is superimposed on each other and the blue box in the box plots represent MECP2 R168X mutant animals, and grey box represents wild type MECP2 animals. FIG.14B shows immunohistochemistry (IHC) images depicting MECP2 protein in wild type mouse (top), mutant MECP2R168X mouse after treatment with vehicle control (middle), and restoration of MECP2 protein in MECP2R168X mouse after treatment with MECP2 gRNA (SEQ ID NO: 34) (bottom). FIG.14C shows quantification from IHC images depicting MECP2 protein in wild type mouse, mutant MECP2R168X mouse after treatment with vehicle control, and MECP2 protein restoration in MECP2R168X mouse after treatment with MECP2 gRNA (SEQ ID NO: 34). Y axis shows percent MECP2+ of nuclei, and X axis shows treatment group. FIG.14D shows immunofluorescence (IF) images depicting MECP2 protein and gRNA FISH detection in mutant MECP2R168X and MECP2WT mouse brains after treatment with control gRNA. FIG.14E shows immunofluorescence (IF) images depicting MECP2 protein and gRNA FISH detection in mutant MECP2R168X and MECP2WT mouse brains after treatment with MECP2 gRNA (SEQ ID NO: 34), including restoration of full-length MECP2 in MECP2R168X animals. [0034] FIGs.15A - FIG.15D shows the results of targeting MECP2 from in vitro experiments. FIG.15A shows percent on-target editing of a target adenosine in MECP2 mutant (R168X) or wild type (WT) transcripts in human iPSC-derived neurons following treatment with exemplary engineered guide RNAs (SEQ ID NO: 34, SEQ ID NO: 55, & SEQ ID NO: 56). Y axis shows percent editing (target A), and X axis shows treatment group. The data is superimposed on each other and the white and colored boxes in the box plots represent MECP2 R168X mutant, and
Attorney Docket No.199235.769601 grey boxes represent wild type MECP2. FIG.15B shows percent MECP2 protein restoration after treatment with exemplary engineered guide RNAs (SEQ ID NO: 34, SEQ ID NO: 55, & SEQ ID NO: 56) in human iPSC-derived neurons. Y axis shows percent MECP2+ of live cells, and X axis shows treatment group. The data is superimposed on each other and the white and colored boxes in the box plots represent MECP2 R168X mutant, and grey boxes represent wild type MECP2. FIG.15C shows MECP2 mean fluorescent intensity after treatment with exemplary engineered guide RNAs (SEQ ID NO: 34, SEQ ID NO: 55, & SEQ ID NO: 56) in human iPSC-derived neurons. Y axis shows geometric mean (gMFI) (MECP2 of Live), and X axis shows treatment group. The data is superimposed on each other and the white and colored boxes in the box plots represent MECP2 R168X mutant, and grey boxes represent wild type MECP2. FIG.15D provides immunofluorescence images of human iPSC-derived neurons transduced with scAAV PHP.eB encoding a guide RNA (SEQ ID NO: 34 (bottom) as compared to untreated WT (top), and untreated mutant (middle). Images show localization of MECP2 (left), DAPI staining of cell nuclei (middle), and MAP2 (right). [0035] FIG.16 shows an exemplary tandem and bidirectional ITR-to-ITR AAV vector construct design with cassette 1 and cassette 2. [0036] FIGs.17A – FIG.17D shows the results of targeting MECP2 using different ITR-to-ITR constructs comprising the engineered guide RNA of SEQ ID NO: 34 and various controls in in vivo mouse experiments. ITR-to-ITR construct 3 and ITR-to-ITR construct 5 comprised the engineered guide RNA of SEQ ID NO: 34, ITR-to-ITR construct 4 comprised a control guide RNA, and the vehicle control did not comprise any guide RNA. FIG.17A shows the percent on- target editing of MECP2 gRNAs or the respective control gRNA’s target in different brain regions as measured by Sanger sequencing. Y axis shows percent editing, and X axis shows treatment group. FIG.17B shows the guide expression in different brain regions as measured by ddPCR. Y axis shows gRNA/U1, and X axis shows mouse genotype, and brain region. FIG.17C shows the mean AAV genome copy number per gDNA (vg/dg) in different brain regions. Y axis shows vg/dg, and X axis shows mouse genotype, and brain region. FIG.17D shows the correlation between vg/dg and guide expression in different brain regions. Y axis shows gRNA/U1, and X axis shows vg/dg. [0037] FIGs.18A – FIG.18D shows the results of targeting MECP2 using different ITR-to-ITR constructs comprising the engineered guide RNA of SEQ ID NO: 34 and various controls in in vivo mouse experiments. ITR-to-ITR construct 3 and ITR-to-ITR construct 5 comprised the engineered guide RNA of SEQ ID NO: 34, ITR-to-ITR construct 4 comprised a control guide RNA, and the vehicle control did not comprise any guide RNA. FIG.18A shows the percent
Attorney Docket No.199235.769601 respective on-target editing of MECP2 or control gRNAs in liver samples as measured by Sanger sequencing. Y axis shows percent respective on-target editing, and X axis shows treatment group in wild type and mutant mice. FIG.18B shows the guide expression in liver samples as measured by ddPCR. Y axis shows gRNA/U1, and X axis shows treatment group in wild type and mutant mice. FIG.18C shows correlation between the guide abundance and percent on- target editing in individual wild type and MECP2 R168X mice (“mutant”) after treatment with ITR-to-ITR construct 3. Y axis shows percent on-target editing, and X axis shows gRNA/U1. FIG.18D shows correlation between the guide abundance and percent on-target editing in individual wild type and MECP2 R168X mice (“mutant”) after treatment with ITR-to-ITR construct 4. [0038] FIGs.19A - FIG.19C show the results of targeting MECP2 from in vivo mouse experiments in a MECP2 R168X mouse model. FIG.19A shows the percent RNA editing in the general cortex (frontal brain), brainstem, and general midbrain after a single systemic injection of AAV PHP.eB (1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively), and a control guide RNA. Y axis shows percent RNA editing, and X axis shows the brain region. FIG.19B shows immunofluorescence (IF) images depicting MECP2 protein restoration in a mutant MECP2R168X mouse after treatment with the vector (SEQ ID NO: 70) (bottom), as compared to a wild type mouse (top), and a mutant MECP2R168X mouse after treatment with vehicle control (middle). FIG.19C shows quantification from IF images depicting MECP2 protein detection in wild type mouse, and mutant MECP2R168X mouse after treatment with vehicle control, and MECP2 protein restoration after treatment of a MECP2R168X mouse after treatment with the vectors comprising SEQ ID NO: 69, or SEQ ID NO: 70 encoding the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51). Y axis shows percent MECP2+ protein by Nuclei, and X axis shows mouse genotype group (R168X (mutant), or wild type). [0039] FIGs.20A - FIG.20G show the results of targeting MECP2 in human Rett-patient iPSC- derived neurons. FIG.20A shows the percent RNA editing of target adenosine in MECP2 in human Rett-patient iPSC-derived neurons at a high dose of 1e5 vg/cell. Y axis shows percent (%) editing (target A), and X axis shows the treatment group. FIG.20B shows immunofluorescent (IF) images depicting protein restoration in human Rett-patient iPSC-derived neurons after treatment with the engineered guide RNA of SEQ ID NO: 56 (far right), as compared to a wild type MECP2 neurons (far left), and human Rett-patient iPSC-derived neurons after treatment with vehicle control (middle). FIG.20C shows quantification from IF images depicting protein restoration in human Rett-patient iPSC-derived MECP2R168X neurons after treatment with vehicle
Attorney Docket No.199235.769601 control, a control guide RNA, and the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51). Y axis shows percent MECP2+ protein by Nuclei, and X axis shows treatment group. FIG.20D shows immunofluorescent (IF) images depicting protein restoration in human Rett- patient iPSC-derived neurons after treatment with the engineered guide RNA of SEQ ID NO: 51 (far right), as compared to a wild type MECP2 neurons (far left), and human Rett-patient iPSC- derived neurons after treatment with vehicle control (middle). FIG.20E shows quantification from IF images depicting protein restoration in WT iPSC WT MECP2 neurons after treatment with vehicle control, a control guide RNA, and the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51). FIG.20F shows average MECP2 intensity within DAPI+ nuclei in human Rett-patient iPSC-derived MECP2R168X neurons after treatment with vehicle control, a control guide RNA, and the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51). Y axis shows mean grey value, and X axis shows the treatment group. FIG.20G shows average MECP2 intensity within DAPI+ nuclei in human MECP2WT neurons after treatment with vehicle control, a control guide RNA, and the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51). Y axis shows mean grey value, and X axis shows the treatment group. [0040] FIG.21 shows the results of targeted editing of MECP2 in human Rett-patient iPSC- derived neurons at a low dose (5e4 vg/cell) and a high dose (1e5 vg/cell). FIG.21 shows the percent RNA editing of target adenosine in MECP2 in human neurons. Y axis shows percent (%) editing (“target A”), and X axis shows the treatment group. The bar graphs are superimposed on each other, not stacked (e.g., both bars for the R168X and WT transcript editing are the full percent editing value as shown on the y-axis). [0041] FIGs.22A - FIG.22B show the guide RNA quantification in human neurons. FIG.22A shows the guide expression in human MECP2R168X neurons. Y axis shows gRNA/U1, and X axis shows the treatment group (scramble control, SEQ ID NO: 56, or SEQ ID NO: 51). FIG.22B shows the guide expression in human MECP2WT neurons. Y axis shows percent (%) editing (target A), and X axis shows the treatment group. [0042] FIGs.23A – FIG.23E show the guide RNA editing in the canonical exon 4 of MECP2 in human neurons. FIG.23A shows a representative schematic of an alternate splice isoform of human MECP2 that results from 5 potential exons. FIG.23B shows frequency of rare isoforms (alternate exons 4-5) in human MECP2R168X neurons after treatment with scramble control, SEQ ID NO: 56, or SEQ ID NO: 51, as compared to untreated control. Y axis shows % Novel splice variant” (NSV), and X axis shows the treatment group. FIG.23C shows frequency of rare isoforms (alternate exons 4-5) in human MECP2WT neurons after treatment with scramble control, SEQ ID NO: 56, or SEQ ID NO: 51, as compared to untreated control. Y axis shows %
Attorney Docket No.199235.769601 NSV, and X axis shows the treatment group. FIG.23D shows MECP2 transcript abundance in human MECP2R168X neurons after treatment with scramble control, SEQ ID NO: 56, or SEQ ID NO: 51, as compared to untreated control. Y axis shows Mecp2/GapdH, and X axis shows the treatment group. FIG.23E shows MECP2 transcript abundance in human MECP2WT neurons after treatment with scramble control, SEQ ID NO: 56, or SEQ ID NO: 51, as compared to untreated control. Y axis shows Mecp2/GapdH, and X axis shows the treatment group. [0043] FIGs.24 and FIG.25 show restoration of full-length MECP2 using the engineered guide RNAs disclosed herein. FIG.24 shows normalized % MECP2+ of live cells. Y axis shows relative % MECP2+ of live cells, and X axis shows the treatment group and different doses. Values are normalized to the average MECP2+ of live cells detected in MECP2WT cells without treatment (“No Tdxn”, grey bars). FIG.25 show normalized gMFI MECP2 of live cells. Y axis shows relative gMFI (MECP2 of live cells), and X axis shows the treatment group and different doses. Values are normalized to the average gMFI of MECP2 detected in MECP2WT cells without treatment (“No Tdxn”, grey bars). [0044] FIGs.26A – FIG.26C show results of target editing Mecp2 transcripts in mouse ex vivo cultures. FIG.26A shows editing rate at target adenosine in mouse MECP2R168X and MECP2 WT neurons after treatment with low or high doses of control, or engineered guide RNAs of SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, or SEQ ID NO: 428, as compared to untreated control. Y axis shows % editing (target A), and X axis shows treatment group. FIG.26B shows % MECP2+ of live cells in mouse MECP2R168X neurons, and mouse MECP2WT neurons after treatment with low or high doses of control, or engineered guide RNAs of SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, or SEQ ID NO: 428, as compared to untreated control as assessed by flow cytometry. Y axis shows % MECP2+ of live cells, and X axis shows treatment group. FIG.26C shows gMFI (MECP2 of live cells) in mouse MECP2R168X neurons, and mouse MECP2WT neurons after treatment with low or high doses of control, or engineered guide RNAs of SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, or SEQ ID NO: 428, as compared to untreated control as assessed by flow cytometry. Y axis shows % MECP2+ of live cells, and X axis shows treatment group. [0045] FIGs.27A - FIG.27E show the results of targeted editing of Mecp2 transcripts from in vivo mouse experiments in a MECP2R168X mouse model. FIG.27A shows the percent RNA editing in the frontal brain, brainstem, and midbrain region after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled control guide RNA (“scrambled gRNA control”), as compared to
Attorney Docket No.199235.769601 vehicle control. Y axis shows percent Mecp2 transcript editing, and X axis shows the treatment group. FIG.27B shows the VG/DG expression in the frontal brain, brainstem, and midbrain region after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, which encodes the engineered guide RNAs (SEQ ID NO: 56) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows percent Vg/Dg (normalized to Tfrc), and X axis shows the treatment group. FIG.27C shows the guide RNA expression in the frontal brain, brainstem, and midbrain region after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, which encodes the engineered guide RNAs (SEQ ID NO: 56) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows percent gRNA/U1, and X axis shows the treatment group. FIG.27D shows the VG/DG expression in the frontal brain, brainstem, and midbrain region after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 70, which encodes the engineered guide RNAs (SEQ ID NO: 51) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows percent Vg/Dg (normalized to Tfrc), and X axis shows the treatment group. FIG.27E shows the guide RNA expression in the frontal brain, brainstem, and midbrain region after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 70, which encodes the engineered guide RNAs (SEQ ID NO: 51) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows percent gRNA/U1, and X axis shows the treatment group. [0046] FIGs.28A - FIG.28H show MECP2 protein restoration in in vivo mouse experiments in a MECP2 R168X mouse model. FIG.28A shows the % MECP2+ nuclei in brainstem MECP2 R168X mice and MECP2 WT mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclei, and X axis shows genotype. FIG.28B shows the % MECP2+ nuclei in midbrain MECP2 R168X mice and MECP2 WT mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclei, and X axis shows genotype. FIG.28C shows the % MECP2+ nuclei in frontal brain MECP2 R168X mice and MECP2 WT mice after a single systemic injection of
Attorney Docket No.199235.769601 AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclei, and X axis shows genotype. FIG.28D shows the % MECP2+ nuclei in the frontal brain, brainstem, and midbrain region of MECP2 R168X mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclei, and X axis shows the treatment group. FIG.28E shows the % MECP2+ nuclei in the frontal brain, brainstem, and midbrain region of MECP2WT mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a control scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclei, and X axis shows genotype. FIG.28F shows the normalized MECP2+ nuclear MFI in mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % MECP2+ nuclear MFI (relative to WT Vehicle % MECP2+ nuclear MFI), and X axis shows the genotype. FIG.28G shows % gRNA+ nuclei across hemisphere in mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows % gRNA+ nuclei, and X axis shows the genotype. FIG.28H shows gRNA MFI across hemisphere in mice after a single systemic injection of AAV PHP.eB (5E11vg/animal or 1E12vg/animal) comprising SEQ ID NO: 69, or SEQ ID NO: 70 which encode the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51, respectively) or a scrambled gRNA control (“scrambled gRNA control”), as compared to vehicle control. Y axis shows MFI, and X axis shows the genotype. DETAILED DESCRIPTION Overview
Attorney Docket No.199235.769601 [0047] Rett syndrome is a neurological and developmental disorder affecting children. Rett syndrome is typically first identified in infancy and occurs primarily in females. This genetic disorder is characterized by language and motor skill deficiencies, with an estimated prevalence of 1 in 10,000 female births. Mutations of the methyl-CpG binding protein 2 (MECP2) gene are associated with Rett syndrome. In humans, MECP2 plays an integral role in development and function of nerve cells. MECP2 plays a role in gene regulation in nerve cells and loss of function mutations such as R168X (arginine to opal stop codon; CGA -> UGA) are associated with Rett syndrome. Disclosed herein are target sites in MECP2 mRNA and guide RNAs for RNA editing. Editing of MECP2 mRNA can restore function of MECP2 protein and treat Rett syndrome. [0048] Several nonsense mutations within the MECP2 gene lead to a truncated form of the protein and are penetrant for Rett Syndrome. Due to its location on the X chromosome, this leads to mosaicism in females, with cells expressing either WT or mutant MECP2. Additionally, over- or under-expression of MECP2 can lead to disease. For this reason, gene replacement requires tight control of expression, since any delivery to the cells expressing WT protein can lead to over expression. And it is very challenging to design a promoter that is fine-tuned to express the right dose in diseased cells while remaining inactive in healthy cells. Thus, RNA editing can be viable strategy, since it can correct the endogenous transcript without perturbing its expression and editing of the WT transcript can lead to a synonymous mutation. [0049] There are many common nonsense mutations within MECP2 that lead to Rett syndrome. In some cases, the mutated nucleotide causes an arginine to opal replacement (CGA -> TGA or CGA -> UGA for RNA). ADAR-mediated RNA editing of the proximal adenosine can restore full-length protein by changing the codon to tryptophan (UGI). A tryptophan substitution can be tolerated in mice and can lead to an improved phenotype compared to diseased animals (e.g., R168X mice). For these reasons, RNA editing with the use of engineered guide RNAs herein can be used to treat Rett Syndrome. RNA Editing [0050] RNA editing can refer to a process by which RNA can be enzymatically modified post synthesis at specific nucleosides. RNA editing can comprise any one of an insertion, deletion, or substitution of a nucleotide(s). Examples of RNA editing include chemical modifications, such as pseudouridylation (the isomerization of uridine residues) and deamination (removal of an amine group from cytidine to give rise to uridine, or C-to-U editing or from adenosine to inosine, or A- to-I editing). RNA editing can be used to introduce mutations, correct missense mutations, correct
Attorney Docket No.199235.769601 nonsense mutations, or edit coding or non-coding regions of RNA to inhibit RNA translation and effect protein knockdown. [0051] Described herein are engineered guide RNAs that facilitate RNA editing by an RNA editing entity (e.g., an adenosine Deaminase Acting on RNA (ADAR)) or biologically active fragments thereof. In some instances, ADARs can be enzymes that catalyze the chemical conversion of adenosines to inosines in RNA. Because the properties of inosine mimic those of guanosine (inosine will form two hydrogen bonds with cytosine, for example), inosine can be recognized as guanosine by the translational cellular machinery. “Adenosine-to-inosine (A-to-I) RNA editing”, therefore, effectively changes the primary sequence of RNA targets. In general, ADAR enzymes share a common domain architecture comprising a variable number of amino-terminal dsRNA binding domains (dsRBDs) and a single carboxy-terminal catalytic deaminase domain. Human ADARs possess two or three dsRBDs. Evidence suggests that ADARs can form homodimer as well as heterodimer with other ADARs when bound to double-stranded RNA, however it can be currently inconclusive if dimerization is needed for editing to occur. The engineered guide RNAs disclosed herein can facilitate RNA editing by any of or any combination of the three human ADAR genes that have been identified (ADARs 1–3). In some embodiments the engineered guide RNAs disclosed herein facilitate RNA editing by ADAR1. In some embodiments the engineered guide RNAs disclosed herein facilitate RNA editing by ADAR2. In some embodiments the engineered guide RNAs disclosed herein facilitate RNA editing by a combination of ADAR1 and ADAR2. In some embodiments, the engineered guide RNA facilitates allele-specific editing by ADAR1, ADAR2, or a combination thereof of a mutant allele. ADARs have a typical modular domain organization that includes at least two copies of a dsRNA binding domain (dsRBD; ADAR1with three dsRBDs; ADAR2 and ADAR3 each with two dsRBDs) in their N-terminal region followed by a C-terminal deaminase domain. The engineered guide RNAs of the present disclosure facilitate RNA editing by endogenous ADAR enzymes. In some embodiments, exogenous ADAR can be delivered alongside the engineered guide RNAs disclosed herein. [0052] The present disclosure, in some embodiments, provides engineered guide RNAs that facilitate edits at particular regions in a target RNA (e.g., mRNA or pre-mRNA). For example, the engineered guide RNAs of the present disclosure can target an adenosine in the coding region of a target RNA. [0053] Target site mutations. In some embodiments, the engineered guide RNAs of the present disclosure target the adenosines in the coding region of MECP2. The engineered guide RNAs facilitate ADAR-mediated RNA editing of premature stop codons (nonsense mutations), and missense mutations. In some embodiments, the engineered guide RNAs disclosed facilitate
Attorney Docket No.199235.769601 ADAR-mediated RNA editing of nucleotides coding the R168X, R255X, R270X, R294, or a combination thereof, in the coding region of MECP2. In some embodiments, the engineered guide RNAs disclosed facilitate ADAR-mediated RNA editing of nucleotides coding the R168X in the coding region of MECP2. For example, engineered guide RNAs can be used to edit nucleotides coding the R168X mutation and restore function of the MECP2 protein. In some cases, the engineered guide RNAs herein can facilitate editing in the 4th exon of MECP2. In some cases, the engineered guide RNAs herein do not substantially result in exon skipping of MECP2. In some cases, engineered guide RNAs can be used to facilitate editing of a UGA stop codon to a UGI tryptophan codon. [0054] FIG.2 shows a schematic of wild type (WT) and mutant transcripts of MECP2. The WT sequence has a CGA codon encoding an arginine, while the mutant transcript has a TGA (UGA for RNA) codon encoding an opal stop codon. RNA editing of the mutated MECP2 transcript with an engineered guide RNA results in a TGI (UGI for RNA) codon encoding a tryptophan and RNA editing of the WT MECP2 transcript with an engineered guide RNA can result in a synonymous edit to a CGI codon encoding an arginine. The mutated MECP2 transcript results in a truncated protein while editing of the mutated transcript to a tryptophan codon results in a rescued protein. [0055] The target site is identified by the reference dbSNP_id: rs61748421. Further coordinates for the target site are hg38 coordinates: chrX:154031326; macFas5 coordinates: chrX:151031525; and m39 coordinates: chrX:73079976. FIG.3 shows an alignment of exon 4 of MECP2 in human wild type (WT), MECP2 cynomolgus (cyno) WT, MECP2 human mutant, MECP2 cyno mutant, Mecp2 mouse WT, and Mecp2 mouse mutant cells. The target adenosine for editing is bolded and underlined. In the present disclosure, MECP2 transcripts (e.g., the human MECP2 transcript, the cyno MECP2 transcript, or the mouse Mecp2 transcript) may be described with various italicization and capitalization of the text. Similarly, the MECP2 protein (e.g., human, mouse, or cyno MECP2 protein) may be described with various capitalization. [0056] In some cases, the WT DNA encoding for an WT MECP2 can comprise AAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAAT GATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACC ACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCC CCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 29). In some cases, the WT RNA of MECP2 can comprise AAGUGGAGUUGAUUGCGUACUUCGAAAAGGUAGGCGACACAUCCCUGGACCCUA AUGAUUUUGACUUCACGGUAACUGGGAGAGGGAGCCCCUCCCGGCGAGAGCAGAA ACCACCUAAGAAGCCCAAAUCUCCCAAAGCUCCAGGAACUGGCAGAGGCCGGGGA
Attorney Docket No.199235.769601 CGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 30). In some cases, the target RNA of WT MECP2 can comprise GGUAACUGGGAGAGGGAGCCCCUCCCGGCGAGAGCAGAAACCACCUAAGAAGCCC AAAUCUCCCAAAGCUCCAGGAACUGGCAGAGGCCGGGGACGCCCC (SEQ ID NO: 53). In some cases, the WT DNA encoding for a WT MECP2 can comprise GGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCC AAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCC (SEQ ID NO: 67). [0057] In some cases, the target DNA encoding for an R168X MECP2 mutation can comprise AAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAAT GATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGTGAGAGCAGAAACC ACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCC CCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 49), where the codon encoding the R168X nonsense mutation is shown in bold and the target adenosine is shown underlined and in bold. In some cases, the target DNA encoding for a R168X MECP2 mutation can comprise GGTAACTGGGAGAGGGAGCCCCTCCCGGTGAGAGCAGAAACCACCTAAGAAGCCCA AATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCC (SEQ ID NO: 68). In some cases, the target RNA can comprise AAGUGGAGUUGAUUGCGUACUUCGAAAAGGUAGGCGACACAUCCCUGGACCCUA AUGAUUUUGACUUCACGGUAACUGGGAGAGGGAGCCCCUCCCGGUGAGAGCAGA AACCACCUAAGAAGCCCAAAUCUCCCAAAGCUCCAGGAACUGGCAGAGGCCGGGG ACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 50), where the codon encoding the R168X MECP2 nonsense mutation is shown in bold and the target adenosine is shown underlined and in bold. Editing of the target adenosine converts the UGA stop codon to UGI, read as UGG encoding tryptophan, encoding a full-length protein. In some cases, the target RNA can comprise GUAACUGGGAGAGGGAGCCCCUCCCGGUGAGAGCAGAAACCACCUAAGA AGCCCAAAUCUCCCAAAGCUCCAGGAACUGGCAGGGGUCGGGGACGCCCC (SEQ ID NO: 54), where the codon encoding the R168X MECP2 nonsense mutation is shown in bold and the target adenosine is shown underlined and in bold. Editing of the target adenosine converts the UGA stop codon to UGI, read as UGG encoding tryptophan, encoding a full-length protein. [0058] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms
Attorney Docket No.199235.769601 with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. [0059] Throughout this application, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. [0060] As used herein, the term “about” a number can refer to that number plus or minus 10% of that number. [0061] As used herein, the term “engineered guide RNA” can be used interchangeable with “guide RNA” and refers to a designed polynucleotide that is at least partially complementary to a target RNA. An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA. Modification of the target RNA includes alteration of RNA splicing, reduction or enhancement of protein translation, target RNA knockdown, target RNA degradation, and/or ADAR mediated RNA editing of the target RNA. In some cases, guide RNAs facilitate ADAR mediated RNA editing for the purpose of target mRNA knockdown, downstream protein translation reduction or inhibition, downstream protein translation enhancement, correction of mutations (including correction of any G to A mutation, such as missense or nonsense mutations), introduction of mutations (e.g., introduction of an A to I (read as a G by cellular machinery) substitution), or alter the function of any adenosine containing a regulatory motif (e.g., polyadenylation signal, miRNA binding site, etc.). In some cases, a guide RNA can effect a functional outcome (e.g., target RNA modulation, downstream protein translation) via a combination of mechanisms, for example, ADAR-mediated RNA editing and binding and/or degrading target RNA. In some cases, a guide RNA can facilitate introduction of mutations at sites targeted by enzymes in order to modify the affinity of such enzymes for targeting and cleaving such sites. The guide RNAs of this disclosure can contain one or more structural features. The guide RNAs of this disclosure can contain two or more structural features. A structural feature can be formed from latent structure in latent (unbound) guide RNA upon hybridization of the engineered latent guide RNA to a target RNA. Latent structure refers to a structural feature that
Attorney Docket No.199235.769601 forms or substantially forms only upon hybridization of a guide RNA to a target RNA. For example, upon hybridization of the guide RNA to the target RNA, the latent structural feature is formed in the resulting double stranded RNA (also referred herein as guide-target RNA scaffold). In such cases, a structural feature can include, but is not limited to, a mismatch, a wobble base pair, a symmetric internal loop, an asymmetric internal loop, a symmetric bulge, or an asymmetric bulge. In other instances, a structural feature can be a pre-formed structure (e.g., a GluR2 recruitment hairpin, or a hairpin from U7 snRNA). [0062] As used herein, the term “targeting sequence” can be used interchangeable with “targeting domain” or “targeting region” and refers to a polynucleotide sequence within an engineered guide RNA sequence that is at least partially complementary to a target polynucleotide. The target polynucleotide (e.g., a target RNA or a target DNA) may be a region of a polynucleotide of interest, such as a gene or a messenger RNA. As used herein, a “complementary” sequence refers to a sequence that is a reverse complement relative to a second sequence. [0063] As disclosed herein, a “bulge” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand. A bulge can independently have from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair – a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a “mismatch.” Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an “internal loop.” A “symmetrical bulge” refers to a bulge where the same number of nucleotides is present on each side of the bulge. An “asymmetrical bulge” refers to a bulge where a different number of nucleotides are present on each side of the bulge. [0064] The term “complementary” or “complementarity” refers to the ability of a nucleic acid to form one or more bonds with a corresponding nucleic acid sequence by, for example, hydrogen bonding (e.g., traditional Watson-Crick), covalent bonding, or other similar methods. In Watson- Crick base pairing, a double hydrogen bond forms between nucleobases T and A, whereas a triple
Attorney Docket No.199235.769601 hydrogen bond forms between nucleobases C and G. For example, the sequence A-G-T can be complementary to the sequence T-C-A. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary, respectively). “Perfectly complementary” can mean that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein can refer to a degree of complementarity that can be at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%.97%, 98%, 99%, or 100% over a region of 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides, or can refer to two nucleic acids that hybridize under stringent conditions (i.e., stringent hybridization conditions). Nucleic acids can include nonspecific sequences. As used herein, the term “nonspecific sequence” or “not specific” can refer to a nucleic acid sequence that contains a series of residues that may not be designed to be complementary to or can be only partially complementary to any other nucleic acid sequence. [0065] The terms “determining,” “measuring,” “evaluating,” “assessing,” “assaying,” and “analyzing” can be used interchangeably herein to refer to forms of measurement. The terms include determining if an element is present or not (for example, detection). These terms can include quantitative, qualitative or quantitative and qualitative determinations. Assessing can be relative or absolute. “Detecting the presence of” can include determining the amount of something present in addition to determining whether it is present or absent depending on the context. [0066] The term “encode,” as used herein, refers to an ability of a polynucleotide to provide information or instructions sequence sufficient to produce a corresponding gene expression product. In a non-limiting example, mRNA can encode a polypeptide during translation, whereas DNA can encode an mRNA molecule during transcription. [0067] An “engineered latent guide RNA” refers to an engineered guide RNA that comprises a portion of sequence that, upon hybridization or only upon hybridization to a target RNA, substantially forms at least a portion of a structural feature, other than a single A/C mismatch feature at the target adenosine to be edited. [0068] As used herein, the term “facilitates RNA editing” by an engineered guide RNA refers to the ability of the engineered guide RNA when associated with an RNA editing entity and a target RNA to provide a targeted edit of the target RNA by the RNA edited entity. In some instances, the engineered guide RNA can directly recruit or position/orient the RNA editing entity to the proper location for editing of the target RNA. In other instances, the engineered guide RNA when hybridized to the target RNA forms a guide-target RNA scaffold with one or more structural
Attorney Docket No.199235.769601 features as described herein, where the guide-target RNA scaffold with structural features recruits or positions/orients the RNA editing entity to the proper location for editing of the target RNA. [0069] A “guide-target RNA scaffold” as disclosed herein, is the resulting double stranded RNA formed upon hybridization of a guide RNA, with latent structure, to a target RNA. A guide-target RNA scaffold has one or more structural features formed within the double stranded RNA duplex upon hybridization. For example, the guide-target RNA scaffold can have one or more structural features selected from a bulge, mismatch, internal loop, hairpin, or wobble base pair. [0070] As disclosed herein, a “hairpin” includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion. [0071] The term percent “identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full- length of the two sequences to be compared. [0072] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows: [(Number of Identical Positions)/(Total Number of Positions in the Test Sequence)] × 100%
Attorney Docket No.199235.769601 [0073] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match/mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023, are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows: Percent Sequence Identity = (“Percent Identity” output value) × (“Query Coverage” output value) [0074] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions)/(total number of nucleotides in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 1: AAAAAGGGGG (SEQ ID NO: 22) (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (SEQ ID NO: 23) (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5)/(10)] ×100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 24) (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (SEQ ID NO: 25) (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10)/(20)] ×100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (SEQ ID NO: 25) (length =
Attorney Docket No.199235.769601 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (SEQ ID NO: 24) (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10)/(10)] ×100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6. [0075] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions)/(total number of amino acids in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (SEQ ID NO: 26) (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (SEQ ID NO: 27) (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5)/(10)] ×100% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 28) (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (SEQ ID NO: 26) (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10)/(20)] ×100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11: FFFFFYYYYY (SEQ ID NO: 26) (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (SEQ ID NO: 28) (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10)/(10)] ×100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12. [0076] Latent structure refers to a structural feature that substantially forms only upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed, and the latent structure provided in the guide RNA is, thus, unmasked. The formation and structure of a latent structural feature upon binding to the target RNA depends on the guide RNA sequence. For example, formation and structure of the latent structural feature may depend on a pattern of complementary and mismatched residues in the guide RNA sequence relative to the target RNA. The guide RNA sequence may be engineered to have a latent structural feature that forms upon binding to the target RNA. [0077] As disclosed herein, a “macro-footprint” sequence can be positioned such that it flanks a micro-footprint sequence. Further, while a macro-footprint sequence can flank a micro-footprint sequence, additional latent structures can be incorporated that flank either end of the macro-
Attorney Docket No.199235.769601 footprint as well. In some embodiments, such additional latent structures are included as part of the macro-footprint. In some embodiments, such additional latent structures are separate, distinct, or both separate and distinct from the macro-footprint. In some embodiments, a macro-footprint sequence can comprise a barbell macro-footprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop. [0078] As disclosed herein, an “internal loop” refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a “bulge” or a “mismatch,” depending on the size of the structural feature. A “symmetrical internal loop” is formed when the same number of nucleotides is present on each side of the internal loop. An “asymmetrical internal loop” is formed when a different number of nucleotides is present on each side of the internal loop. [0079] “Messenger RNA” or “mRNA” are RNA molecules comprising a sequence that encodes a polypeptide or protein. In general, RNA can be transcribed from DNA. In some cases, precursor mRNA containing non-protein coding regions in the sequence can be transcribed from DNA and then processed to remove all or a portion of the non-coding regions (introns) to produce mature mRNA. As used herein, the term “pre-mRNA” can refer to the RNA molecule transcribed from DNA before undergoing processing to remove the non-protein coding regions. [0080] As disclosed herein, a “mismatch” refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a “bulge” or an “internal loop,” depending on the size of the structural feature. [0081] As used herein, the term “polynucleotide” refers to a single or double-stranded polymer of deoxyribonucleotide (DNA) or ribonucleotide (RNA) bases read from the 5’ to the 3’ end. The term “RNA” is inclusive of dsRNA (double stranded RNA), snRNA (small nuclear RNA), lncRNA (long non-coding RNA), mRNA (messenger RNA), miRNA (microRNA) RNAi (inhibitory RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), tRNA (transfer RNA), rRNA (ribosomal RNA), snoRNA (small nucleolar RNA), and cRNA (complementary RNA). The term
Attorney Docket No.199235.769601 DNA is inclusive of cDNA, genomic DNA, and DNA-RNA hybrids. A sequence of a polynucleotide may be provided interchangeably as an RNA sequence (containing U) or a DNA sequence (containing T). A sequence provided as an RNA sequence is intended to also cover the corresponding DNA sequence and the reverse complement RNA sequence or DNA sequence. A sequence provided as a DNA sequence is intended to also cover the corresponding RNA sequence and the reverse complement RNA sequence or DNA sequence. [0082] The term “protein”, “peptide” and “polypeptide” can be used interchangeably and in their broadest sense can refer to a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits can be linked by peptide bonds. In another embodiment, the subunit can be linked by other bonds, e.g., ester, ether, etc. A protein or peptide can contain at least two amino acids and no limitation can be placed on the maximum number of amino acids which can comprise a protein’s or peptide's sequence. As used herein the term “amino acid” can refer to either natural amino acids, unnatural amino acids, or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs and peptidomimetics. As used herein, the term “fusion protein” can refer to a protein comprised of domains from more than one naturally occurring or recombinantly produced protein, where generally each domain serves a different function. In this regard, the term “linker” can refer to a protein fragment that can be used to link these domains together – optionally to preserve the conformation of the fused protein domains, prevent unfavorable interactions between the fused protein domains which can compromise their respective functions, or both. [0083] The term “structured motif” refers to a combination of two or more structural features in a guide-target RNA scaffold. [0084] The terms “subject,” “individual,” or “patient” can be used interchangeably herein. A “subject” refers to a biological entity containing expressed genetic materials. The biological entity can be a plant, animal, or microorganism, including, for example, bacteria, viruses, fungi, and protozoa. The subject can be tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro. The subject can be a mammal. The mammal can be a non-human primate such as a cynomolgus macaque. The mammal can be a mouse. The mammal can be a human. The subject can be diagnosed or suspected of being at high risk for a disease. In some cases, the subject is not necessarily diagnosed or suspected of being at high risk for the disease. [0085] The term “in vivo” refers to an event that takes place in a subject’s body. [0086] The term “ex vivo” refers to an event that takes place outside of a subject’s body. An ex vivo assay may not be performed on a subject. Rather, it can be performed upon a sample separate from a subject. An example of an ex vivo assay performed on a sample can be an “in vitro” assay.
Attorney Docket No.199235.769601 [0087] The term “in vitro” refers to an event that takes places contained in a container for holding laboratory reagent such that it can be separated from the biological source from which the material can be obtained. In vitro assays can encompass cell-based assays in which living or dead cells can be employed. In vitro assays can also encompass a cell-free assay in which no intact cells can be employed. [0088] The term “wobble base pair” refers to two bases that weakly pair. For example, a wobble base pair can refer to a G paired with a U. [0089] The term “substantially forms” or “substantially formed” as described herein, when referring to a particular secondary structure, refers to formation of at least 80% of the structure under physiological conditions (e.g., physiological pH, physiological temperature, physiological salt concentration, etc.). [0090] As disclosed herein, a structured motif comprises two or more structural features in a guide- target RNA scaffold. [0091] As used herein, the terms “treatment” or “treating” can be used in reference to a pharmaceutical or other intervention regimen for obtaining beneficial or desired results in the recipient. Beneficial or desired results include but are not limited to a therapeutic benefit and/or a prophylactic benefit. A therapeutic benefit can refer to eradication or amelioration of one or more symptoms of an underlying disorder being treated. Also, a therapeutic benefit can be achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement can be observed in the subject, notwithstanding that the subject can still be afflicted with the underlying disorder. A prophylactic effect includes delaying, preventing, or eliminating the appearance of a disease or condition, delaying or eliminating the onset of one or more symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof. For prophylactic benefit, a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease can undergo treatment, even though a diagnosis of this disease may not have been made. Engineered Guide RNAs [0092] Disclosed herein are engineered guide RNAs and engineered polynucleotides encoding the same for site-specific, selective editing of a target RNA, for example MECP2 target RNA (e.g., a human or cyno MECP2 RNA or a Mecp2 mouse RNA) via an RNA editing entity or a biologically active fragment thereof. An engineered guide RNA of the present disclosure can comprise latent structures, such that when the engineered guide RNA is hybridized to the target RNA to form a
Attorney Docket No.199235.769601 guide-target RNA scaffold, at least a portion of the latent structure manifests as at least a portion of a structural feature as described herein. [0093] An engineered guide RNA as described herein comprises a targeting domain with complementarity to a target RNA. As such, a guide RNA can be engineered to site- specifically/selectively target and hybridize to the target RNA, thus facilitating editing of specific nucleotide in the target RNA via an RNA editing entity or a biologically active fragment thereof. [0094] An engineered guide RNA as described herein comprises a targeting domain with complementarity to a MECP2 target RNA described herein. As such, a guide RNA can be engineered to site-specifically/selectively target and hybridize to the MECP2 target RNA, thus facilitating editing of specific nucleotide in the MECP2 target RNA via an RNA editing entity or a biologically active fragment thereof. The targeting domain can include a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, the nucleotide opposes a base to be edited by the RNA editing entity or biologically active fragment thereof and does not base pair, or does not fully base pair, with the base to be edited. This mismatch can help to localize editing of the RNA editing entity to the desired base of the target RNA. However, in some instances there can be some, and in some cases significant, off target editing in addition to the desired edit. [0095] Hybridization of the target RNA and the targeting domain of the guide RNA produces specific secondary structures in the guide-target RNA scaffold that manifest upon hybridization, which are referred to herein as “latent structures.” Latent structures when manifested become structural features described herein, including mismatches, bulges, internal loops, and hairpins. Without wishing to be bound by theory, the presence of structural features described herein that are produced upon hybridization of the guide RNA with the target RNA configure the guide RNA to facilitate a specific, or selective, targeted edit of the target RNA via the RNA editing entity or biologically active fragment thereof. Further, the structural features in combination with the mismatch described above generally facilitate an increased amount of editing of a target adenosine, fewer off target edits, or both, as compared to a construct comprising the mismatch alone or a construct having perfect complementarity to a target RNA. Accordingly, rational design of latent structures in engineered guide RNAs of the present disclosure to produce specific structural features in a guide-target RNA scaffold can be a powerful tool to promote editing of the target RNA with high specificity, selectivity, and robust activity. FIG.1 illustrates a target RNA scaffold with exemplary structural features. [0096] Provided herein are engineered guides and polynucleotides encoding the same; as well as compositions comprising said engineered guide RNAs or said polynucleotides. As used herein, the term “engineered” in reference to a guide RNA or polynucleotide encoding the same refers to a
Attorney Docket No.199235.769601 non-naturally occurring guide RNA or polynucleotide encoding the same. For example, the present disclosure provides for engineered polynucleotides encoding engineered guide RNAs. In some embodiments, the engineered guide comprises RNA. In some embodiments, the engineered guide comprises DNA. In some examples, the engineered guide comprises modified RNA bases or unmodified RNA bases. In some embodiments, the engineered guide comprises modified DNA bases or unmodified DNA bases. In some examples, the engineered guide comprises both DNA and RNA bases. [0097] In some examples, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target RNA molecule, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA molecule, wherein the guide- target RNA scaffold comprises at least one structural feature, and wherein the guide-target RNA scaffold recruits an RNA editing entity and facilitates a chemical modification of a base of a nucleotide in the target RNA molecule by the RNA editing entity. In some cases, engineered guide RNAs can be developed by machine learning and/or a high throughput screen, for example in a cell line described herein or in an in vitro assay. [0098] In some examples, the engineered guides provided herein comprise an engineered guide that can be configured, upon hybridization to a target MECP2 RNA molecule, to form, at least in part, a guide-target RNA scaffold with at least a portion of the target RNA molecule, wherein the guide-target RNA scaffold comprises at least one structural feature, and wherein the guide-target RNA scaffold recruits an RNA editing entity and facilitates a chemical modification of a base of a nucleotide in the target RNA molecule by the RNA editing entity. In some cases, engineered guide RNAs can be developed by machine learning and/or a high throughput screen, for example in a cell line described herein or in an in vitro assay. [0099] A target RNA of an engineered guide RNA of the present disclosure can be a pre-mRNA or mRNA. In some embodiments, the target RNA is MECP2 pre-mRNA. In some embodiments, the target RNA is MECP2 mRNA. In some cases, the target RNA comprises a UGA stop codon. Where the target RNA is MECP2 pre-mRNA, the engineered guide RNA of the present disclosure can facilitate editing of a coding region of MECP2. Where the target RNA is MECP2 mRNA, the engineered guide RNA of the present disclosure can facilitate editing of a coding region of MECP2. In some cases, editing of a coding region of MECP2 can result in the restoration of a full- length protein and/or functional protein. In some cases, editing of a premature stop codon (e.g., UGA) of MECP2 can result in the restoration of a full-length protein and/or functional protein. [00100] In some embodiments, a target RNA of an engineered guide RNA can be a wild type mouse MECP2 transcript, a wild type human MECP2 transcript, a human MECP2 transcript with
Attorney Docket No.199235.769601 a R168X mutation, or a mouse MECP2 transcript with a R168X mutation. In some embodiments, a target RNA of an engineered guide RNA can be a human MECP2 transcript with a R168X mutation, or a mouse MECP2 transcript with a R168X mutation. [00101] In some embodiments, the engineered guide RNA of the present disclosure hybridizes to a sequence of the target RNA. In some embodiments, part of the engineered guide RNA (e.g., a targeting domain) hybridizes to the sequence of the target RNA. The part of the engineered guide RNA that hybridizes to the target RNA is of sufficient complementary to the sequence of the target RNA for hybridization to occur. [00102] In some instances, one or more engineered guide RNA(s) can independently hybridize to (target): (1) different target sequences of the same target RNA, or (2) different target sequences of different target RNAs. For example, a first engineered guide RNA can hybridize to a target sequence of a first target RNA while a second engineered guide RNA can hybridize to a target sequence of a second target RNA, in some instances resulting in ADAR-mediated editing of an adenosine in the target sequence of the first target RNA and an adenosine in the target sequence of the second target RNA. [00103] In some instances, one or more engineered guide RNA(s) can independently hybridize to (target) the same target sequence of a target RNA. For example, the one or more engineered guide RNA(s) encoded by the one or more polynucleotide sequence(s) can each independently hybridize to a target sequence of a target RNA and/or facilitate editing of the same adenosine in the target sequence of the target RNA via ADAR. In some cases, the one or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA have identical sequences (i.e., the one or more engineered guide RNAs are copies of each other). [00104] In some embodiments, an engineered guide RNA herein can be of any length. In some cases, an engineered guide RNA is at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229,
Attorney Docket No.199235.769601 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, or up to about 250 nucleotides in length. In some cases, an engineered guide RNA comprises a length of about 25 to 200, 50 to 150, 75 to 100, 80 to 110, 90 to 120, 95 to 115, 60 to 200, 60 to 180, 60 to 160, 60 to 140, 70 to 200, 70 to 180, 70 to 160, 70 to 140, 80 to 200, 80 to 190, 80 to 170, 80 to 160, 80 to 150, 80 to 140, 80 to 130, 80 to 120, 90 to 200, 90 to 190, 90 to 180, 90 to 170, 90 to 160, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 100 to 200, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 110 to 200, 110 to 190, 110 to 180, 110 to 170, 110 to 160, 110 to 150, 110 to 140, 110 to 120, 120 to 200, 120 to 190, 120 to 180, 120 to 170, 120 to 160, 120 to 150, 120 to 140, 130 to 200, 130 to 190, 130 to 180, 130 to 170, 130 to 160, 130 to 150, 140 to 200, 140 to 190, 140 to 180, 140 to 170, 140 to 160, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 160 to 200, 160 to 190, 160 to 180, 180 to 200, 190 to 250, 200 to 250, 210 to 250, 200 to 230, 220 to 240, or 230 to 250 nucleotides. A. Targeting Domain [00105] Engineered guide RNAs disclosed herein can be engineered in any way suitable for RNA editing. In some examples, an engineered guide RNA generally comprises at least a targeting sequence that allows it to hybridize (i.e. is capable of hybridizing) to a region of a target RNA molecule, for example a target MECP2 RNA molecule. A targeting sequence can also be referred to as a “targeting domain” or a “targeting region”. [00106] The targeting sequence of an engineered guide RNA allows the engineered guide RNA to hybridize to a target polynucleotide (e.g., a target RNA) through base pairing, such as Watson Crick base pairing. A targeting sequence can be located at either the N-terminus or C-terminus of the engineered guide RNA, or both, or the targeting sequence can be within the engineered guide RNA. The targeting sequence can be of any length sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence is at least about: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, or up to about 200 nucleotides in
Attorney Docket No.199235.769601 length. In an embodiment, an engineered polynucleotide comprises a targeting sequence that is about 25 to 200, 50 to 150, 75 to 100, 80 to 110, 90 to 120, 95 to 115, 60 to 200, 60 to 180, 60 to 160, 60 to 140, 70 to 200, 70 to 180, 70 to 160, 70 to 140, 80 to 200, 80 to 190, 80 to 170, 80 to 160, 80 to 150, 80 to 140, 80 to 130, 80 to 120, 90 to 200, 90 to 190, 90 to 180, 90 to 170, 90 to 160, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 100 to 200, 100 to 190, 100 to 180, 100 to 170, 100 to 160, 100 to 150, 100 to 140, 100 to 130, 100 to 120, 110 to 200, 110 to 190, 110 to 180, 110 to 170, 110 to 160, 110 to 150, 110 to 140, 110 to 120, 120 to 200, 120 to 190, 120 to 180, 120 to 170, 120 to 160, 120 to 150, 120 to 140, 130 to 200, 130 to 190, 130 to 180, 130 to 170, 130 to 160, 130 to 150, 140 to 200, 140 to 190, 140 to 180, 140 to 170, 140 to 160, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 160 to 200, 160 to 190 or 160 to 180 nucleotides in length. [00107] A targeting sequence comprises at least partial sequence complementarity to a target polynucleotide. The targeting sequence may have a degree of sequence complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. In some cases, the targeting sequence comprises 95%, 96%, 97%, 98%, 99%, or 100% sequence complementarity to the target polynucleotide. In some cases, the targeting sequence comprises less than 100% complementarity to the target polynucleotide sequence. For example, the targeting sequence may have a single base mismatch relative to the target polynucleotide when bound to the target polynucleotide. In other cases, the targeting sequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 20, 30, 40 or up to about 50 base mismatches relative to the target polynucleotide when bound to the target polynucleotide. In some aspects, nucleotide mismatches can be associated with structural features provided herein. In some aspects, a targeting sequence comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or up to about 15 nucleotides that differ in complementarity from a wild type polynucleotide of a subject target polynucleotide. [00108] A targeting sequence comprises nucleotide residues having complementarity to a target polynucleotide. The targeting sequence may have a number of residues with complementarity to the target polynucleotide sufficient to hybridize with the target polynucleotide. The complementary residues may be contiguous or non-contiguous. In some cases, the targeting sequence comprises at least 50 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 250 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises 50,
Attorney Docket No.199235.769601 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises more than 50 nucleotides total and has at least 50 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 150 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 200 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 250 nucleotides having complementarity to the target polynucleotide. In some cases, the targeting sequence comprises from 50 to 400 nucleotides total and has from 50 to 300 nucleotides having complementarity to the target polynucleotide. In some cases, the at least 50 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 150 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 200 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 250 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. In some cases, the from 50 to 300 nucleotides having complementarity to the target polynucleotide are separated by one or more mismatches, one or more bulges, or one or more loops, or any combination thereof. For example, a targeting sequence comprises a total of 54 nucleotides wherein, sequentially, 25 nucleotides are complementarity to the target polynucleotide,
Attorney Docket No.199235.769601 4 nucleotides form a bulge, and 25 nucleotides are complementarity to the target polynucleotide. As another example, a targeting sequence comprises a total of 118 nucleotides wherein, sequentially, 25 nucleotides are complementarity to the target polynucleotide, 4 nucleotides form a bulge, 25 nucleotides are complementarity to the target polynucleotide, 14 nucleotides form a loop, and 50 nucleotides are complementary to the target polynucleotide. [00109] In some embodiments, a guide RNA or a polynucleotide encoding a guide RNA disclosed herein can comprise a targeting sequence disclosed in Table 1. In some embodiments, a composition can comprise an engineered guide RNA comprising any one of SEQ ID NO: 31 – SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 51. In some embodiments, a composition can comprise an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 31 – SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 51. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, and SEQ ID NO: 52. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, and SEQ ID NO: 52. In some embodiments, a composition can comprise an engineered guide RNA comprising any one of SEQ ID NO: 55 – SEQ ID NO: 60 or SEQ ID NO: 89 – SEQ ID NO: 521. In some embodiments, a composition can comprise an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity to any one of SEQ ID NO: 55 – SEQ ID NO: 60 or SEQ ID NO: 89 – SEQ ID NO: 521. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising any one of SEQ ID NO: 61 – SEQ ID NO: 66 or SEQ ID NO: 522 – SEQ ID NO: 954. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of any one of SEQ ID NO: 61 – SEQ ID NO: 66 or SEQ ID NO: 522 – SEQ ID NO: 954. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 34. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 34. In some embodiments, a composition can comprise a polynucleotide encoding an
Attorney Docket No.199235.769601 engineered guide RNA comprising SEQ ID NO: 51. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 51. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 56. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 56. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 58. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 58. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 59. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 59. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 60. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 60. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 403. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 403. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA comprising SEQ ID NO: 428. In some embodiments, a composition can comprise a polynucleotide encoding an engineered guide RNA with at least about: 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 428. TABLE 1 – MECP2 targeting guide RNA sequences and DNA encoding the guides
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA CCCGACCCCUGCCAGUUCCUGGAGCUAACCC CCCGACCCCTGCCAGTTCCTGGAGCTAACCCT UGAUUUGGGCUUCUUAGGUGGUUUCUGCUCC GATTTGGGCTTCTTAGGTGGTTTCTGCTCCGAC ID T C ID A C Q G T A T A A C T A T T A G T G G C A A G G T A T G A T G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUAAACCCGAUUUGGACUUCUUAGGUGGUU CTAAACCCGATTTGGACTTCTTAGGTGGTTGA T A A C G G A G C T A C C G T A G T A G T A A C C G C T G C G T A G T G A T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAA AAACACGGAGAUUUAAGCUUCUUUUGUGGU AACACGGAGATTTAAGCTTCTTTTGTGGTGTG C A G A C A C A A A A A C C A A T A T A A C A C A G A A C A G G A T A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAA AAACACGGAGAUUUGGCAGCUGAGUAUUUU AACACGGAGATTTGGCAGCTGAGTATTTTGCA A A G G A A G G A T A C A T A A G A G T A C A A T A A A A A A C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAA AAACACGGAGAUUUGGGCGAAUCUUAGGUG AACACGGAGATTTGGGCGAATCTTAGGTGGTT A A C T A G A G T A C A G A C T A G T A T A A T A A A C C A A A G
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAA AAACACGGAGAUUUGCGCUUCGAUUAGGAG AACACGGAGATTTGCGCTTCGATTAGGAGGTC A A T T A G A G A C A G A A T C A C T A A A G T A A C A C T A C T A A C
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAA AAACACGGAGAUUUGGGCUUCAAUUAGGUG AACACGGAGATTTGGGCTTCAATTAGGTGGTC G C A G G T G G T G G T G A T G T T G G T G C T G T T G G T G T A G G T G G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGACCCCUGCCAGUUCCUGGA GGGGCGTCCCCGACCCCTGCCAGTTCCTGGAG GCUAACCCUGAUUUGGGCUACAGUUAGGUGG CTAACCCTGATTTGGGCTACAGTTAGGTGGGC T G A T G A T G C T G T T G G T G T A G T T G C G T A G G T G A C G A G C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUACCCUCGAUUUGGGCUUCUUGGUGGUUU CTACCCTCGATTTGGGCTTCTTGGTGGTTTCAG C G T C G T T G T A G A G A T G C C G C G G C G C G C C G C G C C G C
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUACCCUCAUUUGCCUUACACUGAGGUGG CTTACCCTCATTTGCCTTACACTGAGGTGGCCT A G T T G T A G G A G T G T G G T G G T G A G G G A G G C G C G G C
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUUUCUAAAUUUGGGCUUCAAGGUUGUU CTTTTCTAAATTTGGGCTTCAAGGTTGTTTCGG C G A G C G A T G C G C G A G C G G A T G A G G C G A G T A G A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUUGGGAGAUUCUAAAUUCAACUAGGUG CTTTGGGAGATTCTAAATTCAACTAGGTGGCA G A G T G T G T T G G G G G A G T A G G C G T C G T G G C C G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUUGGGUCUAAAGGGAGCCUAGGUGUUU CTTTGGGTCTAAAGGGAGCCTAGGTGTTTTCT G C G C C G C G T G C A G C G C G G A G T A G T T G G C T G C
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUUGGGAUCUAAAGGCUUCAGUUGGUGG CTTTGGGATCTAAAGGCTTCAGTTGGTGGTTC A G C T G G C G G A G T C G T C G G T G A G C T G T T G T C G C G C G G C T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUUUGGGAGUCUAAAGAUAACCUUAGGUU CTTTGGGAGTCTAAAGATAACCTTAGGTTGTC G C A G C G C A G C G G T G T T G C G G C G T G T T G T A C A G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUCCCUCUGAUUUGGGCUUCUUGGGUGGUU CTCCCTCTGATTTGGGCTTCTTGGGTGGTTCGC C G A A G T T G A G G C G C C G T A G G T G G T G G C G C C G G A G T A G A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUACCCUCGAUUUGGGCUUCUUGGUGGUUU CTACCCTCGATTTGGGCTTCTTGGTGGTTTCGG C G G C G A C G C C G G C G T A G C C G A G T A G C C G T G T G T A G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUACCCUCGAUUUGGGCUUCUUCGGUGGUU CTACCCTCGATTTGGGCTTCTTCGGTGGTTGGG C G C C G A A G C C G C C G C C G C G C C G C C G T A G C C G T A G C C G T A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUCCCUCUGAUUUGGGCUGCUUGGGUGGUU CTCCCTCTGATTTGGGCTGCTTGGGTGGTTCGC C G C C C C A C G C C T C T A C C A C C C A C C C C C C G C C C C T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAC CGAAACGGAGAUUAGGGCUUCUUAGGUGCCG GAAACGGAGATTAGGGCTTCTTAGGTGCCGTC A C T T C A C C C C A C C A C C C G C T G C T G A T G T T G T G T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUAAACCCGAUUUGGGCUUCUUAGGUGGUU CTAAACCCGATTTGGGCTTCTTAGGTGGTTGG T G C T G A T G T G T T G T G T G G G G T G G T G T G T G T T G C T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAG GCUAAACCCGAUUUGGGCUUCUUAUGUGGUU CTAAACCCGATTTGGGCTTCTTATGTGGTTTGG T G T G T G T G T G A T T A C T A T T T C A T A T T T G C T C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUAGCCUUCUUAGGUUUUU TAAAAGGAGATTAGCCTTCTTAGGTTTTTTCTG C T C A T C T C T C T T C T G T A T C C T T A T C A T C T T C G T G A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGGCUUGGUAGGUUCA TAAAAGGAGATTTGGGCTTGGTAGGTTCATTC C T G A T C C T T T C A T A A T T T A C T C T C T C T T A T C T T A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUCGGGCUUCUUAGGUGGU TAAAAGGAGATTCGGGCTTCTTAGGTGGTTTC T C A T T G T C A T C C T C T C T T T G T C C T C A T G A T A T A T C T
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGCCUUCUUUGGUGCC TAAAAGGAGATTTGGCCTTCTTTGGTGCCTGA C T A T A C T T A T C T A T T T C T C A T G T C A T A A T A C T C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUCGGGCUUCUUUGGUGUC TAAAAGGAGATTCGGGCTTCTTTGGTGTCGTA A T T T C G T C A T C T C T A C T C A T C T C T C T A T T G T C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGCCUUCGUUGGUUUC TAAAAGGAGATTTGGCCTTCGTTGGTTTCTGG T A T C T T T C T T T T T C C T A C T C C T C T C A T C A T C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGGCUUCCUAUAUGGU TAAAAGGAGATTTGGGCTTCCTATATGGTTTC A T T T T C A T T T A T C T C A T C T A T C A T T T T T A T T A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGGCUUCUUAGGUGGU TAAAAGGAGATTTGGGCTTCTTAGGTGGTTTC C T T G T C A T C A T C A T C C T T C T A A T C C T T T T A T T T T G T C A
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGGGCUUCCUGUGGUUU TAAAAGGAGATTTGGGCTTCCTGTGGTTTCTA A T C C T A C T A T T C T C T C A T C A T C A T C A T C T A T C A T G C
Attorney Docket No.199235.769601 Engineered Guide RNA Sequence DNA Sequence Encoding the Guide RNA GGGGCGUCCCCGGCCUCUGCCAGUUCCUGGA GGGGCGTCCCCGGCCTCTGCCAGTTCCTGGAT UUAAAAGGAGAUUUGCGCUUCUUAGGUGGU TAAAAGGAGATTTGCGCTTCTTAGGTGGTTTC A T C C T A C T C A T C A T A A T T T T G T C A T C G T C A T T A T C C T A A
Attorney Docket No.199235.769601 [00110] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 31. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 38. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -6 relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 6/6 symmetric internal loop at position 30 relative to the target adenosine at position 0. [00111] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 32. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 39. In some cases, an engineered guide RNA when hybridized to the target RNA herein can comprise a 6/6 symmetric internal loop at position -6 relative to the target adenosine at position 0, a wobble base pair at the -28, -24, -21, -16, 15, 21, 43, 48, 54 and 57 positions relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 6/6 symmetric internal loop at position 30 relative to the target adenosine at position 0. [00112] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 40. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -15 relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 6/6 symmetric internal loop at position 33 relative to the target adenosine at position 0.
Attorney Docket No.199235.769601 [00113] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 34. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 41. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -6 relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 6/6 symmetric internal loop at position 30 relative to the target adenosine at position 0. [00114] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 35. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 42. In some cases, an engineered guide RNA when hybridized to the target RNA herein can comprise a 6/6 symmetric internal loop at position -6 relative to the target adenosine at position 0, a wobble base pair at the -29, -24, -21, -16, 6, 15, 21, 50, 57, 61 and 65 positions relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 12/12 symmetric internal loop at position 30 relative to the target adenosine at position 0. [00115] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 36. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 43. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -15 relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 6/6 symmetric internal loop at position 33 relative to the target adenosine at position 0.
Attorney Docket No.199235.769601 [00116] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 37. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 44. In some cases, an engineered guide RNA when hybridized to the target RNA herein can comprise a 6/6 symmetric internal loop at position -15 relative to the target adenosine at position 0, a wobble base pair at the -29, -24, 6, 15, 21, 28, 48, 54 and 61 positions relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, and a 10/10 symmetric internal loop at position 33 relative to the target adenosine at position 0. [00117] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 47. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 48. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -15 and 33 relative to the target adenosine at position 0, a 1-1 mismatch at position -7, -5, 25, 54, and 57 relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, a 3-1 asymmetric bulge at position 6 relative to the target adenosine at position 0, and a 1-3 asymmetric bulge at position 19 relative to the target adenosine at position 0. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -15 and 33 relative to the target adenosine at position 0, a 1-1 mismatch at position -7 and 25, relative to the target adenosine at position 0, a 2/2 symmetric bulge at position -1 relative to the target adenosine at position 0, a 3-1 asymmetric bulge at position 6 relative to the target adenosine at position 0, and a 1-3 asymmetric bulge at position 19 relative to the target adenosine at position 0. [00118] In some cases, an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence
Attorney Docket No.199235.769601 identity to SEQ ID NO: 51. In some cases, polynucleotide encoding an engineered guide RNA can comprise a sequence with at least: 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 52. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -6 and 30 relative to the target adenosine at position 0, a 0-1 asymmetric bulge at position -2 relative to the target adenosine at position 0, a 1-0 asymmetric bulge at position 0 relative to the target adenosine at position 0, a 2-1 asymmetric bulge at position 5 relative to the target adenosine at position 0, and a 1-1 mismatch at position 15 relative to the target adenosine at position 0. In some cases, an engineered guide RNA herein when hybridized to the target RNA can comprise a 6/6 symmetric internal loop at position -6 and 30 relative to the target adenosine at position 0, a 2/2 symmetric bulge at a position -1 relative to the target adenosine at position 0, , a 2-1 asymmetric bulge at position 5 relative to the target adenosine at position 0, a 1-1 mismatch at position 15 relative to the target adenosine at position 0, a 1-1 wobble base pair at position 54 relative to the target adenosine at position 0, and a 1-1 wobble base pair at position 57 relative to the target adenosine at position 0. B. Engineered Guide RNAs Having a Recruiting Domain [00119] In some examples, a subject engineered guide RNA comprises a recruiting domain that recruits an RNA editing entity (e.g., ADAR), where in some instances, the recruiting domain is formed and present in the absence of binding to the target RNA. A “recruiting domain” can be referred to herein as a “recruiting sequence” or a “recruiting region”. In some examples, a subject engineered guide can be configured to facilitate editing of a base of a nucleotide of a polynucleotide of a region of a subject target RNA, modulation expression of a polypeptide encoded by the subject target RNA, or both. In some cases, an engineered guide can be configured to facilitate an editing of a base of a nucleotide or polynucleotide of a region of an RNA by a subject RNA editing entity. In order to facilitate editing, an engineered guide RNA of the disclosure can recruit an RNA editing entity. Various RNA editing entity recruiting domains can be utilized. In some examples, a recruiting domain comprises: Glutamate ionotropic receptor AMPA type subunit 2 (GluR2), APOBEC, or Alu. [00120] In some examples, more than one recruiting domain can be included in an engineered guide of the disclosure. In examples where a recruiting domain can be present, the recruiting domain can be utilized to position the RNA editing entity to effectively react with a subject target
Attorney Docket No.199235.769601 RNA after the targeting sequence, for example an antisense sequence, hybridizes to a target RNA. In some cases, a recruiting domain can allow for transient binding of the RNA editing entity to the engineered guide. In some examples, the recruiting domain allows for permanent binding of the RNA editing entity to the engineered guide. A recruiting domain can be of any length. In some cases, a recruiting domain can be from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, up to about 80 nucleotides in length. In some cases, a recruiting domain can be no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 80 nucleotides in length. In some cases, a recruiting domain can be about 45 nucleotides in length. In some cases, at least a portion of a recruiting domain comprises at least 1 to about 75 nucleotides. In some cases, at least a portion of a recruiting domain comprises about 45 nucleotides to about 60 nucleotides. [00121] In an embodiment, a recruiting domain comprises a GluR2 sequence or functional fragment thereof. In some cases, a GluR2 sequence can be recognized by an RNA editing entity, such as an ADAR or biologically active fragment thereof. In some embodiments, a GluR2 sequence can be a non-naturally occurring sequence. In some cases, a GluR2 sequence can be modified, for example for enhanced recruitment. In some embodiments, a GluR2 sequence can comprise a portion of a naturally occurring GluR2 sequence and a synthetic sequence. [00122] In some examples, a recruiting domain comprises a GluR2 sequence, or a sequence having at least about 70%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% identity and/or length to: GUGGAAUAGUAUAACAAUAUGCUAAAUGUUGUUAUAGUAUCCCAC (SEQ ID NO: 1). In some cases, a recruiting domain can comprise at least about 80% sequence homology to at least about 10, 15, 20, 25, or 30 nucleotides of SEQ ID NO: 1. In some examples, a recruiting domain can comprise at least about 90%, 95%, 96%, 97%, 98%, or 99% sequence homology and/or length to SEQ ID NO: 1. [00123] Additional, RNA editing entity recruiting domains are also contemplated. In an embodiment, a recruiting domain comprises an apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) domain. In some cases, an APOBEC domain can comprise a non- naturally occurring sequence or naturally occurring sequence. In some embodiments, an APOBEC-domain-encoding sequence can comprise a modified portion. In some cases, an APOBEC-domain-encoding sequence can comprise a portion of a naturally occurring APOBEC-
Attorney Docket No.199235.769601 domain-encoding-sequence. In another embodiment, a recruiting domain can be from an Alu domain. [00124] Any number of recruiting domains can be found in an engineered guide of the present disclosure. In some examples, at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to about 10 recruiting domains can be included in an engineered guide. Recruiting domains can be located at any position of subject guides. In some cases, a recruiting domain can be on an N-terminus, middle, or C- terminus of a polynucleotide. A recruiting domain can be upstream or downstream of a targeting sequence. In some cases, a recruiting domain flanks a targeting sequence of a subject guide. A recruiting sequence can comprise all ribonucleotides or deoxyribonucleotides, although a recruiting domain comprising both ribo- and deoxyribonucleotides can in some cases not be excluded. C. Engineered Guide RNAs with Latent Structure [00125] In some examples, an engineered guide disclosed herein useful for facilitating editing of a target RNA by an RNA editing entity can be an engineered latent guide RNA. An “engineered latent guide RNA” refers to an engineered guide RNA that comprises latent structure. “Latent structure” refers to a structural feature that substantially forms upon hybridization of a guide RNA to a target RNA. For example, the sequence of a guide RNA provides one or more structural features, but these structural features substantially form only upon hybridization to the target RNA, and thus the one or more latent structural features manifest as structural features upon hybridization to the target RNA. Upon hybridization of the guide RNA to the target RNA, the structural feature is formed and the latent structure provided in the guide RNA is, thus, unmasked. [00126] A double stranded RNA (dsRNA) substrate is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. The resulting dsRNA substrate is also referred to herein as a “guide-target RNA scaffold.” Described herein are structural features that can be present in a guide-target RNA scaffold of the present disclosure. Examples of features include a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a non-recruiting hairpin). Engineered guide RNAs of the present disclosure can have from 1 to 50 features. Engineered guide RNAs of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features. In some embodiments, structural features (e.g., mismatches, bulges, internal loops) can be formed from latent structure in
Attorney Docket No.199235.769601 an engineered latent guide RNA upon hybridization of the engineered latent guide RNA to a target RNA and, thus, formation of a guide-target RNA scaffold. In some embodiments, structural features are not formed from latent structures and are, instead, pre-formed structures (e.g., a GluR2 recruitment hairpin or a hairpin from U7 snRNA). [00127] FIG. 1 shows a legend of various exemplary structural features present in guide-target RNA scaffolds formed upon hybridization of a latent guide RNA of the present disclosure to a target RNA. Example structural features shown include an 8/7 asymmetric loop (8 nucleotides on the target RNA side and 7 nucleotides on the guide RNA side), a 2/2 symmetric bulge (2 nucleotides on the target RNA side and 2 nucleotides on the guide RNA side), a 1/1 mismatch (1 nucleotide on the target RNA side and 1 nucleotide on the guide RNA side), a 5/5 symmetric internal loop (5 nucleotides on the target RNA side and 5 nucleotides on the guide RNA side), a 24 bp region (24 nucleotides on the target RNA side base paired to 24 nucleotides on the guide RNA side), and a 2/3 asymmetric bulge (2 nucleotides on the target RNA side and 3 nucleotides on the guide RNA side). Unless otherwise noted, the number of participating nucleotides in a given structural feature is indicated as the nucleotides on the target RNA side over nucleotides on the guide RNA side. Also shown in this legend is a key to the positional annotation of each figure. For example, the target nucleotide to be edited is designated as the 0 position. Downstream (3’) of the target nucleotide to be edited, each nucleotide is counted in increments of +1. Upstream (5’) of the target nucleotide to be edited, each nucleotide is counted in increments of -1. Thus, the example 2/2 symmetric bulge in this legend is at the +12 to +13 position in the guide-target RNA scaffold. Similarly, the 2/3 asymmetric bulge in this legend is at the -36 to-37 position in the guide-target RNA scaffold. As used herein, positional annotation is provided with respect to the target nucleotide to be edited and on the target RNA side of the guide-target RNA scaffold. As used herein, if a single position is annotated, the structural feature extends from that position away from position 0 (target nucleotide to be edited). For example, if a latent guide RNA is annotated herein as forming a 2/3 asymmetric bulge at position -36, then the 2/3 asymmetric bulge forms from -36 position to the -37 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold. As another example, if a latent guide RNA is annotated herein as forming a 2/2 symmetric bulge at position +12, then the 2/2 symmetric bulge forms from the +12 to the +13 position with respect to the target nucleotide to be edited (position 0) on the target RNA side of the guide-target RNA scaffold. [00128] In some examples, the engineered guides disclosed herein lack a recruiting region and recruitment of the RNA editing entity can be effectuated by structural features of the guide-target RNA scaffold formed by hybridization of the engineered guide RNA and the target RNA. In some
Attorney Docket No.199235.769601 examples, the engineered guide, when present in an aqueous solution and not bound to the target RNA molecule, does not comprise structural features that recruit the RNA editing entity (e.g., ADAR). The engineered guide RNA, upon hybridization to a target RNA, form with the target RNA molecule, one or more structural features that recruits an RNA editing entity (e.g., ADAR). [00129] In cases where a recruiting sequence can be absent, an engineered guide RNA can be still capable of associating with a subject RNA editing entity (e.g., ADAR) to facilitate editing of a target RNA and/or modulate expression of a polypeptide encoded by a subject target RNA. This can be achieved through structural features formed in the guide-target RNA scaffold formed upon hybridization of the engineered guide RNA and the target RNA. Structural features can comprise any one of a: mismatch, symmetrical bulge, asymmetrical bulge, symmetrical internal loop, asymmetrical internal loop, hairpins, wobble base pairs, or any combination thereof. [00130] Described herein are structural features which can be present in a guide-target RNA scaffold of the present disclosure. Examples of features include a mismatch, a bulge (symmetrical bulge or asymmetrical bulge), an internal loop (symmetrical internal loop or asymmetrical internal loop), or a hairpin (a recruiting hairpin or a non-recruiting hairpin). Engineered guide RNAs of the present disclosure can have from 1 to 50 features. Engineered guide RNAs of the present disclosure can have from 1 to 5, from 5 to 10, from 10 to 15, from 15 to 20, from 20 to 25, from 25 to 30, from 30 to 35, from 35 to 40, from 40 to 45, from 45 to 50, from 5 to 20, from 1 to 3, from 4 to 5, from 2 to 10, from 20 to 40, from 10 to 40, from 20 to 50, from 30 to 50, from 4 to 7, or from 8 to 10 features. In some embodiments, structural features (e.g., mismatches, bulges, internal loops) can be formed from latent structure in an engineered latent guide RNA upon hybridization of the engineered latent guide RNA to a target RNA and, thus, formation of a guide-target RNA scaffold. In some embodiments, structural features are not formed from latent structures and are, instead, pre-formed structures (e.g., a GluR2 recruitment hairpin or a hairpin from U7 snRNA). [00131] A guide-target RNA scaffold is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. As disclosed herein, a mismatch refers to a single nucleotide in a guide RNA that is unpaired to an opposing single nucleotide in a target RNA within the guide-target RNA scaffold. A mismatch can comprise any two single nucleotides that do not base pair. Where the number of participating nucleotides on the guide RNA side and the target RNA side exceeds 1, the resulting structure is no longer considered a mismatch, but rather, is considered a bulge or an internal loop, depending on the size of the structural feature. In some embodiments, a mismatch in a guide RNA is to a G, a C, or a U in the target RNA. For example, a G in the target RNA can mismatch with a G, an A or a U in the guide RNA. In another example, a C in the target RNA can mismatch with a C, an A, or a U in the guide RNA. In another example,
Attorney Docket No.199235.769601 a U in the target RNA can mismatch with a U, a G, or a C in the guide RNA. In some embodiments, a mismatch in a guide RNA is to an A in the target RNA. For example, an A in the target RNA can mismatch with an A, a G, or a C in the guide RNA. In some embodiments, a mismatch in a guide RNA is to a G, a C, or a U in the MECP2 target RNA. For example, a G in the MECP2 target RNA can mismatch with a G, an A or a U in the guide RNA. In another example, a C in the MECP2 target RNA can mismatch with a C, an A, or a U in the guide RNA. In another example, a U in the MECP2 target RNA can mismatch with a U, a G, or a C in the guide RNA. In some embodiments, a mismatch in a guide RNA is to an A in the MECP2 target RNA. For example, an A in the MECP2 target RNA can mismatch with an A, a G, or a C in the guide RNA. In some embodiments, a mismatch is an A/C mismatch. An A/C mismatch can comprise a C in an engineered guide RNA of the present disclosure opposite an A in a target RNA. An A/C mismatch can comprise an A in an engineered guide RNA of the present disclosure opposite a C in a target RNA. A G/G mismatch can comprise a G in an engineered guide RNA of the present disclosure opposite a G in a target RNA. In some embodiments, a guide RNA of the present disclosure may not have an A/C mismatch and each A of the target RNA is base paired to a U in the engineered guide RNA. [00132] In some embodiments, a mismatch positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited. A mismatch can also help confer sequence specificity. Thus, a mismatch can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00133] In another aspect, a structural feature comprises a wobble base. A wobble base pair refers to two bases that weakly base pair. For example, a wobble base pair of the present disclosure can refer to a G paired with a U. Thus, a wobble base pair can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00134] In some cases, a structural feature can be a hairpin. As disclosed herein, a hairpin includes an RNA duplex wherein a portion of a single RNA strand has folded in upon itself to form the RNA duplex. The portion of the single RNA strand folds upon itself due to having nucleotide sequences that base pair to each other, where the nucleotide sequences are separated by an intervening sequence that does not base pair with itself, thus forming a base-paired portion and non-base paired, intervening loop portion. A hairpin can have from 10 to 500 nucleotides in length of the entire duplex structure. The loop portion of a hairpin can be from 3 to 15 nucleotides long. A hairpin can be present in any of the engineered guide RNAs disclosed herein. The engineered guide RNAs disclosed herein can have from 1 to 10 hairpins. In some embodiments, the engineered guide RNAs disclosed herein have 1 hairpin. In some embodiments, the engineered guide RNAs disclosed herein have 2 hairpins. As disclosed herein, a hairpin can include a recruitment hairpin
Attorney Docket No.199235.769601 or a non-recruitment hairpin. A hairpin can be located anywhere within the engineered guide RNAs of the present disclosure. In some embodiments, one or more hairpins is proximal to or present at the 3’ end of an engineered guide RNA of the present disclosure, proximal to or at the 5’ end of an engineered guide RNA of the present disclosure, proximal to or within the targeting domain (e.g., the targeting sequence) of the engineered guide RNAs of the present disclosure, or any combination thereof. [00135] A recruitment hairpin, as disclosed herein, can recruit at least in part an RNA editing entity, such as ADAR. In some cases, a recruitment hairpin can be formed and present in the absence of binding to a target RNA. In some embodiments, a recruitment hairpin is a GluR2 domain or portion thereof. In some embodiments, a recruitment hairpin is an Alu domain or portion thereof. A recruitment hairpin, as defined herein, can include a naturally occurring ADAR substrate or truncations thereof. Thus, a recruitment hairpin such as GluR2 is a pre-formed structural feature that may be present in constructs comprising an engineered guide RNA, not a structural feature formed by latent structure provided in an engineered latent guide RNA. [00136] In some aspects, a structural feature comprises a non-recruitment hairpin. A non- recruitment hairpin, as disclosed herein, does not have a primary function of recruiting an RNA editing entity. A non-recruitment hairpin, in some instances, does not recruit an RNA editing entity. In some instances, a non-recruitment hairpin has a dissociation constant for binding to an RNA editing entity under physiological conditions that is insufficient for binding. For example, a non-recruitment hairpin has a dissociation constant for binding an RNA editing entity at 25 ºC that is greater than about 1 mM, 10 mM, 100 mM, or 1 M, as determined in an in vitro assay. A non- recruitment hairpin can exhibit functionality that improves localization of the engineered guide RNA to the target RNA. In some embodiments, the non-recruitment hairpin improves nuclear retention. In some embodiments, the non-recruitment hairpin comprises a hairpin from U7 snRNA. Thus, a non-recruitment hairpin such as a hairpin from U7 snRNA is a pre-formed structural feature that can be present in constructs comprising engineered guide RNA constructs, not a structural feature formed by latent structure provided in an engineered latent guide RNA. [00137] A hairpin of the present disclosure can be of any length. In an aspect, a hairpin can be from about 10-500 or more nucleotides. In some cases, a hairpin can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,
Attorney Docket No.199235.769601 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 or more nucleotides. In other cases, a hairpin can also comprise 10 to 20, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 10 to 90, 10 to 100, 10 to 110, 10 to 120, 10 to 130, 10 to 140, 10 to 150, 10 to 160, 10 to 170, 10 to 180, 10 to 190, 10 to 200, 10 to 210, 10 to 220, 10 to 230, 10 to 240, 10 to 250, 10 to 260, 10 to 270, 10 to 280, 10 to 290, 10 to 300, 10 to 310, 10 to 320, 10 to 330, 10 to 340, 10 to 350, 10 to 360, 10 to 370, 10 to 380, 10 to 390, 10 to 400, 10 to 410, 10 to 420, 10 to 430, 10 to 440, 10 to 450, 10 to 460, 10 to 470, 10 to 480, 10 to 490, or 10 to 500 nucleotides. [00138] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. As disclosed herein, a bulge refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where contiguous nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand. The nucleotides in a bulge of the guide RNA can comprise any nucleotide, in any order so long as they are not complementary to their positional counterparts on the target RNA. A bulge can change the secondary or tertiary structure of the guide-target RNA scaffold. A bulge can independently have
Attorney Docket No.199235.769601 from 0 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the target RNA side of the guide-target RNA scaffold or a bulge can independently have from 0 to 4 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 to 4 contiguous nucleotides on the guide RNA side of the guide-target RNA scaffold. However, a bulge, as used herein, does not refer to a structure where a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA do not base pair – a single participating nucleotide of the engineered guide RNA and a single participating nucleotide of the target RNA that do not base pair is referred to herein as a mismatch. Further, where the number of participating nucleotides on either the guide RNA side or the target RNA side exceeds 4, the resulting structure is no longer considered a bulge, but rather, is considered an internal loop. In some embodiments, the guide-target RNA scaffold of the present disclosure has 2 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 3 bulges. In some embodiments, the guide-target RNA scaffold of the present disclosure has 4 bulges. Thus, a bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00139] In some embodiments, the presence of a bulge in a guide-target RNA scaffold can position or can help to position ADAR to selectively edit the target A in the target RNA and reduce off- target editing of non-target A(s) in the target RNA. In some embodiments, the presence of a bulge in a guide-target RNA scaffold can recruit or help recruit additional amounts of ADAR. Bulges in guide-target RNA scaffolds disclosed herein can recruit other proteins, such as other RNA editing entities. In some embodiments, a bulge positioned 5’ of the edit site can facilitate base-flipping of the target A to be edited. A bulge can also help confer sequence specificity for the A of the target RNA to be edited, relative to other A(s) present in the target RNA. For example, a bulge can help direct ADAR editing by constraining it in an orientation that yields selective editing of the target A. [00140] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. A symmetrical bulge is formed when the same number of nucleotides is present on each side of the bulge. For example, a symmetrical bulge in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-
Attorney Docket No.199235.769601 target RNA scaffold target and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical bulge of the present disclosure can be formed by 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00141] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. A bulge can be a symmetrical bulge or an asymmetrical bulge. An asymmetrical bulge is formed when a different number of nucleotides is present on each side of the bulge. For example, an asymmetrical bulge in a guide-target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 1 nucleotide on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 0 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 2 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 2 nucleotides on the engineered guide RNA
Attorney Docket No.199235.769601 side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 1 nucleotide on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 3 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 2 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 4 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical bulge of the present disclosure can be formed by 3 nucleotides on the target RNA side of the guide-target RNA scaffold and 4 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical bulge can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00142] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. As disclosed herein, an internal loop refers to the structure substantially formed only upon formation of the guide-target RNA scaffold, where nucleotides in either the engineered guide RNA or the target RNA are not complementary to their positional counterparts on the opposite strand and where one side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, has 5 nucleotides or more. The nucleotides in an internal loop of the guide RNA can comprise any nucleotide, in any order so long as they are not
Attorney Docket No.199235.769601 complementary to their positional counterparts on the target RNA. Where the number of participating nucleotides on both the guide RNA side and the target RNA side drops below 5, the resulting structure is no longer considered an internal loop, but rather, is considered a bulge or a mismatch, depending on the size of the structural feature. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. Internal loops present in the vicinity of the edit site can help with base flipping of the target A in the target RNA to be edited. [00143] One side of the internal loop, either on the target RNA side or the engineered guide RNA side of the guide-target RNA scaffold, can be formed by from 5 to 150 nucleotides. One side of the internal loop can be formed by 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 120, 135, 140, 145, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 nucleotides, or any number of nucleotides therebetween. One side of the internal loop can be formed by 5 nucleotides. One side of the internal loop can be formed by 10 nucleotides. One side of the internal loop can be formed by 15 nucleotides. One side of the internal loop can be formed by 20 nucleotides. One side of the internal loop can be formed by 25 nucleotides. One side of the internal loop can be formed by 30 nucleotides. One side of the internal loop can be formed by 35 nucleotides. One side of the internal loop can be formed by 40 nucleotides. One side of the internal loop can be formed by 45 nucleotides. One side of the internal loop can be formed by 50 nucleotides. One side of the internal loop can be formed by 55 nucleotides. One side of the internal loop can be formed by 60 nucleotides. One side of the internal loop can be formed by 65 nucleotides. One side of the internal loop can be formed by 70 nucleotides. One side of the internal loop can be formed by 75 nucleotides. One side of the internal loop can be formed by 80 nucleotides. One side of the internal loop can be formed by 85 nucleotides. One side of the internal loop can be formed by 90 nucleotides. One side of the internal loop can be formed by 95 nucleotides. One side of the internal loop can be formed by 100 nucleotides. One side of the internal loop can be formed by 110 nucleotides. One side of the internal loop can be formed by 120 nucleotides. One side of the internal loop can be formed by 130 nucleotides. One side of the internal loop can be formed by 140 nucleotides. One side of the internal loop can be formed by 150 nucleotides. One side of the internal loop can be formed by 200 nucleotides. One side of the internal loop can be formed by 250 nucleotides. One side of the internal loop can be formed by 300 nucleotides. One side of the internal loop can be formed by 350 nucleotides. One side of the internal loop can be formed by 400 nucleotides. One side of the internal loop can be formed by 450 nucleotides. One side of the internal loop can be formed by 500 nucleotides. One side of the internal loop can be formed by 600 nucleotides. One side of the internal loop can be formed by 700 nucleotides. One side of the
Attorney Docket No.199235.769601 internal loop can be formed by 800 nucleotides. One side of the internal loop can be formed by 900 nucleotides. One side of the internal loop can be formed by 1000 nucleotides. Thus, an internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00144] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. A symmetrical internal loop is formed when the same number of nucleotides is present on each side of the internal loop. For example, a symmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have the same number of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 5 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 8 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 9 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 10 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 11 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 11 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 12 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 12 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 13 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 13 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 14 nucleotides on the engineered guide RNA side of
Attorney Docket No.199235.769601 the guide-target RNA scaffold target and 14 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 15 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 15 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 20 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 20 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 30 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 30 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 40 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 40 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 50 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 60 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 60 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 70 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 70 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 80 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 80 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 90 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 90 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 100 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 110 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 110 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 120 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 120 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 130 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 130 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop
Attorney Docket No.199235.769601 of the present disclosure can be formed by 140 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 140 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 150 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 200 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 250 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 250 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 300 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 350 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 350 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 400 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 450 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 450 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 500 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 600 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 600 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 700 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 700 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 800 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 800 nucleotides on the target RNA side of the guide-target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 900 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 900 nucleotides on the target RNA side of the guide- target RNA scaffold. A symmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold target and 1000
Attorney Docket No.199235.769601 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a symmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00145] In some embodiments, a symmetrical internal loop can be positioned upstream (5’) of the target A (0 position), downstream (3’) of the target A, or both. In some embodiments, when referring to a location of a structural feature a “-“ or negative integer indicates a nucleotide upstream (5’) of the target A or of a specified position, while a positive integer indicates a
nucleotide downstream (3’) of the target A, or a specified position. In some instances, a first symmetrical internal loop can be downstream of the target A and a second symmetrical internal loop can be upstream of the target A. In some cases, a symmetric internal loop can be from position: -1 to -25, -2 to -10, -4 to -8, -5 to -7, -2 to -15, -4 to -20, -8 to -15, or -10 to -22 relative to the target A. In some cases, a symmetric internal loop can be located at position: -25, -24, -23, -22, -21, -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, or -1 relative to the target A. In some cases, a symmetric internal loop can be from position: +1 to +60, +10 to +50, +10 to +40, +20 to +50, +20 to +40, +25 to +45, +31 to +35, +10 to +20, +15 to +30, +25 to +45, or +45 to +60 relative to the target A. In some cases, a symmetric internal loop can be located at position: 1, +2, +3, +4, +5, +6, +7, +8, +9, +10, +11, +12, +13, +14, +15, +16, +17, +18, +19, +20, +21, +22, +23, +24, +25, +26, +27, +28, +29, +30, +31, +32, +33, +34, +35, +36, +37, +38, +39, +40, +41, +42, +43, +44, +45, +46, +47, +48, +49, +50, +51, +52, +53, +54, +55, +56, +57, +58, +59, or +60 relative to the target A. In some cases, a first symmetric internal loop within about: 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of the 5’ end of the guide RNA, and a second symmetric internal loop within about: 80 bp, 70 bp, 60 bp, 50 bp, 40 bp, 30 bp, 25 bp, 20 bp, 15 bp, 10 bp, or 5 bp of the 3’ end of the guide RNA. [00146] A double stranded RNA (dsRNA) substrate (i.e., a guide-target RNA scaffold) is formed upon hybridization of an engineered guide RNA of the present disclosure to a target RNA. An internal loop can be a symmetrical internal loop or an asymmetrical internal loop. An asymmetrical internal loop is formed when a different number of nucleotides is present on each side of the internal loop. For example, an asymmetrical internal loop in a guide-target RNA scaffold of the present disclosure can have different numbers of nucleotides on the engineered guide RNA side and the target RNA side of the guide-target RNA scaffold. [00147] An asymmetrical internal loop of the present disclosure can be formed by from 5 to 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 150 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the
Attorney Docket No.199235.769601 number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by from 5 to 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and from 5 to 1000 nucleotides on the target RNA side of the guide-target RNA scaffold, wherein the number of nucleotides is the different on the engineered side of the guide-target RNA scaffold target than the number of nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides on the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 7 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides
Attorney Docket No.199235.769601 on the target RNA side of the guide-target RNA scaffold and 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 6 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 8 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 7 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 9 nucleotides internal loop the target RNA side of the guide-target RNA
Attorney Docket No.199235.769601 scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 8 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold and 10 nucleotides internal loop the target RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 9 nucleotides on the target RNA side of the guide-target RNA scaffold and 10 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 5 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA
Attorney Docket No.199235.769601 side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 50 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide
Attorney Docket No.199235.769601 RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 50 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 100 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the
Attorney Docket No.199235.769601 target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 100 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 150 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 5 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical
Attorney Docket No.199235.769601 internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 150 nucleotides on the engineered guide RNA side of the guide- target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide- target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 200 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 200 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 300 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide
Attorney Docket No.199235.769601 RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 300 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 400 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 400 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 500 nucleotides on the target RNA side of the guide-target RNA scaffold and 1000 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. An asymmetrical internal loop of the present disclosure can be formed by 1000 nucleotides on the target RNA side of the guide-target RNA scaffold and 500 nucleotides on the engineered guide RNA side of the guide-target RNA scaffold. Thus, an asymmetrical internal loop can be a structural feature formed from latent structure provided by an engineered latent guide RNA. [00148] As disclosed herein, a base paired (bp) region refers to a region of the guide-target RNA scaffold in which bases in the guide RNA (e.g., the bases in the targeting sequence of the guide RNA) are paired with opposing bases in the target polynucleotide. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to the other end of the guide-target RNA scaffold. Base paired regions can extend between two structural features. Base paired regions can extend from one end or proximal to one end of the guide-target RNA scaffold to or proximal to a structural feature. Base paired regions can extend from a structural feature to the other end of the guide-target RNA scaffold. In some embodiments, a base paired region has from 1 to 50, 1 to 75, 1 to 100, 1 to 125, 1 to 150, 1 to 175, 1 to 200, 1 to 225, 1 to 250, 1 to 275, 1 to 300, 50 to 75, 50 to 100, 50 to 125, 50 to 150, 50 to 175, 50 to 200, 50 to 225, 50 to 250, 50 to 275, 50 to 300, 60 to 75, 60 to 100, 60 to 125, 60 to 150, 60 to 175, 60 to 200, 60 to 225, 60 to 250, 60 to 275, 60 to 300, 70 to 100, 70 to 125, 70 to 150, 70 to 175, 70 to 200, 70 to 225, 70 to 250, 70 to 275, 70 to 300, 80 to 100, 80 to 125, 80 to 150, 80 to 175, 80 to 200, 80 to 225, 80 to 250, 80 to 275, 80 to 300, 90 to 125, 90 to 150, 90 to 175, 90 to 200, 90 to
Attorney Docket No.199235.769601 225, 90 to 250, 90 to 275, 90 to 300, 100 to 125, 100 to 150, 100 to 175, 100 to 200, 100 to 225, 100 to 250, 100 to 275, 100 to 300, 150 to 200, 150 to 225, 150 to 250, 150 to 275, or 150 to 300 base pairs. In some embodiments, a base paired region has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300 base pairs. D. Guides with Macro-Footprints [00149] Guide RNAs of the present disclosure can further comprise a macro-footprint. In some embodiments, the macro-footprint comprises a barbell macro-footprint. A micro-footprint can serve to guide an RNA editing enzyme and direct its activity towards the target adenosine to be edited. A “barbell” as described herein refers to a pair of internal loop latent structures that manifest upon hybridization of the guide RNA to the target RNA. In some embodiments, each internal loop is positioned towards the 5′ end or the 3′ end of the guide-target RNA scaffold formed upon hybridization of the guide RNA and the target RNA. In some embodiments, each internal loop flanks opposing sides of the micro-footprint sequence. Insertion of a barbell macro-footprint sequence flanking opposing sides of the micro-footprint sequence, upon hybridization of the guide RNA to the target RNA, results in formation of barbell internal loops on opposing sides of the micro-footprint. In some cases, barbell internal loops can comprise at least one structural feature that facilitates editing of a specific target RNA. [00150] As described herein, a “micro-footprint” sequence refers to a sequence with latent structures that, when manifested, facilitate editing of the adenosine of a target RNA via an adenosine deaminase enzyme. A macro-footprint can serve to guide an or focus RNA editing entity (e.g., ADAR) and direct its activity towards a micro-footprint. In some embodiments, included
Attorney Docket No.199235.769601 within the micro-footprint sequence is a nucleotide that is positioned such that, when the guide RNA is hybridized to the target RNA, said nucleotide is opposite the adenosine to be edited by the ADAR enzyme and does not base pair with the adenosine to be edited. This nucleotide is referred to herein as the “mismatched position” or “mismatch” and can be a cytosine. Micro-footprint sequences as described herein have upon hybridization of the engineered guide RNA and target RNA, at least one structural feature selected from the group consisting of: a bulge, an internal loop, a mismatch, a hairpin, and any combination thereof. Engineered guide RNAs with superior micro- footprint sequences can be selected based on their ability to facilitate editing of a specific target RNA. Engineered guide RNAs selected for their ability to facilitate editing of a specific target are capable of adopting various micro-footprint latent structures, which can vary on a target-by-target basis. [00151] In some embodiments, the presence of barbells flanking the micro-footprint can improve one or more aspects of editing. For example, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a higher amount of on-target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Additionally, and or alternatively, the presence of a barbell macro-footprint in addition to a micro-footprint can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA lacking the barbells. Further, while the effect of various micro-footprint structural features can vary on a target-by-target basis based on selection in a high throughput screen, the increase in the one or more aspects of editing provided by the barbell macro-footprint structures can be independent of the particular target RNA. For example, macro-footprints (e.g., barbell macro-footprints) and micro-footprints can provide an increased amount of on-target adenosine editing relative to an otherwise comparable guide RNA lacking the barbells. In other embodiments, the presence of the barbell macro-footprint in addition to the micro-footprint described here can result in a lower amount of local off-target adenosine editing, relative to an otherwise comparable guide RNA, upon hybridization of the guide RNA and target RNA to form a guide-target RNA scaffold lacking the barbells. [00152] A dumbbell design in an engineered guide RNA comprises two symmetrical internal loops, wherein the target A to be edited is positioned between the two symmetrical loops for selective editing of the target A. The two symmetrical internal loops are each formed by 6 nucleotides on the guide RNA side of the guide-target RNA scaffold and 6 nucleotides on the target RNA side of the guide-target RNA scaffold. Thus, a dumbbell can be a structural feature formed from latent structure provided by an engineered latent guide RNA.
Attorney Docket No.199235.769601 [00153] In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell is positioned at least about 5 bases (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 bases) away from the A/C mismatch with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A/C mismatch. In some embodiments, the first internal loop of the barbell or the second internal loop of the barbell is positioned at most about 50 bases away from the A/C mismatch (e.g., 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5) with respect to the base of the first internal loop or the second internal loop that is the most proximal to the A/C mismatch. [00154] In some embodiments, a first internal loop or a second internal loop independently comprises a number of bases of at least about 5 bases or greater (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150); about 150 bases or fewer (e.g., 145, 135, 125, 115, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g., 5-150, 6-145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-115, 13-110, 14-105, 15-100, 16-95, 17-90, 18-85, 19-80, 20-75, 21- 70, 22-65, 23-60, 24-55, 25-50) of the engineered guide RNA and a number of bases of at least about 5 bases or greater (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150); about 150 bases or fewer (e.g., 145, 135, 125, 115, 95, 85, 75, 65, 55, 45, 35, 25, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5); or at least about 5 bases to at least about 150 bases (e.g., 5-150, 6-145, 7-140, 8-135, 9-130, 10-125, 11-120, 12-115, 13-110, 14-105, 15-100, 16-95, 17-90, 18-85, 19-80, 20-75, 21-70, 22-65, 23-60, 24-55, 25-50) of the target RNA. [00155] In some embodiments, provided herein are engineered guide RNAs comprising a barbell macro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a micro-footprint. In some embodiments, provided herein are engineered guide RNAs comprising a macro-footprint and a micro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a micro-footprint in the absence of a macro-footprint. In some cases, an engineered guide RNA disclosed herein can comprise a macro-footprint in the absence of a micro-footprint. [00156] In some embodiments, a macro-footprint sequence can comprise a barbell macro-footprint sequence comprising latent structures that, when manifested, produce a first internal loop and a second internal loop. [00157] In some examples, a first internal loop is positioned near the 5′ end of the guide-target RNA scaffold and a second internal loop is positioned near the 3′ end of the guide-target RNA
Attorney Docket No.199235.769601 scaffold. The length of the dsRNA comprises a 5′ end and a 3′ end, where up to half of the length of the guide-target RNA scaffold at the 5′ end can be considered to be “near the 5′ end” while up to half of the length of the guide-target RNA scaffold at the 3′ end can be considered “near the 3′ end.” Non-limiting examples of the 5′ end can include about 50% or less of the total length of the dsRNA at the 5′ end, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. Non-limiting examples of the 3′ end can include about 50% or less of the total length of the dsRNA at the 3′ end about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%. [00158] In some embodiments, the engineered guide RNAs of the disclosure comprising a barbell macro-footprint sequence (that manifests as a first internal loop and a second internal loop) can improve RNA editing efficiency, increase the amount or percentage of RNA editing generally, as well as for on-target nucleotide editing, such as on-target adenosine. In some embodiments, the engineered guide RNAs of the disclosure comprising a first internal loop and a second internal loop can also facilitate a decrease in the amount of or reduce off-target nucleotide editing, such as off-target adenosine or unintended adenosine editing. The decrease or reduction in some examples can be of the number of off-target edits or the percentage of off-target edits. [00159] Each of the first and second internal loops of the barbell macro-footprint can independently be symmetrical or asymmetrical, where symmetry is determined by the number of bases or nucleotides of the engineered guide RNA and the number of bases or nucleotides of the target RNA, that together form each of the first and second internal loops. E. Engineered Polynucleotides Encoding Engineered Guide RNAs [00160] An engineered polynucleotide as described herein can comprise one or more polynucleotide sequence(s) that encode one or more engineered guide RNA(s). For example, an engineered polynucleotide can comprise 1, 2, 3, 4, or more than 4 polynucleotide sequence(s) that encode 1, 2, 3, 4, or more than 4 engineered guide RNAs. [00161] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target): (1) different target sequences of the same target RNA, or (2) different target sequences of different target RNAs. For example, a first engineered guide RNA encoded by a first polynucleotide sequence can hybridize to a target sequence of a first target RNA while a second engineered guide RNA encoded by a second polynucleotide sequence can hybridize to a target sequence of a second target RNA, in some instances resulting in ADAR-
Attorney Docket No.199235.769601 mediated editing of an adenosine in the target sequence of the first target RNA and an adenosine in the target sequence of the second target RNA. [00162] In some instances, the engineered polynucleotide can comprise one or more polynucleotide sequence(s) encoding one or more engineered guide RNA(s) that independently hybridize to (target) the same target sequence of a target RNA. For example, the one or more engineered guide RNA(s) encoded by the one or more polynucleotide sequence(s) can each independently hybridize to a target sequence of a target RNA and/or facilitate editing of the same adenosine in the target sequence of the target RNA via ADAR. In some cases, the one or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA have identical sequences (i.e., the one or more engineered guide RNAs are copies of each other). [00163] Alternatively, two or more engineered guide RNA(s) that hybridize to (target) the same target sequence of a target RNA can comprise different sequences. For example, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to: 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some instances, a first engineered guide RNA encoded by an engineered polynucleotide can have at least about 70% to about 99% sequence identity, at least about 60% to about 99% sequence identity, at least about 80% to about 99% sequence identity, at least about 60% to about 70% sequence identity, at least about 70% to about 80% sequence identity, at least about 75% to about 85% sequence identity, at least about 85% to about 99% sequence identity, at least about 85% to about 90% sequence identity, at least about 88% to about 93% sequence identity, at least about 90% to about 95% sequence identity, at least about 92% to about 99% sequence identity, or at least about 95% to about 99% sequence identity to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 60% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of
Attorney Docket No.199235.769601 less than, greater than, or equal to about 61% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 62% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 63% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 64% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 65% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 66% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 67% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 68% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 69% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the
Attorney Docket No.199235.769601 same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 70% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 71% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 72% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 73% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 74% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 75% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 76% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 77% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of
Attorney Docket No.199235.769601 less than, greater than, or equal to about 78% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 79% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 80% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 81% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 82% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 83% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 84% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 85% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 86% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the
Attorney Docket No.199235.769601 same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 87%, to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 88% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 89% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 90% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 91% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 92% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 93% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 94% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of
Attorney Docket No.199235.769601 less than, greater than, or equal to about 95% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 96% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 97% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 98% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some cases, a first engineered guide RNA encoded by an engineered polynucleotide can have a sequence identity of less than, greater than, or equal to about 99% to a second engineered guide RNA encoded by the engineered polynucleotide, where the second engineered guide RNA hybridizes to (targets) the same target sequence of a target RNA as the first engineered guide RNA. In some embodiments, polynucleotides encoding a first engineered guide RNA, a second engineered guide RNA, or both can be delivered via an AAV. In some instances, the AAV can be formulated in a composition, such as any of the pharmaceutical compositions disclosed herein. F. Additional Engineered Guide RNA Components [00164] The present disclosure provides for engineered guide RNAs with additional structural features and components. For example, an engineered guide RNA described herein can be circular. In another example, an engineered guide RNA described herein can comprise a U7, an SmOPT sequence, or a combination of both. [00165] In some cases, an engineered guide RNA can be circularized. In some cases, an engineered guide RNA provided herein can be circularized or in a circular configuration. In some aspects, an at least partially circular guide RNA lacks a 5’ hydroxyl or a 3’ hydroxyl. [00166] In some examples, an engineered guide RNA can comprise a backbone comprising a plurality of sugar and phosphate moieties covalently linked together. In some examples, a backbone of an engineered guide RNA can comprise a phosphodiester bond linkage between a
Attorney Docket No.199235.769601 first hydroxyl group in a phosphate group on a 5’ carbon of a deoxyribose in DNA or ribose in RNA and a second hydroxyl group on a 3’ carbon of a deoxyribose in DNA or ribose in RNA. [00167] In some embodiments, a backbone of an engineered guide RNA can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to a solvent. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to nucleases. In some embodiments, a backbone of an engineered guide can lack a 5’ reducing hydroxyl, a 3’ reducing hydroxyl, or both, capable of being exposed to hydrolytic enzymes. In some instances, a backbone of an engineered guide can be represented as a polynucleotide sequence in a circular 2-dimensional format with one nucleotide after the other. In some instances, a backbone of an engineered guide can be represented as a polynucleotide sequence in a looped 2-dimensional format with one nucleotide after the other. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be joined through a phosphorus-oxygen bond. In some cases, a 5’ hydroxyl, a 3’ hydroxyl, or both, can be modified into a phosphoester with a phosphorus-containing moiety. [00168] As described herein, an engineered guide can comprise a circular structure. An engineered polynucleotide can be circularized from a precursor engineered polynucleotide. Such a precursor engineered polynucleotide can be a precursor engineered linear polynucleotide. In some cases, a precursor engineered linear polynucleotide can be a precursor for a circular engineered guide RNA. For example, a precursor engineered linear polynucleotide can be a linear mRNA transcribed from a plasmid, which can be configured to circularize within a cell using the techniques described herein. A precursor engineered linear polynucleotide can be constructed with domains such as a ribozyme domain and a ligation domain that allow for circularization when inserted into a cell. A ribozyme domain can include a domain that is capable of cleaving the linear precursor RNA at specific sites (e.g., adjacent to the ligation domain). A precursor engineered linear polynucleotide can comprise, from 5’ to 3’: a 5’ ribozyme domain, a 5’ ligation domain, a circularized region, a 3’ ligation domain, and a 3’ ribozyme domain. In some cases, a circularized region can comprise a guide RNA described herein. In some cases, the precursor polynucleotide can be specifically processed at both sites by the 5’ and the 3’ ribozymes, respectively, to free exposed ends on the 5’ and 3’ ligation domains. The free exposed ends can be ligation competent, such that the ends can be ligated to form a mature circularized structure. For instance, the free ends can include a 5’-OH and a 2’, 3’-cyclic phosphate that are ligated via RNA ligation in the cell. The linear polynucleotide with the ligation and ribozyme domains can be transfected into a cell where it can circularize via endogenous cellular enzymes. In some cases, a polynucleotide can encode an engineered guide RNA comprising the ribozyme and ligation domains described herein, which can circularize within
Attorney Docket No.199235.769601 a cell. For example, PCT/US2021/034301 provides a description of circular guide RNAs and their structures, sequences of circular guide RNAs, and methods of engineering circularized polynucleotide domains, and each of these descriptions in PCT/US2021/034301 is herein incorporated by reference. [00169] An engineered polynucleotide as described herein (e.g., a circularized guide RNA) can include spacer domains. As described herein, a spacer domain can refer to a domain that provides space between other domains. A spacer domain can be used to between a region to be circularized and flanking ligation sequences to increase the overall size of the mature circularized guide RNA. Where the region to be circularized includes a targeting domain as described herein that is configured to associate to a target sequence, the addition of spacers can provide improvements (e.g., increased specificity, enhanced editing efficiency, etc.) for the engineered polynucleotide to the target polynucleotide, relative to a comparable engineered polynucleotide that lacks a spacer domain. In some instances, the spacer domain is configured to not hybridize with the target RNA. In some embodiments, a precursor engineered polynucleotide or a circular engineered guide, can comprise, in order of 5’ to 3’: a first ribozyme domain; a first ligation domain; a first spacer domain; a targeting domain that can be at least partially complementary to a target RNA, a second spacer domain, a second ligation domain, and a second ribozyme domain. In some cases, the first spacer domain, the second spacer domain, or both are configured to not bind to the target RNA when the targeting domain binds to the target RNA. [00170] A circular or looped RNA can be formed by employing a self-cleaving entity, such as a ribozyme, tRNA, aptamer, catalytically active fragment of any of these, or any combination thereof. For example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ end, a 5’ end, or both of a precursor engineered RNA. In another example, a ribozyme, a tRNA, an aptamer, a catalytically active fragment of any of these, or any combination thereof can be added to a 3’ terminal end, a 5’ terminal end, or both of a precursor engineered RNA. A self-cleaving ribozyme can comprise, for example, an RNase P RNA a Hammerhead ribozyme (e.g., a Schistosoma mansoni ribozyme), a glmS ribozyme, an HDV-like ribozyme, an R2 element, a peptidyl transferase 23S rRNA, a GIR1 branching ribozyme, a leadzyme, a group II intron, a hairpin ribozyme, a VS ribozyme, a CPEB3 ribozyme, a CoTC ribozyme, or a group I intron. In some cases, the self-cleaving ribozyme can be a trans-acting ribozyme that joins one RNA end on which it is present to a separate RNA end. In some embodiments, an aptamer can be added to each end of the engineered guide RNA. A ligase can be contacted with the aptamers at each end of the engineered guide RNA to form a covalent linkage between the aptamers thereby forming a circular engineered guide RNA. In some cases, a
Attorney Docket No.199235.769601 self-cleaving element or an aptamer can be configured to facilitate self-circularization of an engineered polynucleotide or a pro-polynucleotide (e.g., from a precursor engineered polypeptide) after transcription in a cell. In some instances, circularization of a guide RNA can be shown by PCR. For example, primers can by developed that bind to the end of a guide RNA and are directed outward such that a product is only formed when guides are circularized. [00171] In some cases, circularization can occur by back-slicing and ligation of an exon. For example, an RNA can be engineered from 5’ to 3’ to comprise a forward complementary sequence intron, an exon (which can comprise the guide sequence), followed by a reverse complementary sequence intron. Once transcribed, the complementary sequence introns can hybridize and form dsRNA. The internal exon containing the guide sequence can be removed by splicing and ligated by an endogenous ligase to form a circular guide. In one example, an engineered guide RNA can initiate circularization in a cell by autocatalytic reactions of encoded ribozymes. After cleavage by one or more ribozymes, the linear polynucleotide will undergo intracellular RNA ligation of the 5’ and the 3’ end of ligation sequences by an endogenous ligase to circularize the guide RNA. [00172] A suitable self-cleaving molecule can include a ribozyme. For example, a ribozyme domain can create an autocatalytic RNA. A ribozyme can comprise an RNase P, an rRNA (such as a Peptidyl transferase 23S rRNA), Leadzyme, Group I intron ribozyme, Group II intron ribozyme, a GIR1 branching ribozyme, a glmS ribozyme, a hairpin ribozyme, a Hammerhead ribozyme, an HDV ribozyme, a Twister ribozyme, a Twister sister ribozyme, a VS ribozyme, a Pistol ribozyme, a Hatchet ribozyme, a viroid, or any combination thereof. A ribozyme can include a P3 twister U2A ribozyme. A ribozyme can comprise 5’ GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT 3’ (SEQ ID NO: 2). A ribozyme can comprise 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCGCC U 3’ (SEQ ID NO: 3). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT 3’ (SEQ ID NO: 2). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GCCAUCAGUCGCCGGUCCCAAGCCCGGAUAAAAUGGGAGGGGGCGGGAAACCGCC U 3’ (SEQ ID NO: 3). A ribozyme can include a P1 Twister Ribozyme. A ribozyme can include 5’ AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 3’ (SEQ ID NO: 4). A ribozyme can include 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC
Attorney Docket No.199235.769601 3’ (SEQ ID NO: 5). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC 3’ (SEQ ID NO: 4). A ribozyme can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACACUGCCAAUGCCGGUCCCAAGCCCGGAUAAAAGUGGAGGGUACAGUCCACGC 3’ (SEQ ID NO: 5). [00173] A ligation domain can facilitate a linkage, covalent or non-covalent, of a first nucleotide to a second nucleotide. In some embodiments, a ligation domain can recruit a ligating entity to facilitate a ligation reaction. In some cases, a ligation domain can recruit a recombining entity to facilitate a homologous recombination. In some instances, a first ligation domain can facilitate a linkage, covalent or non-covalent, to a second ligation domain. In some embodiments, a first ligation domain can facilitate the complementary pairing of a second ligation domain. In some cases, a ligation domain can comprise 5’ AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 6). In some embodiments, a ligation domain can comprise 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 7). In some cases, a ligation domain can comprise 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 8). In some cases, a ligation domain can comprise 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 9). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACCATGCCGACTGATGGCAG 3’ (SEQ ID NO: 6). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GATGTCAGGTGCGGCTGACTACCGTC 3’ (SEQ ID NO: 7). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ AACCAUGCCGACUGAUGGCAG 3’ (SEQ ID NO: 8). In some cases, a ligation domain can comprise at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to 5’ GAUGUCAGGUGCGGCUGACUACCGUC 3’ (SEQ ID NO: 9). [00174] The compositions and methods of the present disclosure provide engineered polynucleotides encoding guide RNAs that are operably linked to a portion of a small nuclear ribonucleic acid (snRNA) sequence. The engineered polynucleotide can include at least a portion of a small nuclear ribonucleic acid (snRNA) sequence. The U7 and U1 small nuclear RNAs, whose natural role is in spliceosomal processing of pre-mRNA, have for decades been re-engineered to alter splicing at desired disease targets. Replacing the first 18 nt of the U7 snRNA (which naturally hybridizes to the spacer element of histone pre-mRNA) with a short targeting (or antisense) sequence of a disease gene, redirects the splicing machinery to alter splicing around that target site.
Attorney Docket No.199235.769601 Furthermore, converting the wild type U7 Sm-domain binding site to an optimized consensus Sm- binding sequence (SmOPT) can increase the expression level, activity, and subcellular localization of the artificial antisense-engineered U7 snRNA. Many subsequent groups have adapted this modified U7 SmOPT snRNA chassis with antisense sequences of other genes to recruit spliceosomal elements and modify RNA splicing for additional disease targets. [00175] An snRNA is a class of small RNA molecules found within the nucleus of eukaryotic cells. They are involved in a variety of important processes such as RNA splicing (removal of introns from pre-mRNA), regulation of transcription factors (7SK RNA) or RNA polymerase II (B2 RNA), and maintaining the telomeres. They are always associated with specific proteins, and the resulting RNA-protein complexes are referred to as small nuclear ribonucleoproteins (snRNP) or sometimes as snurps. There are many snRNAs, which are denominated U1, U2, U3, U4, U5, U6, U7, U8, U9, and U10. [00176] The snRNA of the U7 type is normally involved in the maturation of histone mRNA. This snRNA has been identified in a great number of eukaryotic species (56 so far) and the U7 snRNA of each of these species should be regarded as equally convenient for this disclosure. [00177] Wild-type U7 snRNA includes a stem-loop structure, the U7-specific Sm sequence, and a sequence antisense to the 3′ end of histone pre-mRNA. [00178] In addition to the SmOPT domain, U7 comprises a sequence antisense to the 3′ end of histone pre-mRNA. When this sequence is replaced by a targeting sequence that is antisense to another target pre-mRNA, U7 is redirected to the new target pre-mRNA. Accordingly, the stable expression of modified U7 snRNAs containing the SmOPT domain and a targeting antisense sequence has resulted in specific alteration of mRNA splicing. [00179] The engineered polynucleotide can comprise at least in part an snRNA sequence. The snRNA sequence can be U1, U2, U3, U4, U5, U6, U7, U8, U9, or a U10 snRNA sequence. [00180] In some instances, an engineered polynucleotide that comprises at least a portion of an snRNA sequence (e.g., an snRNA promoter, an snRNA hairpin, and the like) can have superior properties for treating or preventing a disease or condition, relative to a comparable polynucleotide lacking such features. Further, as described herein an engineered polynucleotide that comprises at least a portion of an snRNA sequence can facilitate an editing of a base of a nucleotide in a target RNA (e.g., a pre-mRNA or a mature RNA) at a greater efficiency than a comparable polynucleotide lacking such features. Promoters and snRNA components are described in PCT/US2021/028618 and PCT/US2022/078801, and each of these descriptions in PCT/US2021/028618 and PCT/US2022/078801 are herein incorporated by reference.
Attorney Docket No.199235.769601 [00181] Disclosed herein are engineered RNAs comprising (a) an engineered guide RNA as described herein, and (b) a U7 snRNA hairpin sequence, a SmOPT sequence, or a combination thereof. In some embodiments, the U7 hairpin comprises a human U7 Hairpin sequence, or a mouse U7 hairpin sequence. In some cases, a human U7 hairpin sequence comprises TAGGCTTTCTGGCTTTTTACCGGAAAGCCCCT (SEQ ID NO: 10 or RNA: UAGGCUUUCUGGCUUUUUACCGGAAAGCCCCU (SEQ ID NO: 11). In some cases, a mouse U7 hairpin sequence comprises CAGGTTTTCTGACTTCGGTCGGAAAACCCCT (SEQ ID NO: 12 or RNA: CAGGUUUUCUGACUUCGGUCGGAAAACCCCU SEQ ID NO: 13). In some embodiments, the SmOPT sequence has a sequence of AATTTTTGGAG (SEQ ID NO: 14 or RNA: AAUUUUUGGAG SEQ ID NO: 15). In some embodiments, a guide RNA can comprise a guide RNA comprising a U7 hairpin sequence (e.g., a human or a mouse U7 hairpin sequence), an SmOPT sequence, or a combination thereof. In some cases, a combination of a U7 hairpin sequence and a SmOPT sequence can comprise a SmOPT U7 hairpin sequence, wherein the SmOPT sequence is linked to the U7 sequence. In some cases, a U7 hairpin sequence, an SmOPT sequence, or a combination thereof is downstream (e.g., 3’) of the engineered guide RNA disclosed herein. [00182] Also disclosed herein are promoters for driving the expression of a guide RNA disclosed herein. In some cases, the promoters for driving expression can be 5’ to the guide RNA sequence disclosed herein. In some cases, a promoter can comprise a U1 promoter, a U7 promoter, a U6 promoter or any combination thereof. In some cases, a promoter can comprise a CMV promoter. In some cases, a U7 promoter, or a U6 promoter can be a mouse U7 promoter, or a mouse U6 promoter. In some cases, a U1 promoter, a U7 promoter, or a U6 promoter can be a human U1 promoter, a human U7 promoter, or a human U6 promoter. In some cases, a human U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGA GAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTG ACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATG GACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCT TGTGGAAAGGACGAAACACC (SEQ ID NO: 16) In some cases, a mouse U6 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: GTACTGAGTCGCCCAGTCTCAGATAGATCCGACGCCGCCATCTCTAGGCCCGCGCCG GCCCCCTCGCACAGACTTGTGGGAGAAGCTCGGCTACTCCCCTGCCCCGGTTAATTT
Attorney Docket No.199235.769601 GCATATAATATTTCCTAGTAACTATAGAGGCTTAATGTGCGATAAAAGACAGATAAT CTGTTCTTTTTAATACTAGCTACATTTTACATGATAGGCTTGGATTTCTATAAGAGAT ACAAATACTAAATTATTATTTTAAAAAACAGCACAAAAGGAAACTCACCCTAACTGT AAAGTAATTGTGTGTTTTGAGACTATAAATATCCCTTGGAGAAAAGCCTTGTTTG (SEQ ID NO: 17). In some cases, a human U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TTAACAACAACGAAGGGGCTGTGACTGGCTGCTTTCTCAACCAATCAGCACCGAACT CATTTGCATGGGCTGAGAACAAATGTTCGCGAACTCTAGAAATGAATGACTTAAGTA AGTTCCTTAGAATATTATTTTTCCTACTGAAAGTTACCACATGCGTCGTTGTTTATAC AGTAATAGGAACAAGAAAAAAGTCACCTAAGCTCACCCTCATCAATTGTGGAGTTC CTTTATATCCCATCTTCTCTCCAAACACATACGCA (SEQ ID NO: 18). In some cases, a mouse U7 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TTAACAACATAGGAGCTGTGATTGGCTGTTTTCAGCCAATCAGCACTGACTCATTTG CATAGCCTTTACAAGCGGTCACAAACTCAAGAAACGAGCGGTTTTAATAGTCTTTTA GAATATTGTTTATCGAACCGAATAAGGAACTGTGCTTTGTGATTCACATATCAGTGG AGGGGTGTGGAAATGGCACCTTGATCTCACCCTCATCGAAAGTGGAGTTGATGTCCT TCCCTGGCTCGCTACAGACGCACTTCCGC (SEQ ID NO: 19). In some cases, a human U1 promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: TAAGGACCAGCTTCTTTGGGAGAGAACAGACGCAGGGGCGGGAGGGAAAAAGGGA GAGGCAGACGTCACTTCCTCTTGGCGACTCTGGCAGCAGATTGGTCGGTTGAGTGGC AGAAAGGCAGACGGGGACTGGGCAAGGCACTGTCGGTGACATCACGGACAGGGCG ACTTCTATGTAGATGAGGCAGCGCAGAGGCTGCTGCTTCGCCACTTGCTGCTTCGCC ACGAAGGGAGTTCCCGTGCCCTGGGAGCGGGTTCAGGACCGCTGATCGGAAGTGAG AATCCCAGCTGTGTGTCAGGGCTGGAAAGGGCTCGGGAGTGCGCGGGGCAAGTGAC CGTGTGTGTAAAGAGTGAGGCGTATGAGGCTGTGTCGGGGCAGAGCCCGAAGATCT C (SEQ ID NO: 20). In some cases, a CMV promoter can comprise a sequence with at least about: 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to: ATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCAT TAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGC CTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCA TAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAA CTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACG
Attorney Docket No.199235.769601 TCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACT TTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTT TTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCT CCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCC AAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGT GGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGC CATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACT CTAGAGGATCGAACC (SEQ ID NO: 21). G. Chemically modified guide RNAs [00183] An engineered guide RNA as described herein can comprise at least one chemical modification. In some embodiments, the engineered guide RNA can comprise at least one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 50, 100, or more chemical modifications. In some embodiments, the engineered guide RNA described herein may not comprise a chemical modification. In some cases, the engineered guide RNAs disclosed herein with barbell macro-footprints can be manufactured, chemically modified, and delivered directly to a subject in need thereof as RNA (without a vector, such as an AAV). [00184] Exemplary chemical modifications comprise any one of: 5′ adenylate, 5′ guanosine- triphosphate cap, 5′ N7-Methylguanosine-triphosphate cap, 5′ triphosphate cap, 3′ phosphate, 3′thiophosphate, 5′phosphate, 5′thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9,3′-3′ modifications, 5′-5′ modifications, abasic, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3′DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT- DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers, 2′deoxyribonucleoside analog purine, 2′deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2′-O-methyl ribonucleoside analog, sugar modified analogs, wobble/universal bases, fluorescent dye label, 2′fluoro RNA, 2′O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5′- triphosphate, 5-methylcytidine-5′-triphosphate, 2-O-methyl 3phosphorothioate or any combinations thereof. [00185] A chemical modification can be made at any location of the engineered guide RNA. In some cases, a modification may be located in a 5’ or 3’ end, or both. In some cases, a polynucleotide can comprise a modification at a base selected from: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
Attorney Docket No.199235.769601 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150. In some cases, more than one modification can be made to the engineered guide RNA. In some cases, a modification can be permanent. In other cases, a modification can be transient. In some cases, multiple modifications may be made to the engineered guide RNA. The engineered guide RNA modification can alter physio-chemical properties of a nucleotide, such as their conformation, polarity, hydrophobicity, chemical reactivity, base-pairing interactions, or any combination thereof. [00186] In some embodiments, a chemical modification can also be a phosphorothioate substitute. In some cases, a natural phosphodiester bond can be susceptible to rapid degradation by cellular nucleases and a modification of internucleotide linkage using phosphorothioate (PS) bond substitutes can be more stable towards hydrolysis by cellular degradation. A modification can increase stability in a polynucleic acid. A modification can also enhance biological activity. In some cases, a phosphorothioate enhanced RNA polynucleic acid can inhibit RNase A, RNase T1, calf serum nucleases, or any combinations thereof. These properties can allow the use of PS-RNA polynucleic acids to be used in applications where exposure to nucleases may be of high probability in vivo or in vitro. For example, phosphorothioate (PS) bonds can be introduced between the last 3-5 nucleotides at the 5′-or 3′-end of a polynucleic acid which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout an entire polynucleic acid to reduce attack by endonucleases. [00187] In some embodiments, a chemical modification can occur at 3’OΗ, group, 5’OΗ group, at the backbone, at the sugar component, or at the nucleotide base. Chemical modification can include non-naturally occurring linker molecules of interstrand or intrastrand cross links. In one aspect, the chemically modified nucleic acid comprises modification of one or more of the 3’OΗ or 5’OΗ group, the backbone, the sugar component, or the nucleotide base, or addition of non- naturally occurring linker molecules. In some embodiments, a chemically modified backbone comprises a backbone other than a phosphodiester backbone. In some embodiments, a modified sugar comprises a sugar other than deoxyribose (in modified DNA) or other than ribose (modified RNA). In some embodiments, a modified base comprises a base other than adenine, guanine, cytosine, thymine or uracil. In some embodiments, the engineered guide RNA comprises at least
Attorney Docket No.199235.769601 one chemically modified base. In some instances, an engineered guide RNA can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more modified bases. In some cases, chemical modifications to the base moiety include natural and synthetic modifications of adenine, guanine, cytosine, thymine, or uracil, and purine or pyrimidine bases. [00188] In some embodiments, a chemical modification of the engineered guide RNA can comprise a modification of any one of or any combination of: modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage; modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage; modification of a constituent of the ribose sugar; replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring nucleobase; modification of the ribose- phosphate backbone; modification of 5’ end of polynucleotide; modification of 3’ end of polynucleotide; modification of the deoxyribose phosphate backbone; substitution of the phosphate group; modification of the ribophosphate backbone; modifications to the sugar of a nucleotide; modifications to the base of a nucleotide; or stereopure of nucleotide. Chemical modifications to the engineered guide RNA include any modification contained herein, while some exemplary modifications are recited in TABLE 2. TABLE 2 - Exemplary Chemical Modification Modification of engineered guide RNA Examples Modification of one or both of the non-linking sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, , , r, d or
Attorney Docket No.199235.769601 Modification of engineered guide RNA Examples Modification of the deoxyribose phosphate phosphorothioate, phosphonothioacetate, phosphoroselenates, backbone borano phosphates borano phosphate esters hydrogen r, )
[00189] In some embodiments, the chemical modification can comprise modification of one or both of the non-linking phosphate oxygens in the phosphodiester backbone linkage or modification of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage. As used herein, “alkyl” may be meant to refer to a saturated hydrocarbon group which may be straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl or isopropyl), butyl (e.g., n-butyl, isobutyl, or t-butyl), or pentyl (e.g., n- pentyl, isopentyl, or neopentyl). An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms. As used herein, “aryl” may refer to monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl, phenanthrenyl, indanyl, or indenyl. In some embodiments, aryl groups have from 6 to about 20 carbon atoms. As used herein, “alkenyl” may refer to an aliphatic group containing at least one double bond. As used herein, “alkynyl” may refer to a straight or branched hydrocarbon chain containing 2-12 carbon atoms and characterized in having one or more triple bonds. Examples of alkynyl groups can include ethynyl, propargyl, or 3-hexynyl. “Arylalkyl” or “aralkyl” may refer to an alkyl moiety in which an alkyl hydrogen atom may be replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of "arylalkyl" or "aralkyl" include benzyl, 2-phenylethyl, 3- phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups. “Cycloalkyl” may refer to a cyclic,
Attorney Docket No.199235.769601 bicyclic, tricyclic, or polycyclic non- aromatic hydrocarbon groups having 3 to 12 carbons. Examples of cycloalkyl moieties include, but are not limited to, cyclopropyl, cyclopentyl, and cyclohexyl. “Heterocyclyl” may refer to a monovalent radical of a heterocyclic ring system. Representative heterocyclyls include, without limitation, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, pyrrolidonyl, piperidinyl, pyrrolinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, and morpholinyl. “Heteroaryl” may refer to a monovalent radical of a heteroaromatic ring system. Examples of heteroaryl moieties can include imidazolyl, oxazolyl, thiazolyl, triazolyl, pyrrolyl, furanyl, indolyl, thiophenyl pyrazolyl, pyridinyl, pyrazinyl, pyridazinyl, pyrimidinyl, indolizinyl, purinyl, naphthyridinyl, quinolyl, and pteridinyl. [00190] In some embodiments, the phosphate group of a chemically modified nucleotide can be modified by replacing one or more of the oxygens with a different substituent. In some embodiments, the chemically modified nucleotide can include replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Examples of modified phosphate groups can include phosphorothioate, phosphonothioacetate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group can be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. A phosphorous atom in a phosphate group modified in this way may be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In some cases, the engineered guide RNA can comprise stereopure nucleotides comprising S conformation of phosphorothioate or R conformation of phosphorothioate. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 50%, 60%, 70%, 80%, 90%, or more. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 95%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 96%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 97%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of
Attorney Docket No.199235.769601 98%. In some embodiments, the chiral phosphate product may be present in a diastereomeric excess of 99%. In some embodiments, both non-bridging oxygens of phosphorodithioates can be replaced by sulfur. The phosphorus center in the phosphorodithioates can be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non-bridging oxygens can also include the replacement of the non- bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). In some embodiments, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). In some cases, the replacement can occur at either or both of the linking oxygens. [00191] In certain embodiments, nucleic acids comprise linked nucleic acids. Nucleic acids can be linked together using any inter nucleic acid linkage. The two main classes of inter nucleic acid linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus containing inter nucleic acid linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates (P=S). Representative non-phosphorus containing inter nucleic acid linking groups include, but are not limited to, methylenemethylimino (-CH2-N(CH3)-O-CH2-), thiodiester (-O-C(O)-S-), thionocarbamate (-O-C(O)(NH)-S-); siloxane (-O-Si(H)2-O-); and N,N*-dimethylhydrazine (-CH2-N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a chiral atom can be prepared as a racemic mixture, as separate enantiomers, e.g., alkylphosphonates and phosphorothioates. Unnatural nucleic acids can contain a single modification. Unnatural nucleic acids can contain multiple modifications within one of the moieties or between different moieties. [00192] In some cases, backbone phosphate modifications to nucleic acid include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester, phosphorodithioate, phosphodithioate, and boranophosphate, and can be used in any combination. Other non-phosphate linkages may also be used. [00193] In some embodiments, backbone modifications (e.g., methylphosphonate, phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide linkages) can confer immunomodulatory activity on the modified nucleic acid and/or enhance their stability in vivo. [00194] In some instances, a phosphorous derivative (or modified phosphate group) may be attached to the sugar or sugar analog moiety in and can be a monophosphate, diphosphate,
Attorney Docket No.199235.769601 triphosphate, alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the like. [00195] In some cases, backbone modification comprises replacing the phosphodiester linkage with an alternative moiety such as an anionic, neutral or cationic group. Examples of such modifications include: anionic internucleoside linkage; N3’ to P5’ phosphoramidate modification; boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages such as methylphosphonates; amide linked DNA; methylene(methylimino) linkages; formacetal and thioformacetal linkages; backbones containing sulfonyl groups; morpholino oligos; peptide nucleic acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos. A modified nucleic acid may comprise a chimeric or mixed backbone comprising one or more modifications, e.g., a combination of phosphate linkages such as a combination of phosphodiester and phosphorothioate linkages. [00196] In some cases, substitutes for the phosphate include, for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts. It may be also understood in a nucleotide substitute that both the sugar and the phosphate moieties of the nucleotide can be replaced, by for example an amide type linkage (aminoethylglycine) (PNA). It may be also possible to link other types of molecules (conjugates) to nucleotides or nucleotide analogs to enhance for example, cellular uptake. In some cases, conjugates can be chemically linked to the nucleotide or nucleotide analogs. Such conjugates include but are not limited to lipid moieties such as a cholesterol moiety, a thioether, e.g., hexyl-S-tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l-di-O-hexadecyl- rac-glycero-S-H-phosphonate, a polyamine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. [00197] In some embodiments, a chemical modification described herein can comprise modification of a phosphate backbone. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified phosphate backbone. Exemplary chemically modification of the phosphate group or backbone can include replacing one or more
Attorney Docket No.199235.769601 of the oxygens with a different substituent. Furthermore, the modified nucleotide present in the engineered guide RNA can include the replacement of an unmodified phosphate moiety with a modified phosphate as described herein. In some embodiments, the modification of the phosphate backbone can include alterations resulting in either an uncharged linker or a charged linker with unsymmetrical charge distribution. Exemplary modified phosphate groups can include, phosphorothioate, phosphonothioacetate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some embodiments, one of the non-bridging phosphate oxygen atoms in the phosphate backbone moiety can be replaced by any of the following groups: sulfur (S), selenium (Se), BR3 (wherein R can be, e.g., hydrogen, alkyl, or aryl), C (e.g., an alkyl group, an aryl group, and the like), H, NR2 (wherein R can be, e.g., hydrogen, alkyl, or aryl), or OR (wherein R can be, e.g., alkyl or aryl). The phosphorous atom in an unmodified phosphate group may be achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral; that may be to say that a phosphorous atom in a phosphate group modified in this way may be a stereogenic center. The stereogenic phosphorous atom can possess either the "R" configuration (herein Rp) or the "S" configuration (herein Sp). In such case, the chemically modified engineered guide RNA can be stereopure (e.g., S or R confirmation). In some cases, a chemically modified engineered guide RNA comprises stereopure phosphate modification. For example, the chemically modified engineered guide RNA can comprise S conformation of phosphorothioate or R conformation of phosphorothioate. [00198] Phosphorodithioates have both non-bridging oxygens replaced by sulfur. The phosphorus center in the phosphorodithioates may be achiral which precludes the formation of oligoribonucleotide diastereomers. In some embodiments, modifications to one or both non- bridging oxygens can also include the replacement of the non-bridging oxygens with a group independently selected from S, Se, B, C, H, N, and OR (R can be, e.g., alkyl or aryl). [00199] In some cases, the phosphate linker can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens. Replacement of phosphate moiety [00200] In some embodiments, at least one phosphate group of the engineered guide RNA can be chemically modified. In some embodiments, the phosphate group can be replaced by non-
Attorney Docket No.199235.769601 phosphorus containing connectors. In some embodiments, the phosphate moiety can be replaced by dephospho linker. In some embodiments, the charge phosphate group can be replaced by a neutral group. In some cases, the phosphate group can be replaced by methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In some embodiments, nucleotide analogs described herein can also be modified at the phosphate group. Modified phosphate group can include modification at the linkage between two nucleotides with phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3’-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates (e.g., 3’-amino phosphoramidate and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates. In some cases, the phosphate or modified phosphate linkage between two nucleotides can be through a 3’-5’ linkage or a 2’-5’ linkage, and the linkage contains inverted polarity such as 3’-5’ to 5’-3’ or 2’-5’ to 5’-2’. Substitution of phosphate group [00201] In some embodiments, a chemical modification described herein can comprise modification by replacement of a phosphate group. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modification comprising a phosphate group substitution or replacement. Exemplary phosphate group replacement can include non- phosphorus containing connectors. In some embodiments, the phosphate group substitution or replacement can include replacing charged phosphate group can by a neutral moiety. Exemplary moieties which can replace the phosphate group can include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. Modification of the Ribophosphate Backbone [00202] In some embodiments, the chemical modification described herein can comprise modifying ribophosphate backbone of the engineered guide RNA. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified ribophosphate backbone. Exemplary chemically modified ribophosphate backbone can include scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and
Attorney Docket No.199235.769601 ribose sugar may be replaced by nuclease resistant nucleoside or nucleotide surrogates. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. Modification of sugar [00203] In some embodiments, the chemical modification described herein can comprise modifying of sugar. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified sugar. Exemplary chemically modified sugar can include 2’ hydroxyl group (OH) modified or replaced with a number of different "oxy" or "deoxy" substituents. In some embodiments, modifications to the 2’ hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2’- alkoxide ion. The 2’-alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Examples of "oxy"-2’ hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein "R" can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR, wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the "oxy"-2’ hydroxyl group modification can include (LNA, in which the 2’ hydroxyl can be connected, e.g., by a Ci-6 alkylene or Cj-6 heteroalkylene bridge, to the 4’ carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the "oxy"-2’ hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative). In some cases, the deoxy modifications can include hydrogen (i.e. deoxyribose sugars, e.g., at the overhang portions of partially dsRNA); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2-amino (wherein amino can be, e.g., as described herein),NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which can be optionally substituted with e.g., an amino as described herein. In some instances,
Attorney Docket No.199235.769601 the sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The nucleotide "monomer" can have an alpha linkage at the Γ position on the sugar, e.g., alpha-nucleosides. The modified nucleic acids can also include "abasic" sugars, which lack a nucleobase at C-. The abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that may be in the L form, e.g., L-nucleosides. In some aspects, the engineered guide RNA described herein includes the sugar group ribose, which may be a 5-membered ring having an oxygen. Exemplary modified nucleosides and modified nucleotides can include replacement of the oxygen in ribose (e.g., with sulfur (S), selenium (Se), or alkylene, such as, e.g., methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl); ring contraction of ribose (e.g., to form a 4- membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6-or 7- membered ring having an additional carbon or heteroatom, such as for example, anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone). In some embodiments, the modified nucleotides can include multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA or S-GNA, where ribose may be replaced by glycol units attached to phosphodiester bonds), threose nucleic acid. In some embodiments, the modifications to the sugar of the engineered guide RNA comprises modifying the engineered guide RNA to include locked nucleic acid (LNA), unlocked nucleic acid (UNA), or bridged nucleic acid (BNA). Modification of a constituent of the ribose sugar [00204] In some embodiments, the engineered guide RNA described herein can comprise at least one chemical modification of a constituent of the ribose sugar. In some embodiments, the chemical modification of the constituent of the ribose sugar can include 2’-O-methyl, 2’-O- methoxy-ethyl (2’-MOE), 2’-fluoro, 2’-aminoethyl, 2’-deoxy-2’-fuloarabinou-cleic acid, 2′- deoxy, 2′-O-methyl, 3′-phosphorothioate, 3′-phosphonoacetate (PACE), or 3′- phosphonothioacetate (thioPACE). In some embodiments, the chemical modification of the constituent of the ribose sugar comprises unnatural nucleic acid. In some instances, the unnatural nucleic acids include modifications at the 5’-position and the 2’-position of the sugar ring, such as 5’-CH2-substituted 2’-O-protected nucleosides. In some cases, unnatural nucleic acids include amide linked nucleoside dimers that can be prepared for incorporation into oligonucleotides. In some cases, the 3’ linked nucleoside in the dimer (5’ to 3’) comprises a 2’-OCH3 and a 5’-(S)- CH3. Unnatural nucleic acids can include 2’-substituted 5’-CH2 (or O) modified nucleosides.
Attorney Docket No.199235.769601 Unnatural nucleic acids can include 5’-methylenephosphonate DNA and RNA monomers, and dimers. Unnatural nucleic acids can include 5’-phosphonate monomers having a 2’-substitution and other modified 5’-phosphonate monomers. Unnatural nucleic acids can include 5’-modified methylenephosphonate monomers. Unnatural nucleic acids can include analogs of 5’ or 6’- phosphonate ribonucleosides comprising a hydroxyl group at the 5’ and/or 6’-position. Unnatural nucleic acids can include 5’-phosphonate deoxyribonucleoside monomers and dimers having a 5’-phosphate group. Unnatural nucleic acids can include nucleosides having a 6’-phosphonate group wherein the 5’ or/and 6’-position may be unsubstituted or substituted with a thio-tert-butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2) (and analogs thereof) or a cyano group (CN) (and analogs thereof). [00205] In some embodiments, unnatural nucleic acids also include modifications of the sugar moiety. In some cases, nucleic acids can contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property. In certain embodiments, nucleic acids can comprise a chemically modified ribofuranose ring moiety. Examples of chemically modified ribofuranose rings include, without limitation, addition of substituent groups (including 5’ and/or 2’ substituent groups; bridging of two ring atoms to form bicyclic nucleic acids; replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, C1-C12 alkyl or a protecting group); and combinations thereof. [00206] In some instances, the engineered guide RNA described herein can comprise modified sugars or sugar analogs. Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose, deoxypentose, hexose, deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar “analog” cyclopentyl group. The sugar can be in a pyranosyl or furanosyl form. The sugar moiety can be the furanoside of ribose, deoxyribose, arabinose or 2’-O-alkylribose, and the sugar can be attached to the respective heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar modifications include, but are not limited to, 2’-alkoxy-RNA analogs, 2’- amino-RNA analogs, 2’-fluoro-DNA, and 2’-alkoxy-or amino-RNA/DNA chimeras. For example, a sugar modification may include 2’-O-methyl-uridine or 2’-O-methyl-cytidine. Sugar modifications include 2’-O-alkyl-substituted deoxyribonucleosides and 2’-O-ethyleneglycol-like ribonucleosides. [00207] In some cases, modifications to the sugar moiety include natural modifications of the ribose and deoxy ribose as well as unnatural modifications. Sugar modifications include, but are not limited to, the following modifications at the 2’ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be
Attorney Docket No.199235.769601 substituted or unsubstituted C1 to C10, alkyl or C2 to C10 alkenyl and alkynyl.2’ sugar modifications also include but are not limited to-O[(CH2)nO]m CH3,-O(CH2)nOCH3,- O(CH2)nNH2,-O(CH2)nCH3,-O(CH2)nONH2, and-O(CH2)nON[(CH2)n CH3)]2, where n and m may be from 1 to about 10. Other chemical modifications at the 2’ position include but are not limited to: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2 CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties. Similar modifications may also be made at other positions on the sugar, particularly the 3’ position of the sugar on the 3’ terminal nucleotide or in 2’-5’ linked oligonucleotides and the 5’ position of the 5’ terminal nucleotide. Chemically modified sugars also include those that contain modifications at the bridging ring oxygen, such as CH2 and S. Nucleotide sugar analogs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Examples of nucleic acids having modified sugar moieties include, without limitation, nucleic acids comprising 5’-vinyl, 5’-methyl (R or S), 4’-S, 2’-F, 2’-OCH3, and 2’-O(CH2)2OCH3 substituent groups. The substituent at the 2’ position can also be selected from allyl, amino, azido, thio, O-allyl, O-(C1-C1O alkyl), OCF3, O(CH2)2SCH3, O(CH2)2-O-N(Rm)(Rn), and O-CH2- C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1- C10 alkyl. [00208] In certain embodiments, nucleic acids described herein can include one or more bicyclic nucleic acids. In certain such embodiments, the bicyclic nucleic acid comprises a bridge between the 4’ and the 2’ ribosyl ring atoms. In certain embodiments, nucleic acids provided herein can include one or more bicyclic nucleic acids wherein the bridge comprises a 4’ to 2’ bicyclic nucleic acid. Examples of such 4’ to 2’ bicyclic nucleic acids include, but are not limited to, one of the formulae: 4’-(CH2)-O-2’ (LNA); 4’-(CH2)-S-2’; 4’-(CH2)2-O-2’ (ENA); 4’-CH(CH3)-O-2’ and 4’-CH(CH2OCH3)-O-2’, and analogs thereof; 4’-C(CH3)(CH3)-O-2’and analogs thereof. Modifications on the base of nucleotide [00209] In some embodiments, the chemical modification described herein can comprise modification of the base of nucleotide (e.g., the nucleobase). Exemplary nucleobases can include adenine (A), thymine (T), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or replaced to in the engineered guide RNA described herein. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine
Attorney Docket No.199235.769601 analog. In some embodiments, the nucleobase can be naturally-occurring or synthetic derivatives of a base. [00210] In some embodiments, the chemical modification described herein can comprise modifying an uracil. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified uracil. Exemplary chemically modified uracil can include pseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza- uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy- uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl- uridine, 5-methoxy-uridine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine, 5- carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyl-uridine, 5- methoxycarbonylmethyl-2-thio-uridine, 5-aminomethyl-2-thio-uridine, 5-methylaminomethyl- uridine, 5-methylaminomethyl-2-thio-uridine, 5-methylaminomethyl-2-seleno-uridine, 5- carbamoylmethyl-uridine, 5-carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl- 2-thio-uridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1- taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio- pseudouridine, 5-methyl-uridine, 1 methyl-pseudouridine, 5-methyl-2-thio-uridine, l-methyl-4- thio-pseudouridine, 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine, 2-thio-1-methyl- pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydroundine, dihydropseudoundine, 5,6-dihydrouridine, 5-methyl-dihydrouridine, 2-thio- dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4- methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3- amino-3-carboxypropyl) uridine, 1-methyl-3-(3-amino-3-carboxypropy pseudouridine, 5- (isopentenylaminomethyl) uridine, 5-(isopentenylaminomethy])-2-thio-uridine, a-thio-uridine, 2’-O-methyl-uridine, 5,2’-O-dimethyl-uridine, 2’-O-methyl-pseudouridine, 2-thio-2’-O-methyl- uridine, 5-methoxycarbonylmethyl-2’-O-methyl-uridine, 5-carbamoylmethyl-2’-O-methyl- uridine, 5-carboxymethylaminomethyl-2’-O-methyl-uridine, 3,2’-O-dimethyl-uridine, 5- (isopentenylaminomethyl)-2’-O-methyl-uridine, l-thio-uridine, deoxythymidine, 2’-F-ara- uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-( l-E- propenylamino)uridine, pyrazolo[3,4-d]pyrimidines, xanthine, and hypoxanthine. [00211] In some embodiments, the chemical modification described herein can comprise modifying a cytosine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified cytosine. Exemplary chemically modified cytosine can include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetyl-
Attorney Docket No.199235.769601 cytidine, 5-formyl-cytidine, N4-methyl-cytidine, 5-methyl-cytidine, 5-halo-cytidine, 5- hydroxymethyl-cytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1- methyl-pseudoisocytidine, 4-thio-l-methyl-1-deaza-pseudoisocytidine, 1-methyl-l-deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine, a-thio-cytidine, 2’-O- methyl-cytidine, 5,2’-O-dimethyl-cytidine, N4-acetyl-2’-O-methyl-cytidine, N4,2’-O-dimethyl- cytidine, 5-formyl-2’-O-methyl-cytidine, N4,N4,2’-O-trimethyl-cytidine, 1-thio-cytidine, 2’-F- ara-cytidine, 2’-F-cytidine, and 2’-OH-ara-cytidine. [00212] In some embodiments, the chemical modification described herein can comprise modifying an adenine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified adenine. Exemplary chemically modified adenine can include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro- purine), 6-halo-purine (e.g., 6-chloi-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7- deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine, 2-methyl- adenine, N6-methyl-adenosine, 2-methylthio-N6-methyl-adenosine, N6-isopentenyl-adenosine, 2-methylthio-N6-isopentenyl-adenosine, N6-(cis-hydroxyisopentenyl) adenosine , 2-methylthio- N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyl-adenosine, N6- threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6- threonylcarbamoyl-adenosine, N6, N6-dimethyl-adenosine, N6-hydroxynorvalylcarbamoyl- adenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7- methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2’-O-methyl- adenosine, N6, 2’-O-dimethyl-adenosine, N6-Methyl-2’-deoxyadenosine, N6, N6, 2’-O- trimethyl-adenosine, l ,2’-O-dimethyl-adenosine, 2’-O-ribosyladenosine (phosphate) (Ar(p)), 2- amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F- adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine. [00213] In some embodiments, the chemical modification described herein can comprise modifying a guanine. In some embodiments, the engineered guide RNA described herein can comprise at least one chemically modified guanine. Exemplary chemically modified guanine can include inosine, 1-methyl-inosine, wyosine, methylwyosine, 4-demethyl-wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undemriodified hydroxywybutosine, 7- deaza-guanosine, queuosine, epoxyqueuosine, galactosyl-queuosine, mannosyl-queuosine, 7-
Attorney Docket No.199235.769601 cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza- guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7- methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1- methyl-guanosine, N2-methyl-guanosine, N2, N2-dimethyl-guanosine, N2, 7-dimethyl- guanosine, N2, N2, 7-dimethyl-guanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- meththio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, a-thio- guanosine, 2’-O-methyl-guanosine, N2-methyl-2’-O-methyl-guanosine, N2,N2-dimethyl-2’-O- methyl-guanosine, l-methyl-2’-O-methyl-guanosine, N2, 7-dimethyl-2’-O-methyl-guanosine, 2’- O-methyl-inosine, l , 2’-O-dimethyl-inosine, 6-O-phenyl-2’-deoxyinosine, 2’-O- ribosylguanosine, 1-thio-guanosine, 6-O-methyguanosine, O6-Methyl-2’-deoxyguanosine, 2’-F- ara-guanosine, and 2’-F-guanosine. [00214] In some cases, the chemical modification of the engineered guide RNA can include introducing or substituting a nucleic acid analog or an unnatural nucleic acid into the engineered guide RNA. In some embodiments, nucleic acid analog can be any one of the chemically modified nucleic acid described herein. Exemplary nucleic acid analog can be found in PCT/US2021/034272, PCT/US2015/025175, PCT/US2014/050423, PCT/US2016/067353, PCT/US2018/041503, PCT/US2018/041509, PCT/US2004/011786, or PCT/US2004/011833, all of which are expressly incorporated by reference in their entireties. In some cases, the chemically modified nucleotide described herein can include a variant of guanosine, uridine, adenosine, thymidine, and cytosine, including any natively occurring or non-natively occurring guanosine, uridine, adenosine, thymidine or cytidine that has been altered chemically, for example by acetylation, methylation, hydroxylation. Exemplary chemically modified nucleotide can include 1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine, 2,2-dimethyl-guanosine, 2,6- diaminopurine, 2’-amino-2’-deoxyadenosine, 2’-amino-2’-deoxycytidine, 2’-amino-2’- deoxyguanosine, 2’-amino-2’-deoxyuridine, 2-amino-6-chloropurineriboside, 2-aminopurine- riboside, 2’-araadenosine, 2’-aracytidine, 2’-arauridine, 2’-azido-2’-deoxyadenosine, 2’-azido-2’- deoxycytidine, 2’-azido-2’-deoxyguanosine, 2’-azido-2’-deoxyuridine, 2-chloroadenosine, 2’- fluoro-2’-deoxyadenosine, 2’-fluoro-2’-deoxycytidine, 2’-fluoro-2’-deoxyguanosine, 2’-fluoro- 2’-deoxyuridine, 2’-fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine, 2-methyl-thio- N6-isopenenyl-adenosine, 2’-O-methyl-2-aminoadenosine, 2’-O-methyl-2’-deoxyadenosine, 2’- O-methyl-2’-deoxycytidine, 2 ‘-O-methyl-2’-deoxyguanosine, 2,-O-methyl-2’-deoxyuridine, 2’- O-methyl-5-methyluridine, 2’-O-methylinosine, 2’-O-methylpseudouridine, 2-thiocytidine, 2- thio-cytidine, 3-methyl-cytidine, 4-acetyl-cytidine, 4-thiouridine, 5-(carboxyhydroxymethyl)- uridine, 5,6-dihydrouridine, 5-aminoallylcytidine, 5-aminoallyl-deoxyuridine, 5-bromouridine, 5-
Attorney Docket No.199235.769601 carboxymethylaminomethyl-2-thio-uracil, 5-carboxymethylamonomethyl-uracil, 5-chloro-ara- cytosine, 5-fluoro-uridine, 5-iodouridine, 5-methoxycarbonylmethyl-uridine, 5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-azauridine, 6-chloro-7-deaza-guanosine, 6- chloropurineriboside, 6-mercapto-guanosine, 6-methyl-mercaptopurine-riboside, 7-deaza-2’- deoxy-guanosine, 7-deazaadenosine, 7-methyl-guanosine, 8-azaadenosine, 8-bromo-adenosine, 8-bromo-guanosine, 8-mercapto-guanosine, 8-oxoguanosine, benzimidazole-riboside, beta-D- mannosyl-queosine, dihydro-uridine, inosine, N1-methyladenosine, N6-([6-ami nohexyl] carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine, N6-methyl-adenosine, N7-methyl- xanthosine, N-uracil-5-oxyacetic acid methyl ester, puromycin, queosine, uracil-5-oxyacetic acid, uracil-5-oxyacetic acid methyl ester, wybutoxosine, xanthosine, and xylo-adenosine. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 2-amino-6-chloropurineriboside-5’- triphosphate, 2-aminopurine-riboside-5’-triphosphate, 2-aminoadenosine-5’-triphosphate, 2’- amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5’-triphosphate, 2-thiouridine-5’- triphosphate, 2’-fluorothymidine-5’-triphosphate, 2’-O-methyl-inosine-5’-triphosphate, 4- thiouridine-5’-triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5-aminoallyluridine-5’- triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’-triphosphate, 5-bromo-2’- deoxycytidine-5’-triphosphate, 5-bromo-2’-deoxyuridine-5’-triphosphate, 5-iodocytidine-5’- triphosphate, 5-iodo-2’-deoxycytidine-5’-triphosphate, 5-iodouridine-5’-triphosphate, 5-iodo-2’- deoxyuridine-5’-triphosphate, 5-methylcytidine-5’-triphosphate, 5-methyluridine-5’- triphosphate, 5-propynyl-2’-deoxycytidine-5’-triphosphate, 5-propynyl-2’-deoxyuridine-5’- triphosphate, 6-azacytidine-5’-triphosphate, 6-azauridine-5’-triphosphate, 6- chloropurineriboside-5’-triphosphate, 7-deazaadenosine-5’-triphosphate, 7-deazaguanosine-5’- triphosphate, 8-azaadenosine-5’-triphosphate, 8-azidoadenosine-5’-triphosphate, benzimidazole- riboside-5’-triphosphate, N1-methyladenosine-5’-triphosphate, N1-methylguanosine-5’- triphosphate, N6-methyladenosine-5’-triphosphate, 6-methylguanosine-5’-triphosphate, pseudouridine-5’-triphosphate, puromycin-5’-triphosphate, or xanthosine-5’-triphosphate. In some embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-tauri nomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-
Attorney Docket No.199235.769601 methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In some embodiments, the artificial nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-th io-1-methyl- 1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine. In some embodiments, the chemically modified nucleic acid as described herein comprises at least one chemically modified nucleotide selected from 2-aminopurine, 2, 6-diaminopurine, 7-deaza- adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza- 2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In other embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6- thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8- oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio- guanosine, and N2,N2-dimethyl-6-thio-guanosine. In certain embodiments, the chemically modified nucleic acid as described herein can comprise at least one chemically modified nucleotide selected from 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, pseudo-iso-cytidine, 5-aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha-thio- uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, pyrrolo-cytidine, inosine, alpha-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-chloro-purine, N6-methyl-2- amino-purine, pseudo-iso-cytidine, 6-chloro-purine, N6-methyl-adenosine, alpha-thio-adenosine, 8-azido-adenosine, 7-deaza-adenosine.
Attorney Docket No.199235.769601 [00215] In some embodiments, a modified base of a unnatural nucleic acid includes, but may be not limited to, uracil-5-yl, hypoxanthin-9-yl (I), 2-aminoadenin-9-yl, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8- amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7- methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7- deazaadenine and 3-deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids, such as 5-substituted pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted purines, O-6 substituted purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5- methylcytosine, those that increase the stability of duplex formation, universal nucleic acids, hydrophobic nucleic acids, promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic acids, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5- methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil, 5- halocytosine, 5-propynyl (-C≡C-CH3) uracil, 5-propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4- thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine( [5,4-b][l,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][l,4]benzothiazin-2(3H)-one), G-clamps, phenoxazine cytidine (e.g.9-(2- aminoethoxy)-H-pyrimido[5,4-b][l,4]benzoxazin-2(3H)-one), carbazole cytidine (2H- pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3’,2’:4,5]pyrrolo[2,3-d]pyrimidin- 2-one), those in which the purine or pyrimidine base may be replaced with other heterocycles, 7- deaza-adenine, 7-deazaguanosine, 2-aminopyridine, 2-pyridone, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated cytosine, cyclocytosine, cytosine arabinoside, 5- fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-dihydrocytosine, 5-iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, and 5-
Attorney Docket No.199235.769601 iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-thymine, 4-thio-thymine, 5-propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 5-hydroxycytosine, 2’- deoxyuridine, or 2-amino-2’-deoxyadenosine. [00216] In some cases, the at least one chemical modification can comprise chemically modifying the 5’ or 3’ end such as 5’ cap or 3’ tail of the engineered guide RNA. In some embodiments, the engineered guide RNA can comprise a chemical modification comprising 3’ nucleotides which can be stabilized against degradation, e.g., by incorporating one or more of the modified nucleotides described herein. In this embodiment, uridines can be replaced with modified uridines, e.g., 5-(2-amino) propyl uridine, and 5-bromo uridine, or with any of the modified uridines described herein; adenosines and guanosines can be replaced with modified adenosines and guanosines, e.g., with modifications at the 8-position, e.g., 8-bromo guanosine, or with any of the modified adenosines or guanosines described herein. In some embodiments, deaza nucleotides, e.g., 7-deaza-adenosine, can be incorporated into the gRNA. In some embodiments, O-and N-alkylated nucleotides, e.g., N6-methyladenosine, can be incorporated into the gRNA. In some embodiments, sugar-modified ribonucleotides can be incorporated, e.g., wherein the 2’ OH-group may be replaced by a group selected from H,-OR,-R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), halo,-SH,-SR (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or cyano (-CN). In some embodiments, the phosphate backbone can be modified as described herein, e.g., with a phosphothioate group. In some embodiments, the nucleotides in the overhang region of the gRNA can each independently be a modified or unmodified nucleotide including, but not limited to 2’-sugar modified, such as, 2-F 2’-O-methyl, thymidine (T), 2’-O-methoxyethyl-5-methyluridine (Teo), 2’-O- methoxyethyladenosine (Aeo), 2’-O-methoxyethyl-5-methylcytidine (m5Ceo), or any combinations thereof. Targets and Methods of Treatment [00217] The present disclosure provides for compositions of engineered guide RNAs or engineered polynucleotides encoding these engineered guide RNAs. In some embodiments, the compositions of engineered guide RNAs or engineered polynucleotides encoding these engineered guide RNAs target premature stop codon(s) of MECP2, such as the R168X mutation. Also disclosed are methods of using the engineered guide RNAs of the present disclosure, such as methods of treatment. Also disclosed are methods of using the engineered guide RNAs of the
Attorney Docket No.199235.769601 present disclosure that target premature stop codon(s) of MECP2, such as methods of treatment. In some embodiments, engineered guide RNAs disclosed herein can be screened by in vitro and in vivo methods to determine their ability to facilitate ADAR mediated RNA editing of adenosines in a target RNA. In some cases, a screening method can comprise cell-based reporter assay. In some embodiments, disclosed herein can be a method, comprising administering a composition disclosed herein to a subject (e.g., a human) in need thereof. In some instances, the method can treat a disease in the subject, for example a Rett syndrome. MECP2 [00218] The present disclosure provides for engineered guide RNAs that facilitate RNA editing of MECP2 to restore full-length expression of methyl CpG binding protein 2 (MECP2). As used herein the terms “MECP2” “MeCP2” refer to the human and mouse MECP2 protein, respectively: The terms “MECP2” and “MECP2” are used interchangeably herein, and refer to human MECP2 gene. The terms “Mecp2” and “Mecp2” are used interchangeably herein, and refer to human MECP2 gene. Engineered guide RNAs can also facilitate RNA editing of MECP2 to restore function of MECP2. Rett Syndrome is a genetically-driven neurodevelopmental disorder, which can be characterized by loss of motor functions, speaking problems, and a slowing (and/or reversal) of development. Genetic causes of Rett include MECP2 gene mutations leading to reduced expression of MECP2 and disruption the normal function of nerve cells. Said MECP2 mutations primarily affect female children with a prevalence of 1:10,000. In some embodiments, the present disclosure provides compositions of engineered guide RNAs that target MECP2 and facilitated ADAR-mediated RNA editing of MECP2. In some embodiments, the engineered guide RNA of the present disclosure can facilitate editing of the R168X mutation from UGA to UGI in MECP2 pre-mRNA and/or MECP2 mature mRNA. Each edit described herein can individually and collectively result in full-length protein production and/or functional protein production. [00219] In some embodiments, an in vivo model can be used for testing engineered guide RNAs. In some cases, an in vivo model can comprise a Mecp2R168X mouse, a wild type mouse, or both. In some cases, an in vivo model, such as a mouse can comprise an inducible Mecp2 system. In some cases, an in vivo model can comprise a non-human primate. [00220] In some embodiments, a target tissue for a guide RNA targeting MECP2 can be a peripheral nervous system tissue, a central nervous system tissue, or both. In some embodiments, a target cell for a guide RNA targeting MECP2 can comprise a cell such as a somatic cell. In some cases, a somatic cell can comprise a HEK293 cell. In some cases, a cell can be a wild type
Attorney Docket No.199235.769601 cell. In some cases, a cell can be a fibroblast. In some cases, a cell can be a hepatocyte. In some cases, a HEK293 cell can be an engineered cell for example, the cell can be engineered to express mouse Mecp2 R168X-Flag, mouse Mecp2 WT-Flag, human MECP2 R168X-Flag, and/or human MECP2 WT-Flag. In some cases, a somatic cell can comprise a nerve cell. In some cases, a nerve cell can comprise a neuron, a Schwann cell, a glial cell, an astrocyte, an oligodendrocyte, a microglia, an ependymal cell, or any combination thereof. In some cases, a neuron can be a primary neuron. In some cases, a Schwann cell comprises a myelinating Schwann cell or a non-myelinating Schwann cell. In some cases, a cell can be a primary cell. In some cases, a cell can be a primary human hepatocyte cell. In some cases, a cell can be a human cell, a non-human primate cell, or a mouse cell. In some cases, a cell can be a mouse primary cell. In some cases, a cell can be a primary mouse hepatocyte cell. In some cases, a cell can be a immortalized ear tip fibroblast. In some cases, a cell can be a iPSC-derived cell. In some cases, a cell can be a iPSC-derived cardiomyocyte. In some cases, a cell can be a iPSC-derived neuron. In some cases, a cell can be a human iPSC-derived neuron. In some cases, a cell can be a iPSC- derived astrocyte. In some cases, a cell can be a human iPSC-derived neuron. In some cases, a cell can be a Human Rett Syndrome patient iPSC-derived neuron. In some cases, a cell can be a Human Rett Syndrome patient iPSC-derived astrocyte. In some cases, a cell can be a cortical neuron cell. In some cases, a cell can be a mouse Mecp2R168X cortical neuron cell or a wild type cortical neuron cell. In some cases, a cell can be a mouse Mecp2R168X fibroblast or a wild type fibroblast. In some cases, a cell can be a immortalized fibroblast. In some cases, a cell can comprise a non-human primate cell. In some cases, a cell can comprise a cynomolgus cell. In some cases, editing of MECP2 can be determined by droplet digital PCR, next generation sequencing, and/or Sanger sequencing. In some cases, editing of MECP2 and/or other transcripts can be determined by RNA sequencing. In some cases, the restoration of MECP2 protein levels can be determined by measuring protein levels of MECP2 before and after treatment with a guide RNA. In some cases, an increase and/or decrease of protein levels can be measured by Western blot, flow cytometry, and/or immunocytochemistry, immunohistochemistry, or immunofluorescence. In some cases, cell health and cell proliferation can be measured. In some cases, editing of MECP2 can result in increased expression of the MECP2 protein. For example, in some cases editing of MECP2 can result in: at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% increased expression of the MECP2 protein. In some cases, editing of MECP2 can result in about: 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to
Attorney Docket No.199235.769601 100%, 20% to 40%, 30% to 50%, 40% to 60%, 50% to 70%, 60% to 80%, 20% to 50%, or 30% to 60% increased expression of MECP2 protein. [00221] The engineered guide RNAs of the present disclosure can facilitate ADAR-mediated RNA editing of MECP2. In some embodiments, the engineered guide RNAs of the present disclosure facilitate ADAR-mediated RNA editing from 1 to 100% of a target adenosine. In some cases, the target adenosine can be the adenosine in a UGA mutant transcript. The engineered guide RNAs of the present disclosure can facilitate from 20% to 90% editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 40% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 50% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 60% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 70% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 80% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least 90% RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate at least from 5% to 20%, from 20% to 40%, from 40% to 60%, from 60% to 80%, from 80% to 100%, from 60% to 80%, from 70% to 90%, or up to 90% or more RNA editing of a target adenosine. In some embodiments, the engineered guide RNAs of the present disclosure can demonstrate 40% RNA editing of the target adenosine. Optionally, additionally, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 5% editing of an off-target adenosine. Optionally, additionally, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 10% editing of an off-target adenosine. Optionally, additionally, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 15% editing of an off-target adenosine. Optionally, additionally, the engineered guide RNAs of the present disclosure can facilitate these levels of on-target RNA editing while maintaining less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than
Attorney Docket No.199235.769601 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0% editing of an off-target adenosine. [00222] In some cases, the engineered guide RNAs of the present disclosure do not substantially edit a wild type (non-mutant) transcript. In some embodiments, the engineered guide RNAs disclosed herein facilitates an increased level of editing of the target RNA encoded by the mutant allele, as compared to a level of editing of an otherwise comparable target RNA encoded by a wildtype allele. In some embodiments, the engineered guide RNAs has higher specificity for a target RNA encoded by a mutant allele, as compared to a comparable target RNA encoded by a WT allele. In some embodiments, the engineered guide RNAs disclosed herein are allele specific. In some cases, allele specificity is achieved by introduction of one or more structural features in a guide-target scaffold, or by sequence specificity. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate less than 5% editing of an off- target adenosine and/or a wild type transcript. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate less than 10% editing of an off-target adenosine and/or a wild type transcript. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate less than 15% editing of an off-target adenosine and/or a wild type transcript. In some embodiments, the engineered guide RNAs of the present disclosure can facilitate less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or 0% editing of an off-target adenosine and/or a wild type transcript. [00223] In some embodiments, an engineered guide RNA of the present disclosure can facilitate editing of a R168X mutation, a R255X mutation, a R270X mutation, a R294 mutation, or a combination thereof, in the coding region of MECP2. In some embodiments, the engineered guide RNA can facilitate editing of the R168X mutation of the pre-mRNA or mRNA transcript of MECP2. For example, an engineered guide RNA can facilitate editing of the stop codon UGA to TGI, which is a tryptophan codon. In some cases, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the pre-mRNA or mRNA transcripts of MECP2 have an edit in the R168X mutation site. In some cases, 1% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 60%, 60% to 70%, 70% to 80%, 80% to 90%, 90% to 100%, 20% to 40%, 30% to 50%, 40% to 60%, 50% to 70%, 60% to 80%, 20% to 50%, or 30% to 60% of the pre-mRNA or mRNA transcripts of MECP2 have an edit in the R168X mutation site.
Attorney Docket No.199235.769601 [00224] In some embodiments, an engineered guide disclosed herein has complementarity to a region of the MECP2 target RNA, where the region includes the R168X mutation. In some cases, the target RNA comprises the sequence of AAGUGGAGUUGAUUGCGUACUUCGAAAAGGUAGGCGACACAUCCCUGGACCCUA AUGAUUUUGACUUCACGGUAACUGGGAGAGGGAGCCCCUCCCGGUGAGAGCAGA AACCACCUAAGAAGCCCAAAUCUCCCAAAGCUCCAGGAACUGGCAGAGGCCGGGG ACGCCCCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 50) – the codon encoding the R168X nonsense mutation is shown in bold and the target adenosine is shown underlined and in bold. In some cases, the target RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 50. In some cases, the target RNA can comprise a sequence of more than, less than, or equal to about: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, or 201 nucleotides of SEQ ID NO: 50. [00225] In some cases, the target RNA can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 30, but not 100% sequence identity to SEQ ID NO: 30. In some cases, the target RNA can comprise a sequence of more than, less than, or equal to about: 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,
Attorney Docket No.199235.769601 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, or 199 nucleotides of SEQ ID NO: 30, or 200 nucleotides of SEQ ID NO: 30. [00226] An engineered guide RNA of the present disclosure can be used to facilitate modification of the target RNA. In some embodiments, an engineered guide disclosed herein can facilitate ADAR-mediated RNA editing of one or more adenosines in the target RNA sequence of SEQ ID NO: 50. In some cases, the target DNA sequence encoding a target RNA sequence can comprise a sequence of AAGTGGAGTTGATTGCGTACTTCGAAAAGGTAGGCGACACATCCCTGGACCCTAAT GATTTTGACTTCACGGTAACTGGGAGAGGGAGCCCCTCCCGGTGAGAGCAGAAACC ACCTAAGAAGCCCAAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCC CCAAAGGGAGCGGCACCACGAGACCCAAGGCGG (SEQ ID NO: 49) – the codon encoding the R168X nonsense mutation is shown in bold and the target adenosine is shown underlined and in bold. In some cases, a target DNA sequence encoding a target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 49. [00227] In some cases, a target DNA sequence encoding a target RNA sequence can comprise a sequence with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, about 98%, or about 99% sequence identity to SEQ ID NO: 29, but not 100% sequence identity to SEQ ID NO: 29. Assays for Measuring Specificity and Efficacy of Engineered gRNAs [00228] In some embodiments, the engineered guide RNAs of the present disclosure facilitate ADAR-mediated RNA editing of MECP2. In some embodiments, ADAR-mediated RNA editing of MECP2 can result in an increase of protein levels. In some embodiments, ADAR-mediated RNA editing of MECP2 can result in a functional protein. In some embodiments, ADAR- mediated RNA editing of MECP2 can result in an increase in functional protein. In some embodiments, ADAR-mediated RNA editing of MECP2 can ameliorate a disease phenotype for example Rett syndrome. In some embodiments, the engineered guide RNAs disclosed herein can facilitate allele-specific editing by an RNA editing entity such as ADAR1, ADAR2, or a combination thereof.
Attorney Docket No.199235.769601 [00229] In some embodiments, an assay is used to determine the efficacy of a guide RNA disclosed herein. In some cases, an assay can comprise measuring RNA editing, mRNA levels, or protein levels in a cell. In some cases, an assay can comprise measuring RNA editing, mRNA levels, or protein levels in a cell before and after a treatment with a guide RNA disclosed herein. In some cases, cells can be sampled in a time course assay. In some cases, a cell can comprise a cell with a functional ADAR gene. In some cases, a cell can comprise a cell with a nonfunctional ADAR gene. For example, a cell can comprise a truncated or mutated ADAR gene or a cell can comprise a deleted ADAR gene. In some cases, an assay can be used to compare editing levels, levels of mRNA, or levels of protein, in a cell with a functional copy of an ADAR gene and in a cell without a functional ADAR gene. In some cases, an increase of protein levels in the cell can be identified as ADAR dependent increase in protein levels. Protein levels in a cell can be measured by any standard technique, for example by a Western Blot, flow cytometry, or immunohistochemistry. mRNA levels in a cell can be measured by any standard technique, for example by Real-Time Quantitative Reverse Transcription PCR, or droplet digital PCR. In some cases, protein levels can be determined by a functional assay specific to a protein of interest. For example, an assay can be used to determine the amount of a protein by an enzymatic assay measuring the enzyme kinetics of the protein. In another example, an assay can be used to detect the downstream activity of a protein. In some cases, RNA editing can be measured by next generation sequencing and/or Sanger sequencing. In some cases, a result of an assay herein can be measured after administering guide RNAs to cells. In some cases, the measurement of an assay can be quantitating RNA editing and/or protein production. In some cases, the RNA editing and/or increased protein production by a guide RNA can be compared to a control guide RNA, such as a scrambled guide RNA sequence. [00230] In some embodiments, the functional efficacy of a restored MECP2 protein (e.g., R168W) in a cell can be determined with an assay. In some cases, an assay can comprise RNAseq to establish gene activation and/or silencing patterns in MECP2R168X untreated, MECP2R168X treated, and wild type cells. In some cases, an assay can comprise MECP2/H1 co- localization by immunocytochemistry (mutations in MECP2 can disrupt chromatin condensation and the sub-nuclear localization patterns of MECP2 and H1). In some cases, an assay can comprise MECP2 phosphorylation. In some cases, an assay can comprise dendritic arborization and/or synapse counts (mutations in MECP2 can be associated with lower dendritic complexity). In some embodiments, the functional efficacy of a restored MECP2 protein in a mouse by treatment with an engineered guide RNA can result in an increased lifespan as compared to a mouse not treated with an engineered guide RNA. In some embodiments, the functional efficacy
Attorney Docket No.199235.769601 of a restored MECP2 protein in a mouse by treatment with an engineered guide RNA can result in increased weight as compared to a mouse not treated with an engineered guide RNA. In some embodiments, the functional efficacy of a restored MECP2 protein in a mouse by treatment with an engineered guide RNA can result in a reduce cumulative bird score as compared to a mouse not treated with an engineered guide RNA. [00231] In some embodiments, a guide RNA disclosed herein can facilitate an ADAR dependent increase of full-length protein levels of 1% to 100%. In some cases, a guide RNA disclosed herein can facilitate an ADAR dependent increase of full-length protein levels from 1% to 10%, from 10% to 20%, from 20% to 30%, from 30% to 40%, from 40% to 50%, from 50% to 60%, from 60% to 70%, from 70% to 80%, from 80% to 90%, from 90% to 100%, from 20% to 40%, from 30% to 50%, from 40% to 60%, from 50% to 70%, from 60% to 80%, from 20% to 50%, or from 30% to 60% as compared to a cell before treatment with the guide RNA. In some cases, a guide RNA disclosed herein can facilitate an ADAR dependent increase of full-length protein levels from at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% as compared to a cell before treatment with the guide RNA. In some cases, a guide RNA disclosed herein can facilitate an at least: 1.5 times (x), 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, or 10x ADAR dependent increase of full-length protein levels as compared to a cell before treatment with the guide RNA. In some cases, ADAR dependent increase in full-length protein levels can be compared between a cell comprising a functional copy of ADAR and a cell comprising a nonfunctional copy of ADAR. Full-length protein restoration (e.g., restoration of full-length MECP2) can be measured by an assay comparing a sample or subject treated with the engineered guide RNA to a control sample or subject not treated with the engineered guide RNA. In some cases, an increase in protein levels can be measured by comparing the amount of the protein present in a sample or subject before a treatment with a guide RNA disclosed herein and comparing to the amount of the protein after the treatment. Methods of Treatment [00232] An engineered guide RNA of the present disclosure can be used in a method of treating a disorder in a subject in need thereof. A pharmaceutical composition comprising an engineered guide RNA of the present disclosure, a polypeptide encoding an engineered guide RNA of the present disclosure, or a recombinant AAV of the present disclosure can be used in a method of treating a disorder in a subject in need thereof. An AAV virion encapsidating a DNA vector
Attorney Docket No.199235.769601 genome of the present disclosure can be used in a method of treating a disorder in a subject in need thereof. For example, an engineered guide RNA, polynucleotide encoding an engineered guide RNA, AAV virion or pharmaceutical composition disclosed herein can be used to treat a Rett syndrome. In some instances, a Rett syndrome can comprise a classical Rett syndrome. In some instances, a Rett syndrome can comprise an atypical Rett syndrome. In some cases, an atypical Rett syndrome can comprise a congenital Rett Syndrome (a Rolando Variant), an early- onset Rett Syndrome (a Hanefeld Variant), a late-childhood Rett Syndrome, a Forme Fruste Rett Syndrome, a preserved-speech variant of Rett Syndrome (Zappella Variant), or any combination thereof. In some cases, an engineered guide RNA, polynucleotide encoding an engineered guide RNA, AAV virion or pharmaceutical composition can be used to treat a Rett Syndrome phase. In some cases, a phase of Rett syndrome can comprise an early onset phase, a rapid destructive phase, a plateau phase, or a late motor deterioration phase. A syndrome and/or disorder can be a disease, a condition, a genotype, a phenotype, or any state associated with an adverse effect. In some embodiments, treating a syndrome can comprise preventing, slowing progression of, reversing, or alleviating symptoms of the syndrome. A method of treating a disease can comprise delivering an engineered polynucleotide encoding an engineered guide RNA to a cell of a subject in need thereof and expressing the engineered guide RNA in the cell. In some embodiments, an engineered guide RNA of the present disclosure can be used to treat a genetic disorder (e.g., Rett syndrome). In some embodiments, an engineered guide RNA of the present disclosure can be used to treat a condition associated with one or more mutations. Disclosed herein are methods of treating a Rett syndrome with engineered guide RNAs targeting MECP2. [00233] In some embodiments, treatment of a Rett syndrome comprises treatment of the symptoms associated with Rett syndrome. A symptom of a Rett syndrome can comprise a low muscle tone (e.g., a hypotonia), a difficulty feeding, a repetitive hand movement, a jerky limb movement, a delay with development of speech, a mobility problem, a problem sitting, a crawling difficulty, a walking difficulty, a lack of interest in a toy, or any combination thereof. A symptom of a Rett syndrome can comprise a loss of the ability to use the hands purposefully, periods of distress, an irritability and/or sometimes screaming for no obvious reason, a social withdrawal, a loss of interest in people, an avoidance of eye contact, an unsteadiness when walking, an awkwardness when walking, a problem sleeping, a slowing of head growth, a difficulty eating, a chewing difficulty, a swallowing difficulty, a constipation, a heart rate problem, an arrhythmia, or any combination thereof. A symptom of a Rett syndrome can comprise a seizure, an irregular breathing pattern, or both. In some cases, a symptom of a Rett
Attorney Docket No.199235.769601 syndrome can comprise development of a spinal curve, a scoliosis, a muscle weakness, a spasticity, an inability to walk, or any combination thereof. Vector Construct (ITR-to-ITR region) [00234] An engineered guide RNA of the present disclosure (such as an engineered guide RNA that comprises a polynucleotide sequence of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, or SEQ ID NO: 63 – SEQ ID NO: 70) can be encoded in an engineered polynucleotide. In some cases, the engineered polynucleotide can be a DNA vector. In some cases, the DNA vector can be a plasmid. In some cases, the DNA vector or a portion of the DNA vector (e.g., the region in-between ITRs) can be encapsulated in a AAV capsid (e.g., a recombinant AAV capsid. TABLE 3 shows the ITR-ITR components of a MECP2-targeting vector construct. The components in TABLE 3 can be incorporated into a construct in the forward (5’ to 3’) or reverse directions (3’ to 5’). In some cases, the components in TABLE 3 can be in the reverse complement. An ITR-to-ITR sequence comprises two inverted terminal repeat (ITR) sequences, and an internal DNA sequence (between the two ITR sequences). In some cases, the internal DNA sequence includes a sequence encoding an engineered guide RNA (e.g., comprising an antisense sequence). In some cases, the internal DNA sequence includes one or more additional components. For example, an internal DNA sequence may include a promoter, a terminator, an accessory element (e.g., comprising a SmOPT sequence, a hairpin sequence, or both), or a combination thereof. The ITR-to-ITR sequence can be included in a vector (e.g., a DNA vector), such as a plasmid or a viral genome. The vector may be encapsidated in a viral capsid, such as a recombinant AAV capsid. TABLE 3. Sequences for MECP2-Targeting ITR-to-ITR Constructs Construct component Sequence C T A G is C
Attorney Docket No.199235.769601 Construct component Sequence CCTTTACACTCATGGTGACTTATCAAGGTGCCATTTCCACACC G T G T C C C A T A T A C C A
Attorney Docket No.199235.769601 Construct component Sequence AGCATATTCTGTGAAAGTTAGACTTTTGTTTAAACAATACTCT A G A A A G T T C T T T G A C G g ttt tc
Attorney Docket No.199235.769601 Construct component Sequence aagaaacgagcggttttaatagtcttttagaatattgtttatcgaaccgaataaggaactgtgctttgtgatt gt g ca ac ta c ta c aa tc C T ctt a t tc gc g ttt tc tt gt A g gc att aa g ac at
Attorney Docket No.199235.769601 Construct component Sequence aatgcactgctcacagtacaaatttaaaaaggcaaaatcaaacatttttattctaagcatattctgtgaaagt ct a c gt tc c ) A T att G T G A A C A G T g ttt tc tt gt
Attorney Docket No.199235.769601 Construct component Sequence TGCTCCGACCTCCTCCCGCTCCCTCTCCCAGTTACCaatttttggtagtg ct ac tc ga g ta tt t T A a c gt tc c g a C A C a tc a ct a cg tg cc gg g G g
Attorney Docket No.199235.769601 Construct component Sequence ggcggaaaaccccctcccaatttcactggtttcaaaaacagaaaaacagttctcttccccgctccccggt c gt a at t c gt c a ct aa gc A C gc c g ttt tc tt gt G C g ct ac tc ga g ta tt t gg gt
Attorney Docket No.199235.769601 Construct component Sequence ttttcaatttttggaacagggttttctgccttcgggcggaaaaccccctatcatgttttataaaaaaagactta c gt tc g at g g c T G aa cg a a tc at t aa ca g C T G aa gt ta D at g g
Attorney Docket No.199235.769601 Construct component Sequence ggtgtggaaatggcaccttgataagtcaccatgagtgtaaagggagttgatgtccttccctggc T G aa cg a a tc at t aa ca g C T G aa gt ta D at g g c T T gt t g g aa ttt
Attorney Docket No.199235.769601 Construct component Sequence ccagccagccagtcttcggcttcgccccctaacggtgacataaggcactctgtgaaatgctct tt a tt C A G aa gt ta D g C A tc tt cc ca g gt gg a g a ct C C g c at ta
Attorney Docket No.199235.769601 Construct component Sequence ttaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacgggg ctt g t cg cc ctt g a a ac at g g c A C g g at g ca tt tc at a g at g aa
Attorney Docket No.199235.769601 Construct component Sequence aagacttaaagaggaaaacattatggtgcaactttaggcttaagtgattcattgtcactgtttgttt ct t a a
ator sequence. In some cases, the terminator sequence can be located downstream (e.g., 3’) of the sequence encoding the SmOPT and U7 hairpin sequence or a sequence encoding a guide RNA sequence. In some cases, the terminator sequence can be located downstream (e.g., 3’) of the sequence encoding the SmOPT and U5 hairpin sequence or a sequence encoding a guide RNA sequence. In some cases, the terminator sequence comprises a terminator sequence 1, a terminator sequence 2, a terminator sequence 3, a terminator sequence 5 or a terminator sequence 5. In some cases, a terminator sequence is in the forward direction or the reverse direction in a construct encoding a guide RNA. In some cases, the terminator sequence 1 comprises SEQ ID NO: 80. In some cases, the terminator sequence 1 can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 80. In some cases, the terminator sequence 2 comprises SEQ ID NO: 83. In some cases, the terminator sequence 2 can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 83. In some cases, the terminator sequence 3 comprises SEQ ID NO: 84. In some cases, the terminator sequence 3 can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 84. In some cases, the terminator sequence 4 comprises SEQ ID NO: 86. In some cases, the terminator sequence 4 can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 86. In some cases, the terminator sequence 5 comprises SEQ ID NO: 87. In some cases, the terminator sequence 5 can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 87. [00236] In some cases, a polynucleotide encoding a guide RNA can comprise a ITR sequence. In some cases, the ITR sequence can be located at a terminal end of a polynucleotide sequence encoding a guide RNA (e.g., the 5’ end and/or the 3’ end of the polynucleotide sequence). In some cases, the ITR sequence comprises a scITR sequence or a ssITR sequence. In some cases, the
Attorney Docket No.199235.769601 scITR comprises SEQ ID NO: 71. In some cases, the scITR sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 71. In some cases, the ssITR comprises SEQ ID NO: 88. In some cases, the ssITR sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 88. In some cases, a scITR sequence is in the forward direction or the reverse direction in a construct encoding a guide RNA. In some cases, a ssITR sequence is in the forward direction or the reverse direction in a construct encoding a guide RNA. [00237] In some embodiments, a polynucleotide herein can comprise an ITR-to-ITR region. In some cases, the sequence encoding the scITR sequence (SEQ ID NO: 71), the sequence encoding the engineered mU7 promoter sequence (SEQ ID NO: 72), the sequence encoding the AG dinucleotide motif, the sequence encoding the BamHI site, the sequence encoding the PstI site, the sequence encoding a MECP2 guide (for example, guide 16 (SEQ ID NO: 70)), the sequence encoding the SmOPT and U7 hairpin sequence (SEQ ID NO: 76), the sequence encoding the terminator sequence 1 (SEQ ID NO: 80), the sequence encoding the engineered promoter sequence (SEQ ID NO: 81), the sequence encoding the terminator sequence 2 (SEQ ID NO: 83), and the ssITR sequence (SEQ ID NO: 88) can be packaged into an AAV virion. [00238] In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the guide RNA sequence (e.g., SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, SEQ ID NO: 52, or SEQ ID NO: 63 – SEQ ID NO: 70) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the SmOPT and U7 hairpin sequence (SEQ ID NO: 76) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the SmOPT and U7 hairpin sequence (SEQ ID NO: 77) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the SmOPT and U5 hairpin sequence (SEQ ID NO: 79) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the engineered mU7 promoter sequence (SEQ ID NO: 72) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the terminator sequence 1 (SEQ ID NO: 80) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction
Attorney Docket No.199235.769601 (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the terminator sequence 2 (SEQ ID NO: 83) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’).In some cases, an ITR-to- ITR sequence comprises one or more sequences (e.g., two sequences) encoding the terminator sequence 3 (SEQ ID NO: 84) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the terminator sequence 4 (SEQ ID NO: 86) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the terminator sequence 5 (SEQ ID NO: 87 each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the AG dinucleotide motif each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the BamHI site GGATCC each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the PstI site CTGCAG each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR- to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the engineered promoter sequence (SEQ ID NO: 81) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). In some cases, an ITR-to-ITR sequence comprises one or more sequences (e.g., two sequences) encoding the engineered promoter sequence 2 (SEQ ID NO: 82) each independently in the forward direction (i.e., 5’ to 3’) or in the reverse direction (i.e., 3’ to 5’). [00239] In some embodiments, the ITR-to-ITR sequence comprises SEQ ID NO: 69. In some cases, the ssITR sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the ITR-to-ITR sequence comprises SEQ ID NO: 70. In some cases, the ssITR sequence can have at least about: 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO: 70. Pharmaceutical Compositions
Attorney Docket No.199235.769601 [00240] The compositions described herein (e.g., compositions comprising an engineered guide RNA or an engineered polynucleotide encoding an engineered guide RNA, or a recombinant AAV, or an AAV virion encapsidating a DNA vector genome) can be formulated with a pharmaceutically acceptable carrier for administration to a subject (e.g., a human or a non-human animal). The compositions described herein (e.g., compositions comprising an engineered guide RNA or an engineered polynucleotide encoding an engineered guide RNA, or a recombinant AAV, or an AAV virion encapsidating a DNA vector genome) can be formulated with a pharmaceutically acceptable: excipient, carrier, diluent or any combination thereof for administration to a subject (e.g., a human or a non-human animal). A pharmaceutically acceptable carrier and/or diluent can include, but is not limited to, phosphate buffered saline solution, water, emulsions (e.g., an oil/water emulsion or a water/oil emulsions), glycerol, liquid polyethylene glycols, aprotic solvents such (e.g., dimethylsulfoxide, N-methylpyrrolidone, or mixtures thereof), and various types of wetting agents, solubilizing agents, anti-oxidants, bulking agents, protein carriers such as albumins, any and all solvents, dispersion media, coatings, sodium lauryl sulfate, isotonic and absorption delaying agents, disintegrants (e.g., potato starch or sodium starch glycolate), and the like. The compositions also can include stabilizers and preservatives. Additional examples of carriers, stabilizers, and adjuvants consistent with the compositions of the present disclosure can be found in, for example, Remington’s Pharmaceutical Sciences, 21st Ed., Mack Publ. Co., Easton, Pa. (2005), incorporated herein by reference in its entirety. Delivery [00241] An engineered guide RNA of the present disclosure or an engineered polynucleotide of the present disclosure (e.g., an engineered polynucleotide encoding an engineered guide RNA) can be delivered via a delivery vehicle. In some embodiments, the delivery vehicle is a vector. A vector can facilitate delivery of the engineered guide RNA or the engineered polynucleotide into a cell to genetically modify the cell. Target tissues and cells include but are not limited to satellite cells, myoblasts, myocytes, and myotubes of the face, shoulders, and upper limbs. In some examples, the vector comprises DNA, such as double stranded or single stranded DNA. In some examples, the delivery vector can be a eukaryotic vector, a prokaryotic vector (e.g., a bacterial vector or plasmid), a viral vector, or any combination thereof. In some cases, a delivery vehicle can comprise a non-viral delivery vehicle. In some embodiments, the vector is an expression cassette. In some embodiments, a viral vector comprises a viral capsid, an inverted terminal repeat sequence, and the engineered polynucleotide can be used to deliver the engineered guide RNA to a cell.
Attorney Docket No.199235.769601 [00242] In some cases, the engineered guide RNA of the present disclosure can be an in vitro transcribed (IVT) RNA. In some cases, an engineered guide RNA can be delivered as a formulation comprising the engineered guide RNA. In some cases, the engineered guide RNA may not be comprised in a vector. In some examples, the engineered guide RNA (e.g., as an oligonucleotide) can be formulated for delivery through direct injection. In some examples, the engineered guide RNA, as an oligo nucleotide can be formulated for delivery through intravenous administration or oral administration. [00243] In some embodiments, the viral vector can be a retroviral vector, an adenoviral vector, an adeno-associated viral (AAV) vector, an alphavirus vector, a lentivirus vector (e.g., human or porcine), a Herpes virus vector, an Epstein-Barr virus vector, an SV40 virus vectors, a pox virus vector, or a combination thereof. In some embodiments, the viral vector can be an AAV vector, a lentiviral vector, or a retroviral vector. In some embodiments, the viral vector can be a recombinant vector, a hybrid vector, a chimeric vector, a self-complementary vector, a single-stranded vector, or any combination thereof. [00244] In some embodiments, the viral vector can be an adeno-associated virus (AAV). In some embodiments, the AAV can be any AAV known in the art. In some embodiments, the AAV can comprise an AAV5 serotype, an AAV6 serotype, an AAV8 serotype, or an AAV9 serotype. In some embodiments, the viral vector can be of a specific serotype. In some embodiments, the viral vector can be an AAV1 serotype, an AAV2 serotype, an AAV3 serotype, an AAV4 serotype, an AAV5 serotype, an AAV6 serotype, an AAV7 serotype, an AAV8 serotype, an AAV9 serotype, an AAV10 serotype, an AAV11 serotype, an AAV12 serotype, an AAV13 serotype, an AAV14 serotype, an AAV15 serotype, an AAV16 serotype, an AAV.rh8 serotype, an AAV.rh10 serotype, an AAV.rh20 serotype, an AAV.rh39 serotype, an AAV.Rh74 serotype, an AAV.RHM4-1 serotype, an AAV.hu37 serotype, an AAV.Anc80 serotype, an AAV.Anc80L65 serotype, an AAV.7m8 serotype, an AAV.PHP.B serotype, an AAV2.5 serotype, an AAV2tYF serotype, an AAV3B serotype, an AAV.LK03 serotype, an AAV.HSC1 serotype, an AAV.HSC2 serotype, an AAV.HSC3 serotype, an AAV.HSC4 serotype, an AAV.HSC5 serotype, an AAV.HSC6 serotype, an AAV.HSC7 serotype, an AAV.HSC8 serotype, an AAV.HSC9 serotype, an AAV.HSC10 serotype, an AAV.HSC11 serotype, an AAV.HSC12 serotype, an AAV.HSC13 serotype, an AAV.HSC14 serotype, an AAV.HSC15 serotype, an AAV.HSC16 serotype, and an AAVhu68 serotype, a derivative of any of these serotypes, a chimera of any of these serotypes, a variant of any of these serotypes or any combination thereof.
Attorney Docket No.199235.769601 [00245] In some embodiments, the AAV vector can be a recombinant vector, a hybrid AAV vector, a chimeric AAV vector, a self-complementary AAV (scAAV) vector, a single-stranded AAV, or any combination thereof. [00246] In some embodiments, the AAV vector can be a recombinant AAV (rAAV) vector. Methods of producing recombinant AAV vectors can be known in the art and generally involve, in some cases, introducing into a producer cell line: (1) DNA necessary for AAV replication and synthesis of an AAV capsid, (b) one or more helper constructs comprising the viral functions missing from the AAV vector, (c) a helper virus, and (d) the plasmid construct containing the genome of the AAV vector, e.g., ITRs, promoter and engineered guide RNA sequences, etc. In some examples, the viral vectors described herein can be engineered through synthetic or other suitable means by references to published sequences, such as those that can be available in the literature. For example, the genomic and protein sequences of various serotypes of AAV, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits can be known in the art and can be found in the literature or in public databases such as GenBank or Protein Data Bank (PDB). [00247] The invention provides a recombinant AAV encapsidating a vector, wherein the vector comprises a sequence with at least: 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of SEQ ID NO: 962 – SEQ ID NO: 964, or SEQ ID NO: 966, and an AAV inverted terminal repeat. In some embodiments, the recombinant AAV comprises a sequence selected from SEQ ID NO: 962, 963, 964 and 966. In some embodiments, the vector encapsidated by the recombinant AAV comprises a sequence selected from SEQ ID NO: 70, 959 and 961. [00248] In some examples, methods of producing delivery vectors herein comprising packaging an engineered polynucleotide of the present disclosure (e.g., an engineered polynucleotide encoding an engineered guide RNA) in an AAV vector. In some examples, methods of producing the delivery vectors described herein comprise, (a) introducing into a cell: (i) a polynucleotide comprising a promoter and an engineered guide RNA payload disclosed herein; and (ii) a viral genome comprising a Replication (Rep) gene and Capsid (Cap) gene that encodes a wild-type AAV capsid protein or modified version thereof; (b) expressing in the cell the wild-type AAV capsid protein or modified version thereof; (c) assembling an AAV particle; and (d) packaging the payload disclosed herein in the AAV particle, thereby generating an AAV delivery vector. In some examples, the recombinant vectors comprise one or more inverted terminal repeats and the inverted terminal repeats can comprise a 5’ inverted terminal repeat, a 3’ inverted terminal repeat, and/or a
Attorney Docket No.199235.769601 mutated inverted terminal repeat. In some examples, the mutated terminal repeat lacks a terminal resolution site, thereby enabling formation of a self-complementary AAV. [00249] In some examples, a hybrid AAV vector can be produced by transcapsidation, e.g., packaging an inverted terminal repeat (ITR) from a first serotype into a capsid of a second serotype, wherein the first and second serotypes may not be the same. In some examples, the Rep gene and ITR from a first AAV serotype (e.g., AAV2) can be used in a capsid from a second AAV serotype (e.g., AAV5 or AAV9), wherein the first and second AAV serotypes may not be the same. As a non-limiting example, a hybrid AAV serotype comprising the AAV2 ITRs and AAV9 capsid protein can be indicated AAV2/9. In some examples, the hybrid AAV delivery vector comprises an AAV2/1, AAV2/2, AAV 2/4, AAV2/5, AAV2/8, or AAV2/9 vector. [00250] In some examples, the AAV vector can be a chimeric AAV vector. In some examples, the chimeric AAV vector comprises an exogenous amino acid or an amino acid substitution, or capsid proteins from two or more serotypes. In some examples, a chimeric AAV vector can be genetically engineered to increase transduction efficiency, selectivity, or a combination thereof. [00251] In some examples, the AAV vector comprises a self-complementary AAV genome. Self- complementary AAV genomes can be generally known in the art and contain both DNA strands which can anneal together to form double-stranded DNA. [00252] In some examples, the delivery vector can be a retroviral vector. In some examples, the retroviral vector can be a Moloney Murine Leukemia Virus vector, a spleen necrosis virus vector, or a vector derived from the Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, or mammary tumor virus, or a combination thereof. In some examples, the retroviral vector can be transfected such that the majority of sequences coding for the structural genes of the virus (e.g., gag, pol, and env) can be deleted and replaced by the gene(s) of interest. [00253] In some examples, the delivery vehicle can be a non-viral vector. In some cases, the delivery vehicle can be a DNA encoding the engineered guide RNA. In some examples, the delivery vehicle can be a plasmid. In some embodiments, the plasmid comprises DNA. In some examples, the plasmid comprises circular double-stranded DNA. In some examples, the plasmid can be linear. In some examples, the plasmid comprises one or more genes of interest and one or more regulatory elements. In some examples, the plasmid comprises a bacterial backbone containing an origin of replication and an antibiotic resistance gene or other selectable marker for plasmid amplification in bacteria. In some examples, the plasmid can be a minicircle plasmid. In some examples, the plasmid contains one or more genes that provide a selective marker to induce a target cell to retain the plasmid. In some examples, the plasmid can be formulated for delivery
Attorney Docket No.199235.769601 through injection by a needle carrying syringe. In some examples, the plasmid can be formulated for delivery via electroporation. In some examples, the plasmids can be engineered through synthetic or other suitable means known in the art. For example, in some cases, the genetic elements can be assembled by restriction digest of the desired genetic sequence from a donor plasmid or organism to produce ends of the DNA which can then be readily ligated to another genetic sequence. [00254] In some embodiments, the vector containing the engineered guide RNA or the engineered polynucleotide is a non-viral vector system. In some embodiments, the non-viral vector system comprises cationic lipids, or polymers. For example, the non-viral vector system can be a liposome or polymeric nanoparticle. In some cases, a non-viral vector system can be a lipid nanoparticle (LNP) or a polymer nanoparticle. In some embodiments, the engineered polynucleotide or a non- viral vector comprising the engineered guide RNA or the engineered polynucleotide is delivered to a cell by hydrodynamic injection or ultrasound. Administration [00255] Administration can refer to methods that can be used to enable the delivery of a composition described herein (e.g., comprising an engineered guide RNA or an engineered polynucleotide encoding the same, a recombinant AAV, or an AAV virion encapsidating a DNA vector genome) to the desired site of biological action. For example, an engineered guide RNA can be comprised in a DNA construct, a viral vector, or both and be administered by intravenous administration. Administration disclosed herein to an area in need of treatment or therapy can be achieved by, for example, and not by way of limitation, oral administration, topical administration, intravenous administration, inhalation administration, or any combination thereof. In some cases, administration disclosed herein can be a systemic administration. In some instances, administration can be systemic administration by an injection (e.g., intravenous administration or any administration by an injection) or oral delivery. In some embodiments, delivery can include inhalation, otic, buccal, conjunctival, dental, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intraabdominal, intraamniotic, intraarterial, intraarticular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavernous, intracavitary, intracerebroventricular, intracisternal, intracorneal, intracoronal, intracoronary, intracorpous cavernaosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intrahippocampal, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular,
Attorney Docket No.199235.769601 intraovarian, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous, intratesticular, intrathoracic, intratubular, intratumor, intratympanic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, ophthalmic, oral, oropharyngeal, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, retrobulbar, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, vaginal, infraorbital, intraparenchymal, intrathecal, intraventricular, stereotactic, or any combination thereof. Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion), oral administration, inhalation administration, intraduodenal administration, rectal administration, or a combination thereof. Delivery can include direct application to the affected tissue or region of the body. In some cases, topical administration can comprise administering a lotion, a solution, an emulsion, a cream, a balm, an oil, a paste, a stick, an aerosol, a foam, a jelly, a foam, a mask, a pad, a powder, a solid, a tincture, a butter, a patch, a gel, a spray, a drip, a liquid formulation, an ointment to an external surface of a surface, such as a skin. Delivery can include a parenchymal injection, an intra-thecal injection, an intra-ventricular injection, or an intra- cisternal injection. A composition provided herein can be administered by any method. A method of administration can be by intra-arterial injection, intracisternal injection, intramuscular injection, intraparenchymal injection, intraperitoneal injection, intraspinal injection, intrathecal injection, intravenous injection, intraventricular injection, stereotactic injection, subcutaneous injection, epidural, or any combination thereof. Delivery can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or infusion administration). In some embodiments, delivery can comprise a nanoparticle, a liposome, an exosome, an extracellular vesicle, an implant, or a combination thereof. In some cases, delivery can be from a device. In some instances, delivery can be administered by a pump, an infusion pump, or a combination thereof. In some embodiments, delivery can be by an enema, an eye drop, a nasal spray, or any combination thereof. In some instances, a subject can administer the composition in the absence of supervision. In some instances, a subject can administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician’s assistant, orderly, hospice worker, etc.). In some embodiments, a medical professional can administer the composition. [00256] In some cases, administering can be oral ingestion. In some cases, delivery can be a capsule or a tablet. Oral ingestion delivery can comprise a tea, an elixir, a food, a drink, a
Attorney Docket No.199235.769601 beverage, a syrup, a liquid, a gel, a capsule, a tablet, an oil, a tincture, or any combination thereof. In some embodiments, a food can be a medical food. In some instances, a capsule can comprise hydroxymethylcellulose. In some embodiments, a capsule can comprise a gelatin, hydroxypropylmethyl cellulose, pullulan, or any combination thereof. In some cases, capsules can comprise a coating, for example, an enteric coating. In some embodiments, a capsule can comprise a vegetarian product or a vegan product such as a hypromellose capsule. In some embodiments, delivery can comprise inhalation by an inhaler, a diffuser, a nebulizer, a vaporizer, or a combination thereof. [00257] In some embodiments, an engineered guide RNA disclosed herein or a polynucleotide encoding the engineered guide RNA can be administered with a second therapeutic. In some cases, the second therapeutic can be administered in an amount sufficient to treat a disease or condition. In some cases, administration of the second therapeutic can be concurrent administration or consecutive administration to administration of the engineered guide RNA disclosed herein or the polynucleotide encoding the engineered guide RNA. In some cases, the second therapeutic can comprise trofinetide or a salt thereof. In some cases, trofinetide or a salt thereof can be administered in an amount of about: 0.0001 gram to about 100 grams, about 1,000 mg to about 4,000 mg, about 5,000 mg to about 12,000 mg, or about 6,000 mg to about 10,000 mg. EMBODIMENTS [00258] Embodiment 1. An engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein: (a) the engineered guide RNA, upon hybridization to a sequence of a target MECP2 RNA that comprises a premature stop codon, forms a guide-target RNA scaffold with the sequence of the target MECP2 RNA, wherein the target MECP2 RNA comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 50 or SEQ ID NO: 54; (b) two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to the sequence of the target MECP2 RNA, wherein the two or more structural features comprises a mismatch, a bulge, an internal loop, or a combination thereof; (c) the structural feature is not present within the engineered guide RNA or the target MECP2 RNA prior to the hybridization of the engineered guide RNA to the target MECP2 RNA; and (d) the engineered guide RNA is from about 60 nucleotides to about 200 nucleotides in length. [00259] Embodiment 2. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 1, wherein upon hybridization of the engineered guide RNA to the
Attorney Docket No.199235.769601 target MECP2 RNA, the engineered guide RNA facilitates RNA editing of one or more target adenosines in the target MECP2 RNA by an RNA editing entity. [00260] Embodiment 3. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 2, wherein the RNA editing entity is an endogenous RNA editing entity. [00261] Embodiment 4. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 3, wherein the RNA editing entity is a human endogenous RNA editing entity. [00262] Embodiment 5. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 4, wherein the endogenous RNA editing entity is a human ADAR1, a human ADAR2, or both. [00263] Embodiment 6. The engineered guide RNA of any of embodiments 1-5, wherein the premature stop codon corresponds to an R168X mutation. [00264] Embodiment 7. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-5, wherein the target MECP2 RNA encodes a MECP2 polypeptide comprising a R168X nonsense mutation. [00265] Embodiment 8. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-7, comprising the two or more structural features which comprise the bulge. [00266] Embodiment 9. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 8, wherein the bulge is an asymmetrical bulge. [00267] Embodiment 10. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 8, wherein the bulge is a symmetrical bulge. [00268] Embodiment 11. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-7, comprising the two or more structural features which comprise the internal loop. [00269] Embodiment 12. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 11, wherein the internal loop is a symmetrical internal loop. [00270] Embodiment 13. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 11, wherein the internal loop is an asymmetrical internal loop. [00271] Embodiment 14. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-7, wherein the engineered guide RNA comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at
Attorney Docket No.199235.769601 least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NO: 31 – SEQ ID NO: 37, SEQ ID NO: 47, and SEQ ID NO: 51. [00272] Embodiment 15. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 14, wherein the guide-target RNA scaffold comprises at least one 6/6 symmetric internal loop. [00273] Embodiment 16. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 15, wherein the guide-target RNA scaffold comprises a symmetric bulge. [00274] Embodiment 17. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 15 or 16, wherein the guide-target RNA scaffold comprises a 12/12 symmetric internal loop or a 10/10 symmetric internal loop. [00275] Embodiment 18. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 17, wherein the engineered guide RNA comprises SEQ ID NO: 35 or SEQ ID NO: 37. [00276] Embodiment 19. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 15 or 16, wherein the guide-target RNA scaffold comprises two 6/6 symmetric internal loops. [00277] Embodiment 20. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 19, wherein the engineered guide RNA comprises SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 47, or SEQ ID NO: 51. [00278] Embodiment 21. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 15 or 16, wherein the guide-target RNA scaffold comprises at least nine wobble base pairs. [00279] Embodiment 22. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 21, wherein the engineered guide RNA comprises SEQ ID NO: 32, SEQ ID NO: 35, or SEQ ID NO: 37. [00280] Embodiment 23. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-7, wherein the polynucleotide encoding the engineered guide RNA comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, and SEQ ID NO: 52.
Attorney Docket No.199235.769601 [00281] Embodiment 24. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-23, wherein the polynucleotide encoding the engineered guide RNA is comprised in a viral vector. [00282] Embodiment 25. The engineered guide RNA or the polynucleotide encoding the engineered guide RNA of embodiment 24, wherein the viral vector is an AAV vector, a lentiviral vector, or a retroviral vector. [00283] Embodiment 26. A pharmaceutical composition comprising: (a) the engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-25, and (b) a pharmaceutically acceptable: excipient, carrier, or diluent. [00284] Embodiment 27. A method of increasing a level of full-length MECP2 in a cell, the method comprising administering to the cell the engineered guide RNA or the polynucleotide encoding the engineered guide RNA of any one of embodiments 1-25, or the pharmaceutical composition of embodiment 26. [00285] Embodiment 28. The method of embodiment 27, wherein the level of full-length MECP2 increases by at least 10%, at least 30%, at least 40%, or at least 50% relative to an otherwise comparable cell that was not administered the engineered guide RNA, the polynucleotide encoding the engineered guide RNA, or the pharmaceutical composition. [00286] Embodiment 29. The method of embodiment 27, wherein the level of full-length MECP2 is restored to functional levels in the cell. [00287] Embodiment 30. A method of treating a disease or a condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of embodiment 26. [00288] Embodiment 31. The method of embodiment 30, wherein the disease or condition comprises a Rett Syndrome. [00289] Embodiment 32. The method of embodiment 31, wherein the Rett Syndrome comprises a MECP2 polypeptide comprising a R168X nonsense mutation. [00290] Embodiment 33. The method of any of embodiments 30-32, wherein the subject is human or a non-human animal. [00291] Embodiment 34. An engineered guide RNA or a polynucleotide encoding the engineered guide RNA, wherein: (a) the engineered guide RNA, upon hybridization to a sequence of a target MECP2 RNA that comprises a premature stop codon, forms a guide-target RNA scaffold with the sequence of the target MECP2 RNA, wherein the engineered guide RNA comprises a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to any one of SEQ ID NOs: 31 – SEQ ID
Attorney Docket No.199235.769601 NO: 37, SEQ ID NO: 47, and SEQ ID NO: 51; (b) two or more structural features are substantially formed in the guide-target RNA scaffold upon hybridization of the engineered guide RNA to the sequence of the target MECP2 RNA, wherein the two or more structural features comprises a mismatch, a bulge, an internal loop, or a combination thereof; (c) the structural feature is not present within the engineered guide RNA or the target MECP2 RNA prior to the hybridization of the engineered guide RNA to the target MECP2 RNA; and (d) the engineered guide RNA is from about 60 nucleotides to about 200 nucleotides in length. [00292] Embodiment 35. An AAV virion encapsidating a DNA vector genome comprising a sequence with at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to any one of SEQ ID NO: 38 – SEQ ID NO: 44, SEQ ID NO: 48, and SEQ ID NO: 52. [00293] Embodiment 36. The AAV virion encapsidating the DNA vector genome of embodiment 35, wherein the AAV virion is an AAV1 virion, AAV2 virion, AAV3 virion, AAV4 virion, AAV5 virion, AAV6 virion, AAV7 virion, AAV8 virion, AAV9 virion, AAV10 virion, AAV11 virion, or a derivative, a chimera, or a variant thereof. [00294] Embodiment 37. The AAV virion encapsidating the DNA vector genome of any one of embodiments 35-36, wherein the AAV virion is a recombinant AAV (rAAV) virion, a hybrid AAV virion, a chimeric AAV virion, a self-complementary AAV (scAAV) virion, or any combination thereof. [00295] Embodiment 38. A pharmaceutical composition comprising: the AAV virion encapsidating the DNA vector genome of any one of embodiments 35-37, and a pharmaceutically acceptable: excipient, carrier, or diluent. [00296] Embodiment 39. A method of administering to a subject an effective amount of the AAV virion encapsidating the DNA vector genome of any one of embodiments 35-37, or the pharmaceutical composition of embodiment 38. [00297] Embodiment 40. The method of embodiment 39, wherein the subject is a mouse, a non- human primate, or a human. [00298] Embodiment 41. The method of any one of embodiments 39-40, wherein the pharmaceutical composition is in unit dose form. [00299] Embodiment 42. A method of treating a Rett syndrome in a subject comprising administering to a subject an effective amount of the AAV virion encapsidating the DNA vector genome of any one of embodiments 35-37, or the pharmaceutical composition of embodiment 38. [00300] Embodiment 43. The method of embodiment 42, wherein the subject is a mouse, a non- human primate, or a human.
Attorney Docket No.199235.769601 [00301] Embodiment 44. The method of any one of embodiments 42-43, wherein the pharmaceutical composition is in unit dose form. [00302] Embodiment 45. A method of editing an MECP2 RNA transcript in a subject comprising administering to a subject an effective amount of the AAV virion encapsidating the DNA vector genome of any one of embodiments 35-37, or the pharmaceutical composition of embodiment 38. [00303] Embodiment 46. The method of embodiment 45, wherein the subject is a mouse, a non- human primate, or a human. [00304] Embodiment 47. The method of any one of embodiments 45-46, wherein the pharmaceutical composition is in unit dose form. [00305] Embodiment 48. The method of any one of embodiments 45-47, wherein the editing of the MECP2 RNA transcript comprises editing of a MECP2 RNA transcript that comprises a R168X nonsense mutation. [00306] Embodiment 49. A kit comprising the AAV virion encapsidating the DNA vector genome of any one of embodiments 35-37, or the pharmaceutical composition of embodiment 38 and a container. EXAMPLES [00307] The following illustrative examples are representative of embodiments of the stimulation, systems, and methods described herein and are not meant to be limiting in any way. EXAMPLE 1: Generation of Engineered Guide RNAs for in-cell validation [00308] This example describes the curation of a panel of AI-derived engineered guide RNAs predicted to match the target product profile for in-cell validation. To generate allelic-specific engineered guide RNA architectures that edit both mouse (Mecp2) and human (MECP2) mutant isoforms of MECP2, yet are not reactive with the WT MECP2/Mecp2 allele, a bit diffusion model is utilized for de novo guide RNA microfootprint generation. This model is trained on guide RNA target interactions for > 5,000 clinically relevant targets. The generated designs are then scored using models trained on the same data but paired with various target sequences to predict cross-species reactivities. Some scoring models are also conditioned on metrics that incorporate in-cell editing data from rationally designed engineered guide RNAs for mouse Mecp2R168X. [00309] All engineered guide RNA architectures are grafted into barbell macrofootprints identified from the target assessment. Exemplary macrofootprints are 100.70 (-6, +30), 100.70 (- 9, +33) and 100.70 (-15, +33). The 100.70 (-6, +30) indicates a guide length of 100 nt and a
Attorney Docket No.199235.769601 mismatch at position 70 with a barbell being located 6 base pairs upstream (-6) of the mismatch, and a barbell being located 30 base pairs downstream (+30) of the mismatch. [00310] Selected engineered guide RNA architectures are tested in cells in batches of about 20- 50 and screened by transfection in a HEK293 cell system comprising four isoforms of interest: mouse Mecp2 R168X-Flag, mouse Mecp2 WT-Flag, human MECP2 R168X-Flag, and human MECP2 WT-Flag. Selected guide RNAs are further optimized to incorporate U-deletions, wobble base pairs with the target RNA and/or guide RNA shortening. Cross reactivity for engineered guide RNAs is developed for targeting MECP2 in human, mouse, and/or primate cells. EXAMPLE 2: Testing of engineered guide RNAs in a mouse model [00311] This example describes testing engineered guide RNAs in a mouse model of Rett syndrome. Engineered guide RNAs are packaged into AAV vectors for a delivery in Mecp2R168X male mice. Heterozygous females may be included to demonstrate mutant allele specificity of the engineered guide RNA designs. The study has molecular endpoints at 2, weeks, 4 weeks, 6 weeks, 6 months, and 12 months post-administration. [00312] After administration, mice are evaluated by Cumulative Bird Scoring metrics throughout the study’s duration to track potential engineered guide RNA-associated changes. The Cumulative Bird Score is derived from observational tests of inertia, gait, hindlimb clasping, tremor, and poor general condition. Each symptom is scored regularly as absent, present, or severe (scores of 0, 1 and 2, respectively). Combined, these traits monitor the specific features of the Rett-like mouse phenotype. A limited behavioral component may be added, and the study’s duration extended if differences are noted between engineered guide RNA-treated and untreated mutant animals (relative to WT controls) at 3 weeks-post engineered guide RNA administration. Behavioral assays may include an elevated plus maze or open field test (anxiety), rotarod (motor control), or nesting evaluations. Mecp2 editing, Mecp2 protein restoration, engineered guide RNA expression, and vg/dg are analyzed to quantify delivery and functionality of the Mecp2 engineered guide RNAs. RNA sequencing is performed to measure engineered guide RNA- induced changes in gene expression in vivo. Body weight measurements are recorded throughout the study as male Mecp2R168X mice are known to have a decreased body weight. Additionally, lifespan measurements are recorded throughout the study. EXAMPLE 3: Testing exemplary MECP2-targeting engineered guide RNAs in HEK Cells
Attorney Docket No.199235.769601 [00313] This example describes testing engineered guide RNAs targeting MECP2 in an engineered reporter HEK cell line. [00314] Seven rationally designed engineered guide RNAs were expressed in HEK reporter cell lines containing human R168X MECP2 or WT MECP2 - both fused with a C-terminal flag tag (in progress) or mouse R168X Mecp2 or WT Mecp2 ORF – both fused with a C-terminal flag tag. As shown in FIG.4, four of the seven designs demonstrated high-target site efficiency (>35%) in editing the mouse R168X transcript, showing allele specific editing by selective editing of the mutant allele (R168XMECP2) and little to no editing of the WT allele (WT MECP2) of the guide RNAs (e.g., SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 34, and SEQ ID NO: 35). The engineered guide RNAs showed variable cross-reactivity with the mouse WT transcript. As shown in FIG.5 and FIG.6, all engineered guide RNAs tested lead to partial restoration of both mouse and human protein in transfected cells as measured by FLAG+ flow- cytometry (FIG.5), with high correlation between on-target editing and full-length MECP2 protein (FIG.6). This data shows the engineered guide RNAs can be used to restore expression of full-length MECP2 carrying the R168X mutation. [00315] Guide RNA sequences used in this example are shown below in TABLE 4. The target RNA sequence are: mouse 92.62 target sequence: GGTAACTGGGAGAGGGAGCCCCTCCAGGTGAGAGCAGAAACCACCTAAGAAGCCC AAATCTCCCAAAGCTCCAGGAACTGGCAGGGGTCGGG (SEQ ID NO: 45) and mouse 100.70 target sequence: GGTAACTGGGAGAGGGAGCCCCTCCAGGTGAGAGCAGAAACCACCTAAGAAGCCC AAATCTCCCAAAGCTCCAGGAACTGGCAGGGGTCGGGGACGCCCC (SEQ ID NO: 46). [00316] For each sequence, the structural features formed in the double stranded RNA substrate upon hybridization of the guide RNA to the target MECP2 RNA, are shown in the last column of TABLE 4. For reference, each structural feature formed within a guide-target RNA scaffold (target RNA sequence hybridized to an engineered guide RNA) is annotated as follows: a) the position of the structural feature with respect to the target A (position 0) of the target RNA sequence, with a negative value indicating upstream (5’) of the target A and a positive value indicating downstream (3’) of the target A; b) the number of bases in the target RNA sequence and the number of bases in the engineered guide RNA that together form the structural feature – for example, 6/6 indicates that six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature;
Attorney Docket No.199235.769601 c) the name of the structural feature (e.g., symmetric bulge, symmetric internal loop, asymmetric bulge, asymmetric internal loop, mismatch, or wobble base pair), and d) the sequences of bases on the target RNA side and the engineered guide RNA side that participate in forming the structural feature. [00317] For example, with reference to SEQ ID NO: 31, -6_6-6_internal_loop- symmetric_CCCUCC-CCUCCC, -1->0_2-2_bulge-symmetric_GA-CG, 30_6-6_internal_loop- symmetric_UCCCAA-AACCCU” is read as a structural feature formed in a guide-target RNA scaffold (target MECP2 RNA sequence hybridized to an engineered guide RNA of SEQ ID NO: 31), where a structural feature starts 6 nucleotides upstream (5’) (the -6 position) from the target A (0 position) of the target RNA sequence; six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; the structural feature is an internal symmetric loop; and a sequence of CCCUCC from the target RNA side and a sequence of CCUCCC from the engineered guide RNA side participate in forming the internal symmetric loop. A structural feature starts at 1 nucleotide upstream (the -1 position) from the target A (0 position) of the target RNA sequence; 2 bases from the target RNA and 2 base from the engineered guide RNA form the structural feature; the structural feature is a symmetric bulge; and the sequence of GA from the target RNA side and a sequence of CG from the engineered guide RNA side participate in forming the symmetric bulge. A structural feature starts 30 nucleotides downstream (3’) (the +30 position) from the target A (0 position) of the target RNA sequence; six contiguous bases from the target RNA sequence and six contiguous bases from the engineered guide RNA form the structural feature; the structural feature is an internal symmetric loop; and a sequence of UCCCAA from the target RNA side and a sequence of AACCCU from the engineered guide RNA side participate in forming the internal symmetric loop. TABLE 4 – MECP2 gRNA Sequences (RNA, DNA) and Structural Features Guide RNA Guide DNA Structural Features Sequence Sequence
Attorney Docket No.199235.769601 Guide RNA Guide DNA Structural Features Sequence Sequence UGGUUUCUGCUCC TTTCTGCTCCGACC -6_6-6_internal_loop-symmetric_CCCUCC-ACUCAA A A A
Attorney Docket No.199235.769601 Guide RNA Guide DNA Structural Features Sequence Sequence (SEQ ID NO: 37) CGGTTATC (SEQ ID 33_10-10_internal_loop-symmetric_CAAAGCUCCA-
mice [00318] The engineered guide RNA design comprising engineered guide RNA having SEQ ID NO: 34 was packaged into scAAV-PHP.eB. Prior to packaging into scAAV-PHP.eB, the guides were designed for ITR-to-ITR configuration. The AAV vectors were delivered by retro-orbital administration at a dose of about 1E12 vg/mouse in adult R168X Mecp2 mutant and WT mice. A vehicle control was included to benchmark editing. The primary endpoints were editing and protein restoration of Mecp2 in the treated mutant mice. Individual brain regions (e.g., brainstem, cortex, hippocampus) from mice were examined for editing and/or protein restoration of Mecp2. Two weeks post-dosing with the AAV vectors or vehicle control, mice were euthanized and the brains were harvested for immunohistochemistry (IHC) with C-terminal Mecp2 mAB, immunofluorescence (IF), and to assess molecular endpoints including percent editing and gRNA abundance. [00319] As shown in FIG.14A, the engineered guide RNA of SEQ ID NO: 34 achieved closed to 40-50% average editing of the target adenosine of the mutant R168X MECP2 transcripts and low levels of editing of the WT MECP2 transcript in the brainstem and remaining brain (ROB) regions, showing allele-specific editing. These results show that the guide RNA having a sequence of SEQ ID NO: 34 shows allelic specific RNA editing for mutant MECP2 transcripts and low levels of local off-target and WT MECP2 transcript editing in vivo. FIG.14B demonstrates that introduction of the guide RNA having a sequence of SEQ ID NO: 34 resulted in widespread protein restoration in the R168X Mecp2 mutant mice brain. Notably, restored MECP2 protein showed expected nuclear localization (FIG.14B). This nuclear localization was quantified from the IHC, as shown in FIG.14C, the MECP2 gRNA produced approximately 20% MECP2+ nuclei in MECP2R168X mouse brainstem. This increase in Mecp2 expression in the MECP2R168X mouse brain was further corroborated in IF experiments. As shown in FIG.14D – FIG.14E, the MECP2 gRNA selectively induced Mecp2 expression in the mouse brain as compared to the control gRNA. Together, these results show that the guide RNA having a sequence of SEQ ID NO: X show broad MECP2 Restoration in the MECP2R168X mouse brain two weeks after systemic administration of gRNA Via AAV PHP.eB.
Attorney Docket No.199235.769601 EXAMPLE 5: In vitro testing of MECP2-targeting engineered guide RNAs in neurons [00320] Mouse primary neurons from 4 different male mice with hemizygous R168X mutations were treated with a low dose (5E4 vg/cell) or high dose (5E5 vg/cell) of an AAV encoding a guide RNA (SEQ ID NO: 34) targeting an MECP2 transcript with an R168X mutation that results in reduced expression of full-length MECP2 protein.7 days after the treatment, MECP2 protein was measured by flow cytometry using a MECP2 specific antibody to measure protein restoration, which is shown in FIG.7. At both the low and high doses, the transduced guide RNA increased MECP2 protein production. At the low dose, about 25% of cells contained the restored MECP2 protein (designated as an MECP2 positive cell), and at the high dose about 60% of cells were MECP2 positive. When the mutant cells were not treated (no tdxn group) no MECP2 positive cells were identified. Further, the high dose resulted in full-length MECP2 protein levels approaching the WT cell control in which MECP2 protein is expressed from a transcript lacking the R168X mutation. These results show guide RNAs targeting MECP2 R168X mutant cells can restore protein production in cells that express the R168X mutation. [00321] The same guide RNA was then tested in human neurons. Human iPSC-derived neurons with R168X mutations were treated with a low dose (1E5 vg/cell) or high dose (5E5 vg/cell) of an AAV encoding the guide RNA (SEQ ID NO: 34) targeting an MECP2 transcript with the R168X mutation. Two different delivery vectors were tested (“Vector 1” and “Vector 2” in FIG. 10). Following 7-day infection with the AAV, editing of the target adenosine was measured by Sanger sequencing. As shown in FIG.10 (top), treatment with the guide RNA resulted in allele- specific editing of the MECP2 R168X mutant transcript. The treated cells expressed full-length MECP2 protein from the MECP2 R168X mutant allele, as measured by flow cytometry (FIG. 10, bottom). Together, these results show that the guide RNA having a sequence of SEQ ID NO: 34 show allelic specificity for mutant MECP2 and can restore protein production in human neurons with the human MECP2 R168X mutation. EXAMPLE 6: Testing exemplary MECP2-targeting engineered guide RNAs in HEK Cells [00322] This example describes validating guide RNAs targeting MECP2 obtained as described in EXAMPLE 1 in four engineered HEK293T cell lines. A HEK293T cell line was engineered to express four MECP2 transgenes (human R168X MECP2, human WT MECP2, mouse R168X MECP2 or mouse WT MECP2) and transfected with 500 ng of guide RNA plasmids into 20k cells and the transfection was carried out for 48 hrs. Controls were non-transfected cells, GFP only, LCOR (a non MECP2 targeting control gRNA to establish dynamic range of editing within the system), and a positive control guide RNA generated by rational design (as indicated by the
Attorney Docket No.199235.769601 stars in FIG.8) to enable a direct comparison with a MECP2 R168X targeting gRNA. cDNA synthesis was carried out with transcript specific primers and editing was evaluated by Sanger sequencing. [00323] As shown in FIG.8, assayed guide RNAs enabled up to ~55% editing of either mouse or human R168X mutant transcripts in cells. Guide RNAs demonstrated cross-reactivity with mouse and human MECPR168X and limited cross-reactivity with mouse or human MECP2WT transcripts. This data shows that the engineered guide RNAs exhibit both allelic and species cross-reactivity. [00324] FIG.9 provides an example of RNA editing by an engineered guide RNA that has the RNA sequence of SEQ ID NO: 47 (encoded by a DNA sequence of SEQ ID NO: 48) as set forth in TABLE 5. TABLE 5 – MECP2 gRNA Sequence (RNA, DNA) and Structural Features Guide RNA Guide DNA Structural Features (human Structural Features (mouse Sequence Sequence MECP2) MECP2) T 1 i l l 1 i l l
[00325] This guide RNA exhibited high-on-target editing efficiency for both human (>40%) and mouse (>50%) MECP2 R168X transcripts while retaining high specificity (limiting bystander editing below 5%). Additionally, this guide RNA demonstrated little to no editing of the human MECP2 wild type (WT) transcript and showed limited editing of the mouse WT transcript. The data shows that the engineered guide RNAs (e.g., SEQ ID NO: 47) achieve allelic specificity for mutant MECP2 as well as transcript specificity (limited bystander editing). EXAMPLE 7: Testing a MECP2-targeting engineered guide RNA [00326] This example describes validating a guide RNA targeting MECP2 in HEK293T cell line. A HEK293T cell line was engineered to express four MECP2 transgenes (human (Hu) R168X
Attorney Docket No.199235.769601 MECP2, human WT MECP2, mouse (Mu) R168X MECP2 or mouse WT MECP2) and transfected with 500 ng of guide RNA plasmids into 20k cells and the transfection was carried out for 48 hrs. [00327] As shown in FIG.11A, the assayed guide RNA enabled >51% editing of either mouse or human R168X mutant transcripts in cells. The guide RNA demonstrated cross-reactivity with mouse and human MECP2R168X and limited cross-reactivity with mouse or human MECP2WT transcripts. This data shows that the engineered guide RNA exhibited both allelic RNA editing specificity and species cross-reactivity. FIG.11B shows the predicted secondary structure of the guide RNA when hybridized to the four MECP2 alleles from FIG.11A. The arrows indicate structural alterations that may allow ADAR to discriminate between the alleles and allow for editing of Mecp2 R168X vs WT Mecp2 alleles. For example, this gRNA creates a GA-GC bulge at the target adenosine when hybridized to the human and mouse mutant transcripts, a secondary structure that facilitates editing of adenosines in a 5′ G context. Due to sequence differences at the -2 position, this structure is predicted to collapse when the gRNA hybridizes to the WT transcripts, forming an A-bulge that is refractory to ADAR editing. [00328] TABLE 6 below depicts the guide RNA used in this example, along with the structural features present when the guide RNA hybridizes to the human MECP2 WT and human MECP2 R168X alleles. TABLE 6 – MECP2 gRNA Sequence (RNA, DNA) and Structural Features Guide RNA Sequence Guide DNA Target RNA Structural Features (human MECP2) Sequence
Attorney Docket No.199235.769601 [00329] This guide RNA exhibited high-on-target editing efficiency for both human (>51%) and mouse (>51%) MECP2 R168X transcripts while retaining high specificity (limiting bystander editing below 5%). Additionally, this guide RNA demonstrated little editing of the human MECP2 wild type (WT) transcript (>15%) and showed limited editing of the mouse WT transcript (>10%). The data shows that the engineered guide RNA achieved allelic specificity for mutant MECP2 as well as transcript specificity (limited bystander editing). EXAMPLE 8: In vitro testing of a MECP2-targeting engineered guide RNA [00330] This example describes validating a guide RNA targeting MECP2 in disease-relevant in vitro models. The disease-relevant in vitro models included ex vivo mouse neurons, and human induced pluripotent stem cells (iPSC)-derived neurons. [00331] The ex vivo mouse neurons were transduced with a scAAV PHP.eB comprising a low dose (5e4 vg/cell) and high dose (5e4 vg/cell), and the human iPSC-derived neurons were transduced with a scAAV PHP.eB comprising a low dose (1e5 vg/cell) and high dose (5e5 vg/cell). Up to 95-97% of live cells were transduced with low and high doses of guide RNA (SEQ ID NO: 34) after 7 days. As shown in FIG.12A, the assayed guide RNA enabled >20% RNA editing of mouse and human R168X mutant transcripts in cells. The guide RNA of SEQ ID NO: 34 demonstrated species cross-reactivity (high levels of editing in both mouse and human disease-relevant in vitro models) and dose-dependent editing. Additionally, allele specificity was maintained in disease-relevant in vitro models, including high doses, shown by high levels of editing of the mutant R168X MECP2 mutant transcript and low levels of WT MECP2 transcript editing. Further, as shown FIG.12B, the assayed guide RNA (SEQ ID NO: 34) led to dose- dependent full-length MECP2 protein production in both mouse and human disease-relevant in vitro models. As shown in FIG.13, full-length MECP2 protein expression was restored in human iPSC-derived neurons after seven days transfection with a high dose of guide RNA (SEQ ID NO: 34 ). The data shows that the engineered guide RNA of SEQ ID NO: 34 achieved allelic specific RNA editing of mutant MECP2 transcripts and demonstrated species cross-reactivity as well as dose-dependent protein restoration and production. EXAMPLE 9: Exemplary gRNA Designs Improve Local Specificity In vitro [00332] This example describes additional guide RNA design and optimization using ML. The additional guides generated herein demonstrated improved activity and local specificity in
Attorney Docket No.199235.769601 disease-relevant in vitro models. The disease-relevant in vitro models was human induced pluripotent stem cells (iPSC)-derived neurons. [00333] The human iPSC-derived neurons were transduced with a scAAV PHP.eB comprising a low dose (1e5 vg/cell) and high dose (5e5 vg/cell) containing SEQ ID NO: 41, SEQ ID NO: 55, or SEQ ID NO: 56. After 7 days of infection, cells were harvested to assess molecular endpoints including percent editing and protein expression. As shown in FIG.15A, all gRNAs tested showed species cross-reactivity and dose-dependent efficiency, and allele specificity was maintained at high doses. The guide RNAs (SEQ ID NO: 55, or SEQ ID NO: 56) had up to 50% editing at the target A at the high dose (FIG.15A). The ML optimized guide RNAs (SEQ ID NO: 55, or SEQ ID NO: 56), as shown in FIG.15B, produced similar or increased MECP2 than the parent gRNA (SEQ ID NO: 41). Thus, the ~ 50% editing observed lead to some MECP2 protein restoration. The infection of WT neurons did not impact the WT protein expression, as demonstrated in FIG.15C. The IF in FIG. 15D shows that treatment with the improved gRNA designs resulted in broad restoration of MECP2 protein in mutant human neurons. Collectively, the results show that guide RNAs disclosed in this example had improved local specificity, local target editing and protein restoration in human iPSC-derived neurons. EXAMPLE 10: MECP2 guide RNA expression in various ITR-to-ITR Vector Construct Design [00334] This example provides exemplary vector constructs comprising engineered MECP2 gRNAs. An initial vector design (a bidirectional vector), exemplified in FIG.16, which comprises three promoters (shown as arrows), regulatory elements (shown as connected boxes), ( two guide RNAs (shown as gRNA) and three terminators (shown as boxes). A tandem vector design can also be used which is also shown in FIG.16 and comprises two promoters (shown as arrows), two guide RNAs (shown as gRNA), regulatory elements (shown as connected boxes), and two terminators (shown as boxes). ITR-to-ITR constructs containing engineered guide RNAs targeting MECP2 obtained as described in EXAMPLE 1 were designed. The constructs are described in TABLE 7. Each construct contained two copies of a polynucleotide encoding the same guide RNA (e.g., SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 41, or a control gRNA). [00335] For vector design 1, the guide RNAs were configured in the ITR-to-ITR region in a tandem configuration (e.g., the guides were oriented such that transcription occurs in the same direction). ITR-to-ITR constructs 1, 2, 3 and 4 were designed using vector design 1, where the polynucleotide encoding for the first guide RNA was operably linked to an engineered mU7 promoter (SEQ ID NO: 72), while the polynucleotide encoding for the second guide RNA was operably linked to an engineered promoter sequence (SEQ ID NO: 81). The first copy of the
Attorney Docket No.199235.769601 gRNA comprised a hairpin 3’ accessory element (SEQ ID NO: 955) or a SmOPT and U7 hairpin sequence 3’ accessory elements (SEQ ID NO: 76), and the second copy of the gRNA comprised a SmOPT and U7 hairpin sequence 3’ accessory elements (SEQ ID NO: 76). The polynucleotide encoding for the first guide RNA was operably linked to a terminator sequence 1 (SEQ ID NO: 80) or terminator sequence 5 (SEQ ID NO: 87), while the polynucleotide encoding for the second guide RNA was operably linked to a terminator sequence 2 (SEQ ID NO: 83). For ITR-to-ITR construct 1, the polynucleotide encoding for the first guide RNA comprised the MECP2 guide having the sequencing according to SEQ ID NO: 62 (RNA sequence of SEQ ID NO: 56), while the polynucleotide encoding for the second guide RNA comprised the MECP2 guide having the sequence according to SEQ ID NO: 62 (RNA sequence of SEQ ID NO: 56). For ITR-to-ITR construct 2, the polynucleotide encoding for the first guide RNA comprised the MECP2 guide having the sequencing according to SEQ ID NO: 52 (RNA sequence of SEQ ID NO: 51), while the polynucleotide encoding for the second guide RNA comprised the MECP2 guide having the sequence according to SEQ ID NO: 52 (RNA sequence of SEQ ID NO: 51). For ITR-to-ITR construct 3, the polynucleotide encoding for the first guide RNA comprised the MECP2 guide having the sequencing according to SEQ ID NO: 41 (RNA sequence of SEQ ID NO: 34), while the polynucleotide encoding for the second guide RNA comprised the MECP2 guide having the sequence according to SEQ ID NO: 41 (RNA sequence of SEQ ID NO: 34). For ITR-to-ITR construct 4, the polynucleotide encoding for the first guide RNA comprised a control gRNA sequence accord to SEQ ID NO: 956, while the polynucleotide encoding for the second guide RNA comprised a control gRNA sequence accord to SEQ ID NO: 957. [00336] For vector design 2, the guide RNAs were configured in the ITR-to-ITR region in a bidirectional configuration (e.g., the guides were oriented such that transcription occurs in the opposite direction) with a CMV-Thy1.1 transduction marker cassette for experimental readout. The polynucleotide encoding for the first guide RNA (SEQ ID NO: 41) was operably linked to an engineered mU7 promoter (SEQ ID NO: 72), while the polynucleotide encoding for the second guide RNA (SEQ ID NO: 41) was operably linked to an engineered promoter sequence 3 (SEQ ID NO: 958). Both the first and second copies of the gRNA comprised a SmOPT and U7 hairpin sequence 3’ accessory element (SEQ ID NO: 76), and were operably linked to a terminator sequence 3 (SEQ ID NO: 84). This example describes different ITR-to-ITR constructs developed with different vector designs that contain different regulatory elements. TABLE 7. ITR-to-ITR Construct Design ITR-to-ITR ITR-to-ITR ITR-to-ITR ITR-to-ITR ITR-to-ITR
Attorney Docket No.199235.769601 Vector Design 1 1 1 1 2 Guide RNA Configuration Tandem Tandem Tandem Tandem Bidirectional er : de : Q : : de : Q : : :
[00337] This example describes the in vitro activities of the different ITR-to-ITR constructs described in TABLE 7. The ITR-to-ITR constructs screened comprised either a guide RNA targeting MECP2, as described in EXAMPLE 4, (ITR-to-ITR construct 3, and ITR-to-ITR construct 5), or a control guide (ITR-to-ITR construct 4). The ITR-to-ITR constructs were packaged into scAAV-PHP.eB vector. The AAV vectors were delivered by retro-orbital administration at a dose of about 1E12 vg/mouse in adult R168X Mecp2 mutant mice. A vehicle control was included to benchmark editing. The primary endpoint was percent editing of target adenosine in Mecp2 in the treated mice. Individual brain regions (e.g., brainstem, cerebellum, rest
Attorney Docket No.199235.769601 of brain (ROB)) from mice were examined for editing and/or protein restoration of Mecp2. As shown in FIG. 17A, two weeks after treatment with the different ITR-to-ITR constructs, ITR-to- ITR constructs 1 and 3 exhibited at least 40% editing in the brainstem and ROB regions. Notably, ITR-to-ITR construct 3 demonstrated 60% editing in the brainstem and ROB regions, but it had an increase in variability compared to ITR-to-ITR construct 5. Both ITR-to-ITR constructs 1 and 3 maintained allele specificity with minimal off-target edits. These results demonstrate that ITR-to- ITR constructs 1 and 3 had increased on-target editing in mice brains. ITR-to-ITR construct 3 and ITR-to-ITR construct 5 resulted in high guide quantification, and mean AAV genome copy number per gDNA (vg/dg) (FIG. 17B & FIG. 17C). Guide expression was highest in the brainstem, followed by the ROB regions, with similar levels observed in both ITR-to-ITR construct 3 and ITR-to-ITR construct 4 (FIG.17B). The highest vg/dg was observed in the brainstem and the ROB regions of R168X Mecp2 mutant mice treated with ITR-to-ITR construct 3, as compared to wild type mice (FIG. 17C). The vg/dg for ITR-to-ITR constructs 2 and 3 were roughly 2-fold lower than ITR-to-ITR construct 3 (FIG.17C). Since both ITR-to-ITR constructs 1 and 2 were designed using vector design 1, both constructs behave similarly as shown in FIG.17C. As shown in FIG. 17D, higher guide expression as observed with ITR-to-ITR construct 3, but no positive correlation was observed with vg/dg levels. This data suggests that the expression of the gRNA does not increase with increased vg/dg, potentially because level of guide expression was at the upper limit. The brainstem exhibited the highest gRNA expression compared to other brain regions (FIG. 17D). [00338] Treatment with either the ITR-to-ITR construct 5 or ITR-to-ITR construct 4 demonstrated measurable editing and guide expression in the liver (FIG.18A – FIG. 18B). Measurable editing of the respective target adenosine (up to 16%) was observed in the liver of R168X Mecp2 mutant mice after treatment with ITR-to-ITR constructs 3 and 4 (control gRNA), despite PHP.eB serotype being sub-optimal for liver transduction (FIG.18A). Both wild type and mutant mice treated with ITR-to-ITR construct 4, exhibited from 5 – 10% editing in liver samples. No on-target editing was observed in liver samples for ITR-to-ITR construct 5 (FIG.18A). Similar to the on-target editing observed, ITR-to-ITR construct 3 and ITR-to-ITR construct 4 (control gRNA) showed guide RNA expression in the liver samples of both wild type and mutant mice (FIG.18B). As shown in FIG. 18C – FIG. 18D, higher guide abundance in liver correlated with increased editing within the ITR-to-ITR construct 3 and ITR-to-ITR construct 4 (control gRNA). As shown in FIG. 18C, R168X Mecp2 mutant mice treated with ITR-to-ITR construct 3 exhibited higher guide RNA abundance showed increased editing, as compared to wild type mice treated the same construct. The wild type mice treated with ITR-to-ITR construct 3 expressed the guide, however, no editing
Attorney Docket No.199235.769601 was observed (FIG. 18C). As shown in FIG. 18D, both wild type and mutant mice treated with ITR-to-ITR construct 4 (control gRNA) exhibited a positive correlation between guide expression and editing for both genotypes. EXAMPLE 11: In vivo testing of exemplary MECP2-targeting engineered guide RNAs in mice [00339] The engineered guide RNAs were packaged into scAAV-PHP.eB (“vector”). Prior to packaging into scAAV-PHP.eB, the guides were designed for ITR-to-ITR configuration (ITR-to- ITR construct 1 and ITR-to-ITR construct 2). The AAV vectors were delivered by retro-orbital administration at a dose of about 1E12 vg/mouse in adult R168X Mecp2 mutant and WT mice. Vehicle and an off target gRNA control were included to benchmark editing. The endpoints assessed were percent RNA editing and protein restoration of Mecp2 in the treated mice, as compared to wild type. To this end, individual brain regions (e.g., general cortex, frontal brain; brainstem; and midbrain) from mice were examined for editing and/or protein restoration of Mecp2. Two to three weeks post-dosing with the AAV vectors or vehicle control, mice were euthanized and the brains were harvested. The harvested brains were then used to assess percent editing and sagittal brain sections were stained for immunohistochemistry (IHC) with a C- terminal Mecp2 mAB. [00340] As shown in FIG.19A, ITR-to-ITR construct 1 (SEQ ID NO: 69), and ITR-to-ITR construct 2 (SEQ ID NO: 70) comprising the sequences encoding the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51) achieved broad distribution across the mouse brain hemisphere (66% gRNA-positive cells (data not shown)) and mutant allele-specific editing of 52- 69% of the target adenosine in the general cortex, brainstem, and general midbrain. These results show that the ITR-to-ITR construct 1 (SEQ ID NO: 69), and ITR-to-ITR construct 2 (SEQ ID NO: 70) comprising the sequences encoding the engineered guide RNAs (SEQ ID NO: 56, and SEQ ID NO: 51) demonstrated allelic specificity for mutant MECP2, and high percent editing in vivo as compared to the vehicle control. FIG.19B demonstrates that introduction of the ITR-to- ITR construct 1 (SEQ ID NO: 69) comprising the sequence encoding the engineered guide RNA (SEQ ID NO: 56) resulted in widespread in vivo protein restoration in the R168X Mecp2 mutant mice brain, as compared to vehicle control. Notably, restored MECP2 protein showed expected nuclear localization (FIG.19B). This nuclear localization was quantified from the IHC. As shown in FIG.19C, the MECP2 gRNA produced approximately 39% MECP2+ nuclei in mutant MECP2R168X mouse, as compared to the vehicle control, showing restoration of the full-length protein in an average of 39% of cells. Lastly, the introduction of ITR-to-ITR construct 1 (SEQ ID NO: 69), and ITR-to-ITR construct 2 (SEQ ID NO: 70) comprising the engineered guide RNAs
Attorney Docket No.199235.769601 (SEQ ID NO: 56, and SEQ ID NO: 51) resulted in widespread in vivo protein restoration in the mutant MECP2R168X mouse, without altering the levels of the wild type MECP2 (FIG.19C). Together, these results show that introduction of the vectors disclosed herein comprising the sequences encoding the guide RNAs disclosed herein into mice resulted in high percent RNA editing, and broad MECP2 restoration in the MECP2R168X mouse brain after systemic administration. EXAMPLE 12: Exemplary gRNA Designs Improve Local Specificity In vitro [00341] This example describes in vitro evaluation of exemplary constructs comprising sequences (SEQ ID NO: 62, SEQ ID NO: 52, SEQ ID NO: 836, SEQ ID NO: 861, SEQ ID NO: 66, SEQ ID NO: 64, or SEQ ID NO: 65) encoding the guide RNAs having a sequence of SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, SEQ ID NO: 428, SEQ ID NO: 60, SEQ ID NO: 58, or SEQ ID NO: 59 in a human Rett-patient iPSC-derived neurons. Briefly, human Rett patient iPSC-derived neurons were used to derive two cell lines: R168X and WT cell lines by selective X-chromosome inactivation from 1 patient. These cell lines were further engineered for uniform, rapid neuronal differentiation. The neurons were differentiated for 8 days, and following the differentiation the cells were infected for 7 days. The Rett-patient iPSC-derived neurons were transduced with a scAAV PHP.eB vector at doses of 5e4 vg/cell and 1e5 vg/cell in triplicate. The DNA sequence of each guide RNA (SEQ ID NO: 62, SEQ ID NO: 52, SEQ ID NO: 836, SEQ ID NO: 861, SEQ ID NO: 66, SEQ ID NO: 64, or SEQ ID NO: 65) was packaged into the scAAV PHP.eB vector construct according to vector design 1 as exemplified in EXAMPLE 10 and EXAMPLE 11 (e.g., ITR-to-ITR construct 1 comprising SEQ ID NO: 69 for the engineered guide RNA sequence of SEQ ID NO: 56 and ITR-to-ITR construct 2 comprising SEQ ID NO: 70 for the engineered guide RNA sequence of SEQ ID NO: 51). A scrambled guide RNA control construct was also tested (“Control gRNA”). After 7 days of infection, percent editing was assessed. FIG.20A and FIG.21 show percent target adenosine editing at the higher dose 1e5 cells (FIG.20A), or both lower dose 5e4 vg/cell and higher dose 1e5 vg/cell (FIG.21), with the editing bars superimposed on each other, not stacked. As shown in FIG.20A and FIG.21, exemplary guide RNA (SEQ ID NO: 56) encoded by SEQ ID NO: 63 demonstrated up to 43% mutant allele-specific editing of MECP2 in the human Rett-patient iPSC-derived neurons, as compared to 18% wild type editing at the higher dose of 1e5 vg/cell. Additionally, both guide RNAs (SEQ ID NO: 403, SEQ ID NO: 428) encoded by SEQ ID NO: 836, or SEQ ID NO: 861 showed remarkable editing of both mutant and wild type MECP2 transcripts in human cells. This
Attorney Docket No.199235.769601 data shows that the exemplary guides disclosed herein edit mutant MECP2 transcript from 27% to 66%, as compared to wild type editing which ranged from 11% to 61%. [00342] FIG.20B demonstrates that the engineered guide RNA of SEQ ID NO: 56 resulted in widespread in vitro protein restoration in the R168X Mecp2 neurons, as compared to vehicle control. Notably, restored MECP2 protein showed expected nuclear localization (FIG.20B). As shown in FIG.20D, the engineered guide RNA of SEQ ID NO: 51 resulted in some in vitro protein restoration in the R168X Mecp2 neurons, as compared to vehicle control. This nuclear localization was quantified from the IF. As shown in FIG.20C, the MECP2 gRNA of SEQ ID NO: 56 produced approximately 38% MECP2+ nuclei in mutant MECP2R168X human neurons, as compared to the vehicle control, showing restoration of the full-length protein in an average of 38% of cells. The MECP2 gRNA of SEQ ID NO: 51 produced approximately 14% MECP2+ nuclei in mutant MECP2R168X human neurons, as compared to the vehicle control, showing restoration of the full-length protein in an average of 14% of cells. [00343] As shown in FIGs.20C and 20F, treatment with the engineered gRNA of SEQ ID NO: 56 resulted in a greater increase in MECP2+ nuclei in MECP2R168X neurons nuclei than compared to engineered gRNA of SEQ ID NO: 51. Each biological replicate of neurons treated with engineered gRNA of SEQ ID NO: 56 demonstrated statistical significance independently. However, both treatments (engineered gRNAs of SEQ ID NO: 56 and SEQ ID NO: 51) cause the majority of neuron nuclei to be positive, but signal was quite dim for engineered gRNA of SEQ ID NO: 51 treated neurons which limited the accuracy of threshold-based counting and caused a general underestimation of percent positive cells. Despite this limitation, treatment with either construct resulted in an increase in MECP2+ nuclei, which indicated protein restoration. A minority of MECP2R168X neurons showed up with very intense positivity, which was reflected in intensity plots. This occurred regardless of treatment with MECP2 gRNA or controls and may be due to re-activation of the inactive WT copy of MECP2 in these cells during iPSC reprogramming. Importantly, treatment with the engineered gRNAs did not significantly alter nuclear MECP2 in MECP2WT neurons (FIGs.20E and 20G). The MECP2WT neurons were 75- 90% positive for MECP2 staining, and treatment with either construct or control did not significantly alter MECP2 nuclear staining intensity or percent positive nuclei. Collectively, this data demonstrated that treatment with the engineered guide RNAs disclosed herein resulted in an increase in MECP2 protein in the nuclei of MECP2R168X neurons, and did not affect MECP2 protein in MECP2WT neurons. [00344] Additionally, flow cytometry was also used to quantify MECP2+ expression in neurons after treatment with either a low dose (5e4 vg/cell) or a high dose (1e5vg/cell) of the engineered
Attorney Docket No.199235.769601 guide RNAs (SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, or SEQ ID NO: 428), as compared to scramble control (NTGC) and no treatment control (No Tdxn). In general, treatment with the engineered guide RNAs at either dose restored some amount of full-length MECP2 protein in MECP2R168X neurons (FIG.24 and FIG.25). [00345] Together, these results show that introduction of the vectors disclosed herein comprising the sequences encoding the guide RNAs disclosed herein into human Rett-patient iPSC-derived neurons resulted in high percent RNA editing, and broad MECP2 restoration in the MECP2R168X cells after systemic administration. [00346] To determine if the differences in editing was a result of a difference in expression, guide RNA expression was then quantified using ddPCR. Based on ddPCR, the engineered guide RNAs displayed similar levels of abundance between the doses in both wild type and mutant human neurons (MECP2R168X) (FIGs.22A -22B). [00347] Engineered guide RNAs of SEQ ID NO: 56 and SEQ ID NO: 51 were also tested for global specificity in human iPSC-derived neurons from a Rett Syndrome patient. No large change in splicing was observed in any of the engineered guide RNA treated samples demonstrating that the engineered guide RNAs have high specificity in human neurons. [00348] There is a rare, naturally occurring isoform of MECP2 that excludes a 109 bp segment of exon 4, and can be targeted by the first 68 bp of the engineered guide RNAs disclosed herein (FIG. 23A). If protein(s) are produced from this transcript variant, the C-terminal end is out-of-frame relative to the common E1 and E2 isoforms. Human Rett patient iPSC-derived neurons were infected for 7 days following 8 days of neuronal differentiation in triplicate. Both mutant (MECP2R168X) and wild type (MECP2WT) neurons were tested. The neurons were then transduced with low dose (5e4 cells) or high dose (1e5 cells) of guide RNAs (SEQ ID NO: 56 or SEQ ID NO: 51), and a scramble control. An increase in novel splice variants (NSV) was identified via human Mecp2 AmpSeq (RQID-482) after treatment with the engineered guide RNAs, as compared to the scramble control and untreated wild type and mutant neurons (FIGs.23B-23C). As demonstrated in FIGs.23B-23C, the frequency of a partial exon exclusion increased with gRNA treatment. The variant resulting from treatment excluded a 109 bp region of the canonical Exon 4, including the target adenosine and partial gRNA hybridization zone. Moreover, the engineered guide RNA (SEQ ID NO: 56) demonstrated more exon exclusion than the engineered guide RNA (SEQ ID NO: 51), and there was an increase in exon exclusion in mutant neurons as compared to wild type neurons. Total MECP2 transcript was assessed using primers and a probe that bind in an exon 3 region conserved across known and predicted MECP2 transcripts. As shown in FIGs.23D-23E, MECP2 transcript abundance was similar in treated and non-treated samples of the same genotype (2-Way
Attorney Docket No.199235.769601 ANOVA). Relative untreated MECP2R168X transcript was present at about half of the untreated MECP2WT transcript level. MECP2R168X neurons showed slightly higher baseline levels of the rare transcript isoform than the wild type counterpart (data not shown). Collectively, these results demonstrated that treatment with the engineered guide RNAs (SEQ ID NO: 56 and SEQ ID NO: 51) was associated with a small increase in the frequency of a rare, naturally occurring MECP2 isoform in both mutant and WT human neurons. Moreover, the engineered guide RNAs were not associated with significant changes in MECP2 transcript relative to untreated cells of the same genotype. EXAMPLE 13: Ex vivo testing of the engineered guide RNAs [00349] This example describes ex vivo testing of the engineered guide RNAs. The ex vivo mouse MECP2R168X neurons and mouse MECP2WT neurons were transduced with a low dose (5E4 vg/cell) and high dose (1E5 vg/cell) of the engineered guide RNAs (SEQ ID NO: 56, SEQ ID NO: 51, SEQ ID NO: 403, or SEQ ID NO: 428) after 7 days. Generally, the engineered guide RNAs edited MECP2R168X transcript more than MECP2WT transcript (FIG. 26A), with MECP2R168X transcript reaching up to 67% and 80% for the high dose for SEQ ID NO: 56 and SEQ ID NO: 428, respectively. Protein restoration was observed after treatment with the engineered guide RNAs, as indicated by an increase in % MECP2+ cells, and gMFI as assessed by flow cytometry (FIGs. 26B-26C). Collectively, these results demonstrated that the engineered guide RNAs were highly efficient in mouse ex vivo cultures. EXAMPLE 14: In vivo editing, guide quantification and viral genome of exemplary MECP2- targeting engineered guide RNAs in mice [00350] This example describes the in vivo testing of the engineered guide RNAs in mutant (Mecp2R168X) and wild type (Mecp2WT) mice. The engineered guide RNAs were packaged into scAAV-PHP.eB (“vector”). Prior to packaging into scAAV-PHP.eB, the guides were designed for ITR-to-ITR configuration (ITR-to-ITR construct 1 and ITR-to-ITR construct 2, SEQ ID NO: 69 and SEQ ID NO: 70). A control vector with a scrambled gRNA sequence that is not capable of RNA-editing of the target RNA was also used (“Scrambled gRNA control”). The AAV vectors were delivered by retro-orbital administration at a dose of about 5E11 or 1E12 vg/mouse in juvenile Mecp2R168X and Mecp2WT mice. A vehicle control was included to benchmark editing. The endpoints assessed were percent RNA editing and protein restoration of Mecp2 in the treated mice, as compared to wild type. To this end, individual brain regions (e.g., frontal brain, brainstem, and midbrain) from mice were examined for editing and/or protein restoration of MECP2. Two to three weeks post-dosing with the AAV vectors or vehicle control, mice were
Attorney Docket No.199235.769601 euthanized and the brains were harvested. The harvested brains were then used to assess percent editing and sagittal brain sections were stained for immunofluorescence (IF) with a C-terminal Mecp2 mAB. Robust MECP2 target adenosine editing was widespread across the different brain regions assessed (FIG.27A). However, minimal differences were observed in editing rates by doses or engineered guide RNAs. Over 50% editing of the target adenosine was observed in the MECP2R168X mice. As shown in FIGs.27B-27E, vg/dg expression was comparable for both engineered guide RNAs and a dose response was observed with both vg/dg expression and gRNA quantification. VG/DG in frontal brain reached over 200 viral genomes, whereas brainstem & midbrain regions have 50-60 VGs (FIGs.27B and 27D). While the highest vg/dg expression levels were observed in the frontal brain region, the opposite was true for gRNA quantification (FIGs.27C and 27E). ITR-to-ITR construct 1 (SEQ ID NO: 69) demonstrated slightly higher gRNA expression as compared to ITR-to-ITR construct 2 (SEQ ID NO: 70). Collectively, these results demonstrated that the frontal brain region achieved over 200 viral genomes, brain stem and midbrain regions reached over 1 copy of gRNA/U1. EXAMPLE 15: In vivo Mecp2 protein restoration using exemplary MECP2-targeting engineered guide RNAs in mice [00351] This example describes in vivo assessment of protein restoration in Mecp2R168X mutant and Mecp2WT mice after treatment with ITR-to-ITR construct 1 and ITR-to-ITR construct 2, SEQ ID NO: 69 and SEQ ID NO: 70, respectively. A control vector with a scrambled gRNA sequence that is not capable of RNA-editing of the target RNA was also used (“Scrambled gRNA control”). Briefly, the AAV vectors were delivered by retro-orbital administration at a dose of either 5E11 vg/mouse or 1E12 vg/mouse in adult Mecp2R168X and Mecp2WT mice. Next, fluorescent detection immunofluorescence (IF) using C-terminal MECP2 mAB and AF-647 secondary nanobody on sagittal tissue sections from mice two to three weeks after treatment. Fluorescent in situ hybridization (FISH) with probe sets specific for the engineered guide RNAs (SEQ ID NO: 56 or SEQ ID NO: 51) encoded by ITR-to-ITR construct 1, and ITR-to-ITR construct 2, respectively. Tissue sections were imaged and tiled on the Zeiss Axio Observer using the same settings for all samples. For each sample for IF, 4 separate fields of view (FOVs) of the same size, within brainstem, midbrain, frontal brain, were analyzed for quantification and the data was combined. For FISH, the entire hemisphere was quantified. Zeiss image analysis software was used to quantify MECP2 or gRNA+ nuclei and MFI of each stain. MECP2 restoration averages 33% across all treatments and brain regions (FIGs.28A-28C). MECP2 signal was the highest in the midbrain with both ITR-to-ITR constructs and doses. Using a
Attorney Docket No.199235.769601 selection of animals (n=2) from each treatment group, MECP2 protein expression was compared by number of positive nuclei in Mecp2R168X and Mecp2WT mice across 3 brain regions (FIGs. 28D-28E). This assay detected an average of 84% MECP2+ nuclei in 2 vehicle Mecp2WT mice assessed. Without normalization to Mecp2WT detection levels, MECP2R168X mice demonstrated the following detection: vehicle and scramble control (SEQ ID NO: 96): <0.5% nuclei, all regions, and an average of 33% nuclei were MECP2+ (FIGs.28D-28E). MECP2R168X mice showed consistency in MECP2 protein detection within a treatment group and brain region. MECP2 protein was restored in 38-41% Mecp2+ nuclei in MECP2R168X midbrain, regardless of dose or gRNA, as compared to 87% Mecp2+ nuclei in Mecp2WT midbrain (FIG.28F). When normalized to the average intensity of MECP2 expression in Mecp2WT mice (vehicle), MECP2R168X animals showed 55-58% of the intensity of MECP2 expression. As shown in FIGs. 28G and 28H, gRNA distribution was broader in high dose condition, but ITR-to-ITR construct (SEQ ID NO: 69) was more highly expressed. Collectively, these results showed that gRNA was expressed across the entire hemisphere, engineered guide RNA distribution increased with increased dose, but it was similar between the Mecp2R168X and Mecp2WT mice. EXAMPLE 16: In vitro screening of MECP2 guide RNAs [00352] This example describes in vitro screening and validation of additional engineered guide RNAs of the current disclosure from the generation of guide RNAs for in-cell validation as described in EXAMPLE 1. The engineered guide RNAs were screened for MECP2 transcript editing of human WT MECP2 transcripts, human MECP2R168X transcripts, mouse WT MECP2 transcripts, or mouse MECP2R168X transcripts. The guide RNAs were screened in a HEK293 cell line engineered to express the following four isoforms of the MECP2 target: the Human WT MECP2 target sequence (encoded by GGTAACTGGGAGAGGGAGCCCCTCCCGGCGAGAGCAGAAACCACCTAAGAAGCCC AAATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCC (SEQ ID NO: 67)), the Human R168X MECP2 target sequence (encoded by GGTAACTGGGAGAGGGAGCCCCTCCCGGTGAGAGCAGAAACCACCTAAGAAGCCCA AATCTCCCAAAGCTCCAGGAACTGGCAGAGGCCGGGGACGCCCC (SEQ ID NO: 68)), the Mouse WT MECP2 target sequence (encoded by GGTAACTGGGAGAGGGAGCCCCTCCAGGAGAGAGCAGAAACCACCTAAGAAGCCC AAATCTCCCAAAGCTCCAGGAACTGGCAGGGGTCGGGGACGCCCC (SEQ ID NO: 967)), and the Mouse R168X MECP2 target sequence (encoded by GGTAACTGGGAGAGGGAGCCCCTCCAGGTGAGAGCAGAAACCACCTAAGAAGCCC
Attorney Docket No.199235.769601 AAATCTCCCAAAGCTCCAGGAACTGGCAGGGGTCGGGGACGCCCC (SEQ ID NO: 46)). Pooled MECP2 cell line expressing all four transcript MECP2 isoforms were also tested. The cells were transfected for 2 days, after which the RNA was extracted and the RNA editing was measured by next generation sequencing after cDNA synthesis and target PCR amplification. [00353] TABLE 8 below depicts the structural features of the exemplary MECP2-targeting guide RNA used in this example when hybridized to the human MECP2 WT and human MECP2 R168X alleles of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 967, and SEQ ID NO: 46. TABLE 8 – MECP2 gRNA Structural Features when Hybridized to MECP2 Human and Mouse MECP2 Alleles Guide Structural Features Structural Features Structural Features Structural Features RNA for Human R168X for Human WT for Mouse R168X for Mouse WT SEQ ID MECP2 Transcri t MECP2 Transcri t MECP2 Transcri t MECP2 Transcri t
Attorney Docket No.199235.769601 Guide Structural Features Structural Features Structural Features Structural Features RNA for Human R168X for Human WT for Mouse R168X for Mouse WT SEQ ID MECP2 Transcript MECP2 Transcript MECP2 Transcript MECP2 Transcript G
Attorney Docket No.199235.769601 Guide Structural Features Structural Features Structural Features Structural Features RNA for Human R168X for Human WT for Mouse R168X for Mouse WT SEQ ID MECP2 Transcript MECP2 Transcript MECP2 Transcript MECP2 Transcript
Attorney Docket No.199235.769601 Guide Structural Features Structural Features Structural Features Structural Features RNA for Human R168X for Human WT for Mouse R168X for Mouse WT SEQ ID MECP2 Transcript MECP2 Transcript MECP2 Transcript MECP2 Transcript
, , , 4.1, Batch 4.2, Batch 4b, Batch 5, Batch 6, Batch 7, and Batch 8 which are detailed further below. For each batch, the editing specificity, target editing, and local specificity target editing were measured. The editing specificity is the sum of editing across all positions in the target divided by the number of positions edited. The target editing is the editing at the target position included in all transcript. The local specificity target editing is the editing on transcripts that only have an edit at the on target adenosine position. For example, an editing profile may have a target editing value of 75% but this is a sum of all sequences for that given gRNA and the gRNA may actually only have 50% of the total sequences having an edit at only the on target adenosine position so that would be a local specificity target editing of 50%. All editing parameters for guide RNAs tested are presented in TABLE 9. TABLE 9 provides the fraction specificity, fraction target editing, and fraction local specificity target editing for each of the batches of guide RNA screens. Batch 1 [00355] In batch 1, 21 guide RNA designs generated by machine learning were assayed in replicate. The highest editing observed was around 25% with observed cross-reactivity across human and mouse R168X targets with limited to no editing observed in the WT targets. Batch 2 [00356] In batch 2, 47 guide RNA designs generated by machine learning were assayed in duplicate. The highest editing observed was around 50% with observed cross-reactivity across human and mouse R168X targets with limited to no editing observed in the WT targets.
Attorney Docket No.199235.769601 Batch 3 [00357] In batch 3, 37 guide RNA designs generated by machine learning were assayed in duplicate. The highest editing observed was around 50% with observed cross-reactivity across human and mouse R168X targets with limited to no editing observed in the WT targets. Batch 4.1 [00358] In batch 4.1, 48 guide RNA designs generated by machine learning were assayed in triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. This experiment also included 16 monoclonal cell population to assess relative isoform abundance. There was similar transcript abundance seen across species and target types. There was also a more uniform distribution of target amplicons observed than the previous batches of guide screening. Batch 4.2 [00359] In batch 4.2, 49 guide RNA designs generated by machine learning were assayed in triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. There was observed cross-reactivity across human and mouse R168X targets. Batch 4b [00360] In batch 4b, 16 guide RNA designs were selected from Batch 4.1 and Batch 4.2 and assayed in triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms and ADAR1. The cells were also co-transfected using a secondary plasmid expressing ADAR2, thus both ADAR1 and ADAR2 were present. There was strong cross- reactivity observed between human and mouse mutant isoforms. Batch 5 [00361] In batch 5, 50 guide RNA designs were assayed by triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. There was cross- reactivity observed between human and mouse mutant isoforms. Batch 6 [00362] In batch 6, 58 guide RNA designs were assayed by triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. There was cross- reactivity observed between human and mouse mutant isoforms. Batch 7 – Second Round Validation [00363] In batch 7, there was a second round validation performed for validating the 15 best MECP2 guide RNA designs by triplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. Each guide RNA and control had three replicates. Similar fraction editing values were observed across samples and their respective
Attorney Docket No.199235.769601 replicates. The highest mutant on-target editing was seen around 45% and strong species cross- reactivity was observed between human and mouse mutant isoforms. SEQ ID NO: 48 achieved greater than 40% editing efficiency across all replicates with less then 15% editing at all off- target positions. Other guide RNAs also consistently showed around 40% editing. Batch 8 [00364] In batch 8, 164 guide RNA designs were assayed by duplicate transient transfection of pooled MECP2 cell line expressing all four transcript MECP2 isoforms. There was cross-reactivity observed between human and mouse mutant isoforms.
1 0 6 9 6 7 Q n . E oit l i a c i t f e g i g r n 8 5 S c c c a i 4 7 1 1 0 8 0 2 6 0 1 4 2 7 0 6 9 3 2 0 7 2 5 0 2 1 1 0 8 4 4 0 2 3 9 0 6 2 1 0 7 2 9 0 3 3 9 0 0 4 9 0 2 3 7 0 1 4 0 3 ( a r o l e p t t y i d e 0 . - E 6 . - E 8 . - E 0 . - E 4 6 . - E 7 8 . - E 3 7 . - E 3 3 . - E 9 2 . - E 2 3 . - E 4 4 . - E 6 7 . - E 1 6 . - E 4 8 . - E 2 2 9 F s t 1 8 3 9 8 3 3 1 1 1 1 2 1 4 9 1. o N t e k c o D y e n r ott A i i l i r i .
P C) 7 n E 6 : o it t e g 4 g n i 1 6 3 1 0 2 1 3 3 0 1 6 4 3 0 5 8 5 2 0 7 0 2 1 0 0 8 4 7 0 1 7 4 6 0 4 5 5 1 0 6 8 3 3 0 8 7 4 9 0 8 4 8 0 7 5 2 0 7 5 4 0 4 4 0 MO c a r r a t t i d e 5 . - 8 7 E . - 2 5 E . - 9 - 2 - 0 - 1 - 1 - 0 - 9 - 3 6 - 5 3 - 1 5 - 9 2 - 8 E . 8 E . 1 E . 1 E . 2 E . 8 E . 6 E . 1 E . 1 E . 5 E . 8 E . 1 E T N F W n a n o i c i 9 6 1 3 3 1 8 2 1 3 9 1 8 1 4 1 2 1 2 1 4 1 6 1 5 1 9 1 2 1 4 1 m it fi u c a c e y t 9 6 0 6 0 5 0 4 0 9 8 0 0 5 0 7 5 0 9 5 0 7 7 0 9 4 0 4 5 0 3 5 0 0 5 0 2 6 0 . - 9 . - 9 . - 9 . - 9 . - 9 . - 9 . - 9 . - 9 . - 9 - 9 - 9 - 9 - 9 - H r F p s 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ct h c h c h c h c h c h c h c h h h h h h h t t t t c c c c c c c t a e B a B a B a B a t B a t a t a t a t a t a t a t a t a t a g B B B B B B B B B B r a T AD I : N O 4 9 0 1 2 3 4 5 6 7 8 9 0 1 RQ E N 3 8 9 9 9 9 9 9 9 9 9 9 0 1 0 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 8 2 5 4 5 4 8 4 2 3 2 4 9 2 8 3 9 2 4 2 9 2 9 4 3 4 9 2 1 3 MO c e a g n r r i a t 6 t i 2 d e 9 0 . - 5 9 6 0- 1 9 2 0- 6 5 2 0- 0 8 2 0- 6 8 5 0- 0 1 0- 5 4 0- 2 9 0- 6 4 0- 3 7 0- 8 7 0- 1 7 0- 7 3 0- 8 5 0- 2 E . 3 E . 4 E . 5 E . 4 E . 7 E 4 . 7 E 1 . 1 E 9 . 2 E 9 . 4 E 1 . 9 8 E . 9 7 E . E 5 . E 8 . E T N F 5 2 5 W n a n o i c i 0 0 0 7 7 1 7 4 1 5 7 1 8 7 1 4 1 1 9 0 1 6 8 1 0 0 1 4 9 0 5 1 0 7 2 1 9 6 1 6 5 1 9 2 1 m it c fi c y t 2 0 5 0 - 5 0 5 0 8 0 3 0 5 0 9 0 8 0 1 0 3 0 6 0 7 0 5 0 3 0 u a H r e F p 0 s . + 9 1 E . 9 9 E . - 9 9 E . - 9 9 E . - 9 9 E . - 4 9 E . - 8 - 2 - 0 + 0 + 8 - 8 - 6 - 9 - 8 E . 9 E . 8 E . 1 E . 1 E . 9 E . 9 E . 9 E . 9 E h 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a B a g B r a T AD I : N O 2 3 4 5 6 7 8 9 0 1 2 RQ 0 0 0 0 0 0 4 0 0 1 1 1 3 1 4 1 5 1 E N 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 8 4 5 3 0 3 9 2 9 1 8 1 0 2 9 3 8 3 0 1 8 3 6 2 9 3 7 2 5 2 MO c e a g n r r i a t 4 t i 4 d e 0 0 . - 6 2 1 0- 6 2 6 0- 4 9 4 0- 0 3 6 0- 4 0 2 0- 1 4 0- 0 3 0- 4 6 0- 8 3 0- 9 9 0- 3 2 0- 9 3 0- 4 5 0- 2 7 0- 4 E . 4 E . 3 E . 4 E . 1 E . 1 E 2 . 5 E 7 . 1 E 5 . 6 E 3 . 1 E 0 . 2 1 E . 6 5 E . E 1 . E 6 . E T N F 4 1 4 W n a n o i c i 2 7 1 9 0 1 4 4 1 4 2 0 6 0 0 2 6 0 7 3 0 2 8 1 0 2 1 8 8 1 5 7 1 9 8 1 4 0 1 6 5 1 7 6 0 m it c fi c y t 2 0 - 0 0 3 0 2 0 7 0 2 0 1 0 8 0 9 0 6 0 2 0 9 0 5 0 1 0 0 0 u a H r e F p 9 s . 9 9 E . - 7 9 E . - 0 9 E . + 0 1 E . + 0 1 E . + 0 1 E . + 6 - 6 - 5 - 9 - 8 - 5 - 7 - 0 + 1 E . 9 E . 8 E . 9 E . 9 E . 9 E . 9 E . 9 E . 1 E h 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a a g B B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 RQ 1 1 1 1 5 2 2 2 2 2 2 7 6 2 7 2 8 2 E N 1 1 1 1 5 1 1 1 1 1 1 4 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 9 3 4 2 5 3 6 3 0 0 7 1 1 3 2 1 9 2 9 3 9 2 9 1 7 2 6 3 5 2 MO c e a g n r r i a t 9 t i 8 d e 0 0 . - 5 5 3 0- 3 6 4 0- 1 9 0 0- 0 0 0 0 4 + 6 7 0- 3 9 0- 8 2 0- 8 6 0- 0 0 0- 8 9 0- 3 2 0- 7 7 0- 6 7 0- 1 2 0- 1 E . 6 E . 6 E . 4 E . 0 E . 1 E 8 . 1 E 1 . 1 E 4 . 8 E 8 . 9 E 2 . 1 E 1 . 2 1 E . E 5 . E 7 . E T N F 2 2 8 W n a n o i c i 0 0 1 1 7 1 9 2 1 2 8 1 4 3 1 6 3 0 3 4 1 1 1 0 8 1 1 6 8 1 3 8 1 5 2 0 8 0 0 6 6 1 2 7 0 m it c fi c y t 3 0 - 9 0 9 0 2 0 0 0 2 0 9 0 4 0 2 0 3 0 4 0 7 0 0 0 6 0 0 0 u a H r e F p 9 s . 7 9 E . - 3 8 E . - 8 9 E . - 6 9 E . - 1 9 E . + 7 1 E . - 0 + 9 - 2 - 4 - 0 + 0 + 2 - 0 + 9 E . 1 E . 9 E . 9 E . 7 E . 1 E . 1 E . 9 E . 1 E h 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a B a g B r a T AD I : N O 9 0 1 2 3 4 5 6 7 8 9 RQ 2 3 3 3 3 7 3 3 3 3 3 3 0 4 1 4 2 4 E N 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 7 3 4 1 1 2 8 2 7 2 8 2 4 1 1 2 8 2 7 3 7 3 8 3 8 4 4 2 4 3 MO c e a g n r r i a t 7 t i 1 d e 5 0 . - 9 8 0 0- 1 8 2 0- 1 2 5 0- 0 7 2 0- 6 8 3 0- 7 5 0- 6 5 0- 9 2 0- 3 5 0- 1 9 0- 0 9 0- 4 2 0- 3 2 0- 3 5 0- 4 E . 1 E . 2 E . 1 E . 8 E . 8 E 2 . 3 E 0 . 8 E 3 . 3 E 5 . 2 E 1 . 5 3 E . 7 3 E . E 6 . E 7 . E T N F 1 4 1 W n a n o i c i 5 1 1 2 5 0 4 4 1 0 6 1 0 5 1 8 7 0 1 0 0 5 2 1 0 2 0 6 9 1 6 1 1 8 5 1 3 9 1 1 2 0 0 6 1 m it c fi c y t 6 0 - 5 0 8 0 5 0 4 0 3 0 0 0 0 0 0 0 4 0 2 0 2 0 9 0 3 0 7 0 u a H r e F p 6 s . 0 9 E . + 7 1 E . - 8 9 E . - 9 9 E . - 0 9 E . + 2 1 E . + 3 - 0 + 3 - 7 - 9 - 8 - 0 + 8 - 1 E . 8 E . 1 E . 9 E . 9 E . 9 E . 9 E . 1 E . 9 E h 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a B a g B r a T AD I : N O 3 4 5 6 7 8 2 9 0 1 2 RQ 4 4 4 4 4 4 0 4 4 5 5 5 3 5 4 5 5 5 E N 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 9 4 5 4 7 2 8 2 0 4 8 3 2 3 7 4 6 3 0 5 5 4 1 4 3 4 7 3 6 4 MO c e a g n r r i a t 2 t i 4 d e 3 0 . - 2 1 1 0- 9 7 1 0- 7 4 5 0- 7 6 7 0- 1 3 3 0- 3 2 0- 1 1 0- 2 2 0- 8 5 0- 9 9 0- 5 3 0- 8 6 0- 8 4 0- 9 1 0- 2 E . 5 E . 5 E . 2 E . 2 E . 2 E 4 . 7 E 7 . 1 E 3 . 1 E 8 . 3 E 9 . 4 E 9 . 0 6 E . 0 6 E . E 1 . E T N F 1 1 W n a n o i i c i 8 0 1 5 1 1 1 0 1 8 4 0 0 0 1 8 8 1 4 6 1 0 0 1 5 8 1 0 0 1 1 7 1 7 3 1 6 0 1 5 4 1 6 1 1 m t c fi c y t 9 0 - 3 0 - 4 0 1 0 4 0 4 0 8 0 2 0 5 0 2 0 7 0 9 0 9 0 2 0 1 0 u a H r e 8 F p s . 9 9 E . 5 9 E . - 0 9 E . + 8 1 E . - 8 9 E . - 4 9 E . - 9 9 E . - 7 - 9 - 8 - 7 - 8 - 9 - 9 - 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 2 9 0 1 2 3 4 5 6 RQ 5 5 5 0 5 6 6 6 6 6 6 6 7 6 8 6 9 6 E N 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 9 4 4 4 6 3 9 3 5 4 8 3 7 4 9 4 6 4 5 4 6 5 3 4 5 4 2 3 4 2 MO c a g n r r i a t 9 t i 7 d e 9 0 . - 4 5 2 0- 5 2 7 0- 9 6 6 0- 9 2 4 0- 2 6 0 0- 0 6 2 0- 6 9 0 0- 9 8 0- 5 3 0- 0 6 0- 5 9 0- 5 0 0- 2 8 0- 6 1 0- 1 E . 5 E . 2 E . 1 E . 1 E . 1 E . 1 E . 2 E 2 . 3 E 0 . 2 E 1 . 9 E 8 . 3 E 3 . 3 E 5 . 2 9 E . 5 E T N F W n a n o i i c t if 2 6 1 2 8 1 3 2 1 0 6 1 1 0 1 4 4 1 1 4 1 2 3 1 0 5 1 3 3 1 1 4 1 7 7 1 8 4 1 4 6 1 5 4 1 m c i c y t 2 0 - 0 0 - 0 0 - 9 0 - 4 0 0 0 4 0 1 0 5 0 3 0 4 0 3 0 1 0 4 0 6 0 u a e 9 . 9 . 9 . 8 . 9 . - 9 . - 9 . - 9 . - 9 . - 9 . - 9 . - 7 - 9 - 3 - 8 - H r F p s 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E 9 E . 9 E . 9 E . 9 E . 8 E h 3 3 3 3 3 3 3 3 3 3 3 3 3 ct t a h c h t c h h h h h h h h h h h h c h e B a t c a t c a t c a t c a t c a t c a t c a t c c c c a t a t a t t t a 1 . 4 c t a 1 . 4 g B B B B B B B B B B B a B a B B B r a T AD I : N O 0 1 2 3 4 5 6 7 8 9 0 1 RQ 7 7 7 7 7 7 7 7 7 7 8 8 2 8 3 8 4 8 E N 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 4 1 2 9 1 2 1 7 3 5 6 3 2 6 2 5 7 2 4 2 3 2 2 3 2 1 3 2 2 2 2 4 2 7 2 9 2 M c g n r r i t 7 0 t d - 7 0 - 9 0 - 4 0 - 5 0 - 7 0 - 2 0- 7 9 0- 8 1 0- 9 9 0- 3 4 0- 9 9 0- 0 0 0- 5 8 0 7 2 0 O a a i e 4 . 3 1 2 0 4 6 3 2 0 5 9 3 0 - 7 - 1 E . 2 E . 1 E . 9 E . 1 E . 1 E . E . E . E . E . E . E . E . E . E T N F 1 5 1 9 1 4 1 1 6 W n a n o i i c t ifi 1 2 1 8 0 5 1 4 0 9 1 5 0 7 1 6 0 7 1 4 0 5 1 0 0 8 1 3 0 9 1 0 3 8 4 5 4 9 0 3 1 0 5 1 0 2 1 0 4 1 0 1 1 0 9 1 0 5 1 m u c a c e y t 0 1 - 0 1 - 6 4 - 8 1 - 7 2 - 8 0 - 6 - 0 - 8 - 9 - 3 - 3 - 9 - 0 - 9 0- H r F p s . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E 4 . 9 E 4 . 9 E 3 . 9 E 3 . 9 E 5 . 9 E 5 . 8 E 4 . 9 E 4 . 9 E 6 . 9 E h c h t a c h t c h c h c h c h h h h h h h h h h a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . c t a 1 . c t a 1 . c t a 1 . c t a 1 . t B B B B B B B B B B B B 4 B 4 B 4 4 4 e g B B r a T AD I : N O 5 6 7 8 9 0 1 2 3 4 RQ 8 8 8 8 8 9 9 9 9 9 5 9 6 9 7 9 8 9 9 9 E N 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 1 3 4 4 4 4 3 4 4 9 5 3 0 3 3 5 7 4 0 2 3 9 3 7 3 6 4 4 3 5 2 1 4 7 4 6 2 M c g n r r i t 2 0 t d - 9 0 - 4 0 - 0 0 - 6 0 - 0 0 - 7 0- 6 8 0- 6 3 0- 7 5 0- 9 6 0- 6 3 0- 2 5 0- 2 9 0 8 1 0 O a a i e 9 . 0 4 5 9 4 4 2 0 9 7 3 0 6 - 2 - 3 E . 3 E . 5 E . 1 E . 2 E . 4 E . E . E . E . E . E . E . E . E . E T N F 3 1 2 5 1 5 2 7 1 W n a n o i i c t ifi 7 6 1 7 0 3 1 3 0 2 1 1 0 7 1 0 0 4 1 1 0 0 1 3 0 8 1 4 0 6 1 9 7 8 9 2 6 8 0 9 1 0 1 1 0 5 1 0 5 1 0 7 1 0 3 1 0 5 1 m u c a c e y t 9 4 - 2 3 - 8 3 - 2 7 - 8 4 - 2 1 - 9 - 7 - 7 - 3 - 7 - 3 - 3 - 5 - 0 0- H r F p s . 9 E . 9 E . 9 E . 8 E . 9 E . 9 E 2 . 9 E 2 . 9 E 0 . 9 E 2 . 9 E 1 . 9 E 5 . 9 E 2 . 9 E 1 . 9 E 4 . 9 E h c h t a c h t c h c h c h c h h h h h h h h h h a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . c t a 1 . c t a 1 . c t a 1 . c t a 1 . t B B B B B B B B B B B B 4 B 4 B 4 4 4 e g B B r a T AD I : N O 0 1 2 3 4 5 6 7 8 9 RQ 0 0 0 0 0 0 0 0 0 0 0 1 1 1 2 1 3 1 4 1 E N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 0 0 4 4 6 2 0 2 4 6 1 4 8 0 3 0 5 4 0 7 3 6 3 6 4 8 2 6 4 7 3 5 3 1 3 3 4 M c g n r r i t 9 0 t d - 4 0 - 0 0 - 4 0 - 1 0 - 6 0 - 5 0- 9 9 0- 5 9 0- 9 7 0- 6 0 0- 4 0 0- 5 0 0- 5 6 0 3 6 0 O a a i e 9 . 6 9 6 1 3 0 9 6 5 7 3 5 0 - 7 - 3 E . 2 E . 7 E . 5 E . 1 E . 9 E . E . E . E . E . E . E . E . E . E T N F 1 1 7 4 6 2 3 1 5 W n a n o i i c t ifi 0 6 1 1 0 7 1 5 0 0 1 4 0 2 1 5 0 1 1 9 0 0 1 9 0 2 1 3 0 3 1 1 4 9 5 2 2 5 0 0 1 0 9 1 0 2 1 0 6 1 0 9 1 0 1 1 0 6 1 m u c a c e y t 1 4 - 1 6 - 8 2 - 2 4 - 5 2 - 5 2 - 0 - 0 - 9 - 3 - 4 - 3 - 6 - 3 - 3 0- H r F p s . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E 1 . 9 E 0 . 9 E 2 . 9 E 7 . 8 E 0 . 9 E 0 . 9 E 0 . 9 E 5 . 8 E 2 . 9 E h c h t a c h t c h c h c h c h h h h h h h h h h a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . 4 c t a 1 . c t a 1 . c t a 1 . c t a 1 . c t a 1 . t B B B B B B B B B B B B 4 B 4 B 4 4 4 e g B B r a T AD I : N O 5 6 7 8 9 0 1 2 3 4 RQ 1 1 1 1 1 2 2 2 2 2 5 2 6 2 7 2 8 2 9 2 E N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 9 8 4 3 7 3 3 9 3 6 0 3 1 4 4 2 1 3 8 3 8 3 6 3 5 2 8 3 4 4 6 3 2 4 1 3 MO c a g n r r i a t t i 5 d e 2 0 . - 8 7 0 - 1 2 0 - 3 9 0 - 9 0 0 - 5 9 0- 9 8 8 0- 1 3 1 0- 4 2 6 0- 3 8 3 0- 2 6 5 0- 4 1 2 0- 5 5 6 0- 9 2 0 0- 3 8 3 0- 5 E . 8 E . 1 E . 3 E . 7 E . E . E . E . E . E . E . E . E . E . E T N F 1 4 7 6 6 1 5 1 8 3 W n a n o i i c t ifi 9 7 1 7 0 4 1 7 0 4 1 5 0 6 1 6 0 2 1 2 0 1 1 1 0 9 1 6 4 6 6 3 9 6 4 0 7 1 0 2 1 0 6 1 0 1 1 0 9 1 0 4 1 0 7 1 0 1 m u c a c e y t 6 3 - 8 3 - 8 3 - 0 8 - 2 5 - 4 - 8 - 3 - 4 - 5 - 7 - 8 - 8 - 6 0 - 8 0- H r F p s . 9 E . 9 E . 9 E . 8 E . 8 E 9 . 8 E 2 . 9 E 8 . 8 E 7 . 8 E 2 . 9 E 2 . 9 E 8 . 8 E 2 . 9 E 3 . 9 9 E . 8 E h c h t a c h t c h c h c h h h h h h h h h h h a 1 . 4 t a 1 . 4 t a 2 . 4 t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . t B B B B B B B B B B B 4 B 4 B 4 4 4 4 e g B B B r a T AD I : N O 0 1 2 3 4 5 6 7 8 RQ 3 4 3 3 3 3 3 3 3 3 9 3 0 4 1 4 2 4 3 4 E N 2 3 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 3 8 3 0 0 4 5 4 4 0 3 4 9 9 4 2 2 4 9 4 4 8 4 5 4 4 4 3 4 3 4 0 4 9 4 9 4 M c g n r r i t 1 0 t d - 4 0 - 8 0 - 6 0 - 6 0 - 3 0 - 8 0- 6 7 0- 6 2 0- 4 8 0- 5 4 0- 6 3 0- 8 1 0- 9 3 0 4 2 0 O a a i e 1 . 1 4 1 0 1 7 4 7 1 0 9 1 9 - 7 - 3 E . 6 E . 8 E . 2 E . 5 E . 1 E . E . E . E . E . E . E . E . E . E T N F 2 8 2 2 2 5 5 1 6 W n a n o i i c t ifi 4 6 1 7 0 9 1 2 0 5 1 9 0 7 1 5 0 8 1 4 0 7 1 7 0 0 1 3 0 3 1 1 7 5 4 2 2 0 0 8 1 0 3 1 0 5 1 0 7 1 0 8 1 0 4 1 0 9 1 m u c a c e y t 3 4 - 4 9 - 5 2 - 2 8 - 7 5 - 9 2 - 4 - 8 - 9 - 1 - 8 - 8 - 4 - 5 - 9 0- H r F p s . 8 E . 8 E . 9 E . 8 E . 8 E . 9 E 4 . 9 E 9 . 8 E 0 . 9 E 2 . 9 E 0 . 9 E 1 . 9 E 3 . 9 E 2 . 9 E 5 . 9 E h c h t a c h t c h c h c h c h h h h h h h h h h a 2 . 4 t a 2 . 4 t a 2 . 4 t a 2 . 4 t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . t B B B B B B B B B B B B 4 B 4 B 4 4 4 e g B B r a T AD I : N O 4 5 6 7 8 9 0 1 2 3 RQ 4 4 4 4 4 4 5 5 5 5 4 5 5 5 6 5 7 5 8 5 E N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 7 1 4 4 1 4 9 3 4 0 0 4 0 0 0 9 2 4 3 2 4 6 3 7 3 5 4 4 4 1 4 3 4 3 4 7 4 M c g n r r i t 3 0 t d - 4 0 - 6 0 - 0 0 - 0 0 0 0 - 0 0- 3 2 0- 8 5 0- 6 0 0- 2 9 0- 8 1 0- 5 8 0- 6 0 0- 7 1 0 O a a i e 8 . 5 6 1 0 + 5 8 2 2 0 5 5 6 8 4 - 3 E . 9 E . 4 E . 1 E . 0 E . 3 E . E . E . E . E . E . E . E . E . E T N F 3 2 1 3 1 1 6 5 6 W n a n o i i c t ifi 8 9 1 0 0 2 1 0 0 8 1 8 0 9 1 4 0 6 1 8 0 4 1 1 0 1 1 1 0 7 1 5 8 5 0 1 2 4 0 5 1 0 1 1 0 4 1 0 0 1 0 3 1 0 2 1 0 7 1 m u c a c e y t 0 6 - 3 4 - 8 4 - 1 4 - 4 6 - 2 3 - 6 - 0 - 5 - 6 - 2 - 9 - 2 - 0 - 1 0- H r F p s . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E 3 . 9 E 7 . 8 E 3 . 9 E 3 . 9 E 3 . 9 E 5 . 9 E 4 . 9 E 3 . 9 E 4 . 9 E h c h t a c h t c h c h c h c h h h h h h h h h h a 2 . 4 t a 2 . 4 t a 2 . 4 t a 2 . 4 t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . t B B B B B B B B B B B B 4 B 4 B 4 4 4 e g B B r a T AD I : N O 9 0 1 2 3 4 5 6 7 8 RQ 5 6 6 6 6 6 6 6 6 6 9 6 0 7 1 7 2 7 3 7 E N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6 c 1 D R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 6 2 4 9 7 4 5 5 4 2 8 4 7 2 4 5 4 2 3 2 4 6 2 0 1 5 2 5 2 2 2 9 2 9 2 MO c a g n r r i a t t i 9 d e 6 0 . - 9 1 0 - 6 5 0 - 6 4 0 - 6 5 0- 7 0 0 0- 4 1 4 0- 8 5 9 0- 7 1 0 0- 1 2 6 0- 1 0 0 0- 8 3 6 0- 5 4 3 0- 9 3 9 0- 2 3 6 0- 8 E . 6 E . 5 E . E . E . E . E . E . E . E . E . E . E . E . E T N F 2 4 5 5 1 9 1 7 2 2 2 1 W n a n o i i c t ifi 2 0 1 6 0 6 1 6 0 2 1 8 0 6 1 9 0 1 1 2 4 0 7 9 8 4 1 4 6 0 5 1 0 8 1 0 0 1 0 8 1 0 5 1 0 2 1 0 8 1 9 1 9 1 0 1 m u c a c e y t 2 1 - 9 0 - 5 - 5 - 7 - 6 - 6 - 4 - 4 - 6 - 0 - 1 0 - 2 0 - 3 0 - 9 0- H r F p s . 9 E . 9 E 1 . 9 E 4 . 9 E 2 . 9 E 4 . 9 E 3 . 9 E 6 . 9 E 1 . 8 E 3 . 7 E 3 . 4 7 E . 3 7 E . 5 7 E . 7 7 E . 8 E h c h h h h h h h h h h h h h h h t t a c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . 4 c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a 2 . c t a b 4 c t a b 4 c t a b 4 c t a b 4 c t a b 4 e B g B B B B B 4 B 4 B 4 B 4 B 4 B 4 B B B B B r a T AD I : N O 4 5 6 7 8 9 0 1 2 RQ 7 7 7 7 7 7 8 8 8 4 4 8 5 8 6 8 0 9 1 9 E N 2 2 2 2 2 2 2 2 2 3 1 1 1 1 1 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6D c 1 R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 5 5 2 3 3 2 0 0 2 7 1 2 2 3 2 7 2 0 2 1 2 5 2 3 1 6 2 0 2 5 2 0 2 MO c a g n r r i a t t i 3 d 1 0 . - E 9 7 0 . - E 9 9 0 . - 3 E 2 0 0 . - 1 E 6 7 0 . - 3 E 9 5 0 . - 9 E 0 4 0 . - 9 E 0 3 0 . - 3 E 5 7 0 . - 1 E 5 3 0 . - 4 E 3 1 0 . - 3 E 1 0 0 . - 8 E 1 7 0 . - 3 E 9 7 0 . - 6 E 9 6 0 . - E T N F e 7 3 6 1 3 1 8 5 1 3 1 1 2 7 6 W n a n o i i c t ifi 9 5 1 2 6 7 1 5 9 9 4 8 1 9 4 4 0 0 1 1 0 6 1 0 7 1 0 4 1 0 1 1 8 1 5 1 9 1 4 1 0 1 5 1 5 1 0 1 6 1 m c c y t 3 - 3 - 6 - 9 - 4 - 0 0 - 4 0 - 4 0 - 2 0 - 3 0 - 5 0 - 9 0 6 0 9 0 6 0 u a e 6 . E 6 . E 8 . E 4 . 5 . 4 . 0 . 0 . 9 . 6 . 2 . 8 . - 2 . - 2 . - 0 . - H r F p s 7 7 6 7 E 7 E 8 E 7 E 8 E 7 E 7 E 6 E 7 E 7 E 7 E 7 E h c h h h h h h h h h t t a c t B a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b 4 e g B r a T AD I : N O 2 5 6 9 5 9 1 6 8 2 4 RQ 9 9 9 9 0 0 1 1 1 2 2 1 3 2 3 6 3 7 3 E N 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6D c 1 R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 2 6 2 3 7 2 8 0 2 2 2 8 2 1 2 4 3 4 2 4 2 3 2 8 2 3 2 0 2 2 2 4 2 MO c a g n r r i a t t i 4 d 9 0 . - E 7 9 0 . - E 9 9 0 . - 2 E 3 7 0 . - 4 E 5 7 0 . - 3 E 9 0 0 . - 8 E 2 1 0 . - 1 E 5 7 0 . - 2 E 8 7 0 . - 5 E 6 2 0 . - 2 E 1 4 0 . - 9 E 7 8 0 . - 4 E 3 4 0 . - 7 E 5 0 0 . - 7 E 5 5 0 . - E T N F e 6 2 3 3 3 3 9 2 1 4 4 1 5 2 3 W n a n o i i c t ifi 9 1 1 2 1 6 1 9 9 0 3 3 9 4 4 8 1 0 8 1 0 3 1 0 2 1 0 4 1 0 6 1 3 1 6 1 1 1 5 1 6 1 0 1 4 1 7 1 3 1 m c c y t 3 - 6 - 4 - 4 - 0 - 7 0 - 8 0 - 8 0 - 7 0 - 9 0 - 7 0 - 0 0 5 0 0 0 0 0 u a e 2 . E 6 . E 8 . E 1 . 5 . 5 . 6 . 2 . 8 . 2 . 2 . 0 . - 4 . - 4 . - 6 . - H r F p s 7 7 6 8 E 8 E 7 E 7 E 8 E 8 E 7 E 7 E 8 E 7 E 7 E 7 E h c h h h h h h h h h t t a c t B a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b h B 4 c t a b 4 e g B r a T AD I : N O 2 3 4 5 6 7 8 9 0 1 2 RQ 4 4 4 4 4 4 4 4 5 5 5 3 5 4 5 5 5 6 5 E N 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P C) 1 6 . E 4 o M : N t XO N e 8 k 6D c 1 R I o D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t e g 8 6 2 8 5 1 3 1 4 1 9 2 5 1 4 1 2 1 4 1 6 1 8 1 9 1 0 2 8 3 6 2 MO c a g n r i a ti 0 d 5 0 . - E 6 1 0 . - 7 E 1 4 0 . - 9 E 1 4 0 . - 3 E 8 7 0 . - 8 E 1 6 0 . - 3 E 6 2 0 . - 5 E 0 3 0 . - 6 E 5 3 0 . - 3 E 6 2 0 . - 3 E 3 6 0 . - 1 E 6 4 0 . - 8 E 0 0 0 . - 3 E 4 4 0 . - 9 E 8 2 0 . - E T N r F t e 2 1 1 1 7 1 1 1 1 1 1 1 2 3 1 W n a n o i i c t ifi 8 5 1 9 7 4 5 9 9 4 4 1 8 9 1 2 9 0 7 1 0 9 1 0 9 1 6 1 4 1 3 1 6 1 8 1 1 1 3 1 6 1 2 1 9 1 2 1 m c c y t 0 - 3 - 4 - 6 0 - 7 0 - 9 0 - 2 0 - 3 0 - 2 0 5 0 3 0 2 0 3 0 0 0 3 0 u a e 9 . 3 . 5 . 7 . 5 . 7 . 9 . 6 . 2 . - 5 . - 1 . - 3 . - 1 . - 7 . - 4 . - H r F p s 7 E 9 E 9 E 9 E 8 E 8 E 8 E 9 E 9 E 9 E 8 E 8 E 9 E 9 E 9 E h c h h h 5 5 5 t t a c t a b 4 c t a b 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t a b h 4 c t b h h h 4 c t c t c t e B B B B B B B B B B B B a B a B a a g B B r a T AD I : N O 7 8 9 0 1 6 7 8 0 1 RQ 5 5 5 6 6 6 6 6 8 8 2 8 4 3 8 4 8 5 8 E N 2 2 2 2 2 2 2 2 2 2 2 3 2 2 2 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 6 2 6 2 7 2 5 3 0 2 4 2 3 2 1 2 5 3 9 3 7 2 2 3 4 2 0 2 3 3 MO c e a g n r r i a t 2 t i 3 d e 2 0 . - 0 6 3 0- 1 0 1 0- 3 5 7 0- 3 5 0 0- 5 7 0 0- 0 9 0- 7 2 0- 2 9 0- 3 7 0- 2 0 0- 5 5 0- 4 7 0- 8 1 0- 4 0 0- 1 E . 3 E . 2 E . 3 E . 1 E . 1 E 2 . 2 E 3 . 1 E 0 . 2 E 3 . 1 E 3 . 2 1 E . 5 3 E . E 0 . E 3 . E T N F 1 1 4 W n a n o i c i 1 1 1 6 8 1 3 4 1 9 3 1 1 9 1 8 2 1 8 4 1 1 6 1 7 5 1 0 4 1 7 5 1 8 8 1 6 2 1 4 7 1 8 3 1 m it c fi c y t 4 0 - 0 0 7 0 1 0 5 0 5 0 3 0 6 0 3 0 8 0 5 0 1 0 2 0 0 0 5 0 u a H r e F p 0 s . 7 9 E . - 6 9 E . - 6 9 E . - 4 9 E . - 1 9 E . - 5 9 E . - 3 - 5 - 5 - 5 - 7 - 5 - 7 - 5 - 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a B a g B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 RQ 8 8 8 8 8 9 9 9 9 9 9 9 7 9 8 9 9 9 E N 2 5 2 2 2 2 2 2 2 2 2 2 2 2 2 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 1 3 1 3 4 3 8 2 6 3 4 3 3 2 1 2 0 2 6 3 7 3 2 2 4 3 1 3 4 3 MO c e a g n r r i a t 1 t i 3 d e 2 0 . - 9 3 2 0- 9 3 7 0- 5 0 4 0- 0 9 3 0- 8 3 8 0- 5 9 0- 6 4 0- 5 7 0- 8 9 0- 6 5 0- 6 3 0- 7 7 0- 5 2 0- 0 3 0- 6 E . 5 E . 3 E . 1 E . 3 E . 7 E 0 . 2 E 1 . 1 E 7 . 1 E 1 . 4 E 2 . 4 5 E . 2 1 E . E 5 . E 7 . E T N F 6 6 8 W n a n o i c i 6 6 1 8 7 1 2 8 1 1 5 1 3 9 1 5 6 1 3 8 1 2 7 1 4 9 1 7 1 1 2 6 1 6 8 1 6 8 1 8 4 1 7 2 1 m it c fi c y t 3 0 - 1 0 1 0 4 0 4 0 4 0 0 0 3 0 4 0 3 0 3 0 3 0 9 0 0 0 7 0 u a H r e F p 6 s . 7 9 E . - 7 9 E . - 3 9 E . - 3 9 E . - 4 9 E . - 7 9 E . - 6 - 1 - 3 - 7 - 6 - 0 - 3 - 6 - 9 E . 9 E . 8 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a a g B B r a T AD I : N O 0 1 2 3 4 5 6 7 8 9 RQ 0 0 0 0 0 1 9 0 0 0 0 0 0 1 1 1 2 1 E N 3 3 3 3 3 5 5 3 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 3 3 6 3 6 3 6 3 4 3 8 3 9 2 1 4 3 2 5 3 4 2 2 2 6 2 0 3 3 3 MO c e a g n r r i a t 1 t i 2 d e 1 0 . - 9 0 1 0- 9 8 2 0- 0 6 1 0- 8 3 9 0- 3 1 0 0- 7 1 0- 0 2 0- 7 3 0- 9 3 0- 4 4 0- 2 7 0- 4 9 0- 1 7 0- 7 9 0- 5 E . 5 E . 1 E . 2 E . 3 E . 2 E 0 . 1 E 7 . 9 E 4 . 1 E 2 . 1 E 5 . 1 E 1 . 5 1 E . 6 1 E . E 6 . E T N F 2 3 W n a n o i i c i 2 7 1 4 7 1 4 9 1 4 1 1 3 6 1 9 1 1 2 7 1 1 4 1 8 9 1 4 2 1 5 1 1 6 1 1 5 5 1 8 9 1 8 0 1 m t c fi c y t 5 0 - 3 0 - 1 0 4 0 5 0 0 0 1 0 0 0 7 0 7 0 2 0 9 0 0 0 3 0 7 0 u a H r e 7 F p s . 5 9 E . 2 9 E . - 7 9 E . - 6 9 E . - 5 9 E . - 8 9 E . - 7 9 E . - 6 - 7 - 8 - 7 - 6 - 6 - 7 - 9 E . 9 E . 9 E . 8 E . 9 E . 9 E . 9 E . 9 E h 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 3 4 5 6 7 8 9 0 1 2 3 4 RQ 1 1 1 1 1 1 1 2 2 2 2 2 5 2 6 2 7 2 E N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 c 1 D I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n o t g 9 2 3 1 7 2 6 2 8 2 9 4 7 1 1 2 6 2 9 2 0 2 1 2 2 1 1 1 4 E 6 : it MO c e a g n r r i a t 7 t i 2 d e 2 0 . - 7 9 0 0- 1 1 4 0- 5 3 6 0- 1 8 0 0- 5 3 0- 1 7 0- 1 0 0- 1 2 0- 7 9 0- 2 5 0- 8 1 0- 4 5 0- 2 1 0- 8 1 1 0- 2 E . 1 E . 7 E . 6 E . 1 E 8 . 8 E 4 . 1 E 3 . 8 E 8 . 4 E 4 . 1 6 E . 2 3 E . E 5 . E 4 . E 0 . E T N F 3 2 2 2 W n a n o i c 9 8 1 4 1 9 5 1 m it i u c fi a H r c e y 6 t 4 1 0 4 2 1 0 3 4 1 0 3 8 1 0 2 7 1 0 6 2 1 0 7 1 1 0 5 0 1 4 0 8 6 1 7 0 3 1 1 5 0 0 6 1 3 0 7 0 1 1 0 8 7 0 0 0 2 3 0 0 8 5 0 0 0 F p 1 s . - 7 9 E . - 5 9 E . - 3 8 E . - 5 8 E . - 5 - 4 - 5 - 2 - 8 - 4 - 5 - 0 + 1 + 0 + 8 E . 9 E . 8 E . 9 E . 9 E . 9 E . 9 E . 9 E . 1 E . 1 E . 1 E h 5 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ct t a h c h h h h h h h h h h h h h h t c e B a t c B a t c B a t c B a t c B a t c c c c c c c c c B a t B a t B a t B a t B a t t t t t B a B a a a a g B B B B r a T AD I : N O 4 8 1 2 3 7 5 9 2 7 RQ 9 9 9 9 1 9 0 0 1 2 8 2 9 2 0 3 E N 3 5 2 2 2 2 5 5 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 1 1 7 1 9 1 6 1 9 1 1 1 6 1 6 1 5 1 9 1 9 1 0 1 9 1 5 1 6 2 MO c e a g n r r i a t 4 t i 8 d e 4 0 . - 5 2 0 0- 1 5 2 0- 8 8 7 0- 8 6 6 0- 2 2 4 0- 7 6 0- 6 6 0- 6 5 0- 7 3 0- 4 4 0- 8 9 0- 4 2 0- 3 8 0- 1 7 0- 1 E . 3 E . 1 E . 1 E . 2 E . 1 E 9 . 2 E 9 . 1 E 2 . 2 E 1 . 1 E 4 . 2 E 0 . 1 1 E . 3 1 E . E 5 . E T N F 1 9 W n a n o i i c i 4 5 1 1 5 0 8 8 1 2 9 0 0 2 1 3 6 0 5 5 0 2 0 1 0 4 0 0 8 0 4 5 0 3 7 0 5 6 1 0 5 0 4 4 0 m t c fi c y t 7 0 - 5 0 6 0 - 7 0 8 0 6 0 1 0 2 0 3 0 4 0 2 0 2 0 7 0 5 0 3 0 u a H r e 7 F p s . 0 9 E . + 8 1 E . 0 9 E . + 1 1 E . - 0 9 E . + 0 1 E . + 0 1 E . - 0 + 0 + 0 + 0 + 7 - 0 + 0 + 9 E . 1 E . 1 E . 1 E . 1 E . 9 E . 1 E . 1 E h 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 1 2 3 4 5 6 7 8 9 0 1 2 RQ 3 3 3 3 3 3 3 3 3 4 4 4 3 4 4 4 5 4 E N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 6 1 2 1 0 1 7 2 9 2 8 2 3 2 0 2 3 2 9 2 4 2 2 2 9 2 0 2 9 2 MO c e a g n r r i a t 7 t i 0 d e 5 0 . - 2 9 7 0- 3 0 2 0- 2 5 3 0- 7 9 3 0- 7 8 1 0- 7 9 0- 7 8 0- 9 2 0- 7 9 0- 5 9 0- 1 5 0- 0 4 0- 4 6 0- 6 1 0- 2 E . 1 E . 2 E . 5 E . 5 E . 3 E 7 . 3 E 4 . 4 E 6 . 3 E 3 . 6 E 0 . 9 E 9 . 8 4 E . 6 4 E . E 5 . E T N F 9 1 W n a n o i i c i 3 6 1 0 6 0 8 8 0 9 8 1 0 6 1 5 0 1 8 7 1 3 2 1 7 0 1 4 5 1 1 5 1 1 6 1 8 8 1 2 2 1 9 1 1 m t c fi c y t 9 0 - 9 0 5 0 9 0 - 8 0 2 0 5 0 5 0 5 0 1 0 2 0 6 0 3 0 4 0 5 0 u a H r e 3 F p s . 0 9 E . + 0 1 E . + 9 1 E . 1 7 E . - 8 8 E . - 7 8 E . - 0 8 E . - 5 - 4 - 9 - 7 - 8 - 1 - 4 - 8 E . 8 E . 8 E . 8 E . 8 E . 7 E . 9 E . 8 E h 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 4 4 4 4 5 5 5 5 5 5 5 5 8 5 9 5 0 6 E N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 7 3 7 2 6 2 0 2 0 3 5 2 0 2 2 2 3 2 0 1 4 2 5 1 0 2 5 1 8 1 MO c e a g n r r i a t 8 t i 7 d e 2 0 . - 0 1 9 0- 1 2 6 0- 1 8 3 0- 6 3 2 0- 3 7 4 0- 3 1 0- 2 3 0- 6 8 0- 4 8 0- 5 6 0- 4 1 0- 2 3 0- 2 0 0- 5 8 0- 2 E . 3 E . 3 E . 2 E . 5 E . 2 E 2 . 3 E 1 . 4 E 9 . 9 E 1 . 1 E 3 . 3 8 E . 6 1 E . E 3 . E 2 . E T N F 4 1 1 W n a n o i c i 2 9 1 7 0 1 3 2 1 8 2 1 4 3 1 4 4 1 9 2 1 6 9 1 6 0 1 7 7 1 2 9 1 0 2 1 9 9 1 0 4 1 6 3 1 m it c fi c y t 1 0 - 8 0 2 0 1 0 3 0 6 0 9 0 1 0 9 0 9 0 9 0 3 0 1 0 7 0 1 0 u a H r e F p 0 s . 8 9 E . - 6 8 E . - 4 8 E . - 9 8 E . - 2 8 E . - 0 9 E . - 9 - 6 - 5 - 9 - 1 - 3 - 6 - 6 - 9 E . 7 E . 8 E . 8 E . 7 E . 9 E . 9 E . 9 E . 7 E h 6 6 6 6 6 6 6 6 6 6 6 6 6 6 6 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a a g B B r a T AD I : N O 1 6 2 6 3 6 4 6 5 6 6 6 7 6 8 6 9 6 0 7 1 7 0 2 7 3 7 4 RQ E N 3 3 3 3 3 3 3 3 3 3 3 6 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 c 1 D I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n o t g 2 1 3 1 4 1 4 1 2 1 9 1 3 1 1 1 9 1 7 1 5 1 0 3 6 1 3 1 5 E 6 : it MO c e a g n r r i a t 6 t i 2 d e 6 0 . - 3 8 2 0- 8 7 1 0- 1 8 4 0- 2 5 5 0- 5 5 0- 7 2 0- 6 8 0- 6 7 0- 3 4 0- 8 7 0- 3 5 0- 0 8 0- 9 5 0- 6 1 1 0- 1 E . 1 E . 1 E . 1 E . 2 E 3 . 1 E 0 . 1 E 7 . 1 E 2 . 1 E 1 . 9 1 E . 6 2 E . E 7 . E 3 . E 9 . E T N F 1 2 2 1 W n a n o i c 5 2 8 1 5 7 2 0 m it i u c fi a H r c e y 3 t 9 1 0 1 1 0 0 4 5 0 0 9 0 0 0 7 9 0 0 2 3 0 0 6 2 0 0 7 9 1 3 0 1 6 1 5 0 9 0 0 0 7 1 1 0 0 1 1 3 1 1 0 4 6 0 0 2 9 8 1 9 0 5 2 0 0 F p 3 s . - 0 7 E . + 0 1 E . + 0 1 E . + 0 1 E . + 0 + 0 + 2 - 6 - 0 + 2 + 6 - 0 + 5 - 1 + 1 E . 1 E . 1 E . 9 E . 9 E . 1 E . 1 E . 9 E . 1 E . 9 E . 1 E h 7 7 7 7 7 7 7 7 7 7 7 7 7 7 7 ct t a h c h h h h h h h h h h h h h h t c e B a t c B a t c B a t c B a t c B a t c c c c c c c c c B a t B a t B a t B a t B a t t t t t B a B a a a a g B B B B r a T AD I : N O 4 9 0 1 0 1 4 8 4 RQ 4 1 1 5 2 2 2 7 2 7 3 5 3 7 4 2 0 E N 3 1 1 1 5 1 1 1 4 1 5 1 1 1 1 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 0 2 5 2 1 2 8 2 7 1 8 1 7 1 0 1 7 1 4 1 8 1 9 2 4 1 2 1 3 1 MO c e a g n r r i a t 7 t i 8 d e 9 0 . - 0 7 7 0- 7 9 4 0- 2 0 0 0- 4 0 0 0- 1 3 7 0- 5 2 0- 5 3 0- 6 5 0- 8 2 0- 4 9 0- 0 3 0- 2 6 0- 3 3 0- 3 3 0- 7 E . 1 E . 1 E . 8 E . 2 E . 1 E 6 . 1 E 4 . 1 E 3 . 1 E 5 . 2 E 9 . 3 1 E . 8 9 E . E 3 . E 6 . E T N F 1 2 2 W n a n o i c i 4 3 1 3 4 1 0 4 1 7 6 0 3 4 0 0 6 0 4 3 0 4 3 0 0 8 0 0 6 0 8 6 0 8 9 0 2 6 0 8 0 0 0 9 0 m it c fi c y t 9 0 - 5 0 9 0 3 0 4 0 3 0 5 0 5 0 3 0 7 0 0 0 0 0 0 0 6 0 5 0 u a H r e F p 9 s . 6 9 E . - 8 9 E . - 0 9 E . + 1 1 E . + 0 1 E . + 0 1 E . + 0 + 0 + 1 + 1 + 0 + 1 + 1 + 1 + 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h h h h h h h h h h h h e B a t B a t c B a t c B a t c B a t c B a t c B a t c c c c c c c B a t B a t B a t B a t B a t t t B a B a B a g B r a T AD I : N O 8 5 9 4 5 6 7 8 9 0 1 2 RQ 0 0 7 7 7 7 7 7 8 8 8 3 8 4 8 5 8 E N 5 3 3 3 3 3 3 3 3 3 3 3 3 3 3 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 3 1 0 1 3 1 0 1 3 1 0 1 3 1 8 1 7 1 3 1 4 1 0 1 4 1 8 1 2 1 MO c e a g n r r i a t 3 t i 0 d e 5 0 . - 7 9 5 0- 2 5 3 0- 5 4 9 0- 9 0 1 0- 7 6 4 0- 5 0 0- 2 8 0- 2 3 0- 0 9 0- 9 9 0- 3 0 0- 6 8 0- 4 2 0- 0 4 0- 2 E . 1 E . 2 E . 1 E . 3 E . 1 E 8 . 2 E 9 . 1 E 8 . 1 E 3 . 3 E 5 . 2 E 7 . 6 2 E . 8 1 E . E 1 . E T N F 2 3 W n a n o i i c i 9 2 0 3 5 1 2 9 0 1 2 0 0 4 0 9 0 1 0 5 0 6 3 0 2 0 0 0 5 0 8 2 0 9 3 0 9 3 0 8 5 0 1 3 0 m t c fi c y t 5 0 5 0 - 4 0 1 0 0 0 5 0 - 7 0 8 0 0 0 1 0 9 0 9 0 1 0 9 0 6 0 u a H r e 0 F p s . + 8 1 E . 1 9 E . + 1 1 E . + 0 1 E . + 9 1 E . 1 9 E . + 0 1 E . + 0 + 2 + 0 + 0 + 0 + 0 + 0 + 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 8 8 8 8 9 9 9 9 9 9 9 9 8 9 9 9 0 0 E N 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 6 1 9 1 3 1 3 1 8 1 3 1 5 1 1 1 5 1 6 1 9 1 7 1 2 1 5 1 1 1 MO c e a g n r r i a t 6 t i 6 d e 6 0 . - 7 1 2 0- 6 5 5 0- 4 1 3 0- 8 0 4 0- 3 3 1 0- 8 6 0- 5 9 0- 6 9 0- 9 9 0- 2 4 0- 0 2 0- 4 7 0- 2 4 0- 7 8 0- 1 E . 2 E . 3 E . 3 E . 3 E . 2 E 3 . 2 E 8 . 2 E 3 . 1 E 5 . 3 E 9 . 1 E 7 . 9 2 E . 7 2 E . E 5 . E T N F 2 3 W n a n o i i c i 8 5 0 7 4 1 5 2 0 0 4 0 3 2 0 7 1 0 2 7 0 6 4 0 5 6 0 1 0 0 1 2 0 7 3 0 6 7 0 5 0 0 2 9 0 m t c fi c y t 8 0 + 8 0 - 7 0 3 0 2 0 2 0 6 0 3 0 5 0 3 0 6 0 4 0 2 0 9 0 2 0 u a H r e 0 F p s . 4 1 E . 2 9 E . + 2 1 E . + 1 1 E . + 0 1 E . + 0 1 E . + 0 1 E . + 0 + 2 + 0 + 1 + 0 + 1 + 1 + 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 1 2 3 4 5 6 7 8 9 0 1 2 RQ 0 0 0 0 0 0 0 0 0 1 1 1 3 1 4 1 5 1 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 8 1 3 1 0 1 1 1 2 1 0 1 1 1 0 2 0 1 0 1 5 1 0 1 2 1 7 1 6 1 MO c e a g n r r i a t 3 t i 6 d e 4 0 . - 7 2 2 0- 9 4 2 0- 1 4 0 0- 9 3 3 0- 6 7 1 0- 1 8 0- 9 4 0- 2 0 0- 4 8 0- 1 7 0- 0 6 0- 4 5 0- 2 8 0- 0 5 0- 3 E . 2 E . 2 E . 1 E . 2 E . 3 E 0 . 3 E 8 . 7 E 9 . 1 E 4 . 3 E 6 . 2 E 3 . 7 1 E . 1 3 E . E 9 . E T N F 3 1 W n a n o i i c i 2 6 0 5 0 1 6 7 1 0 0 1 7 6 0 1 9 0 5 8 0 0 3 1 0 3 0 0 3 0 2 9 0 4 3 0 3 2 0 1 7 0 3 4 1 m t c fi c y t 3 0 4 0 - 8 0 - 3 0 7 0 3 0 9 0 3 0 7 0 1 0 4 0 1 0 2 0 3 0 3 0 u a H r e 0 F p s . + 2 1 E . 7 9 E . 6 8 E . - 0 8 E . + 0 1 E . + 1 1 E . + 6 1 E . - 0 + 0 + 0 + 0 + 2 + 1 + 2 - 9 E . 1 E . 1 E . 1 E . 1 E . 1 E . 1 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 1 1 1 1 2 2 2 2 2 2 2 2 8 2 9 2 0 3 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 8 1 3 1 6 1 6 1 5 1 9 1 2 1 2 1 6 1 0 1 9 1 3 1 3 1 1 1 1 1 MO c e a g n r r i a t 2 t i 0 d e 5 0 . - 2 5 2 0- 0 6 1 0- 0 3 4 0- 3 7 6 0- 4 3 5 0- 7 3 0- 8 4 0- 7 9 0- 2 6 0- 9 7 0- 1 9 0- 8 3 0- 7 5 0- 1 4 0- 3 E . 3 E . 2 E . 2 E . 2 E . 3 E 5 . 3 E 4 . 2 E 3 . 3 E 0 . 2 E 1 . 3 E 2 . 1 1 E . 0 3 E . E 0 . E T N F 2 2 W n a n o i i c i 9 0 0 0 1 0 4 7 0 0 0 0 6 3 0 4 3 0 3 1 0 0 0 0 9 8 0 8 7 0 9 0 0 8 2 1 9 2 1 5 7 0 8 6 0 m t c fi c y t 3 0 7 0 3 0 7 0 9 0 2 0 6 0 7 0 8 0 2 0 4 0 7 0 - 2 0 - 5 0 1 0 u a H r e 1 F p s . + 1 1 E . + 1 1 E . + 0 1 E . + 1 1 E . + 1 1 E . + 1 1 E . + 1 1 E . + 0 + 1 + 1 + 4 5 0 + 1 + 1 E . 1 E . 1 E . 1 E . 9 E . 9 E . 1 E . 1 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 1 2 3 4 5 6 7 8 9 0 1 2 RQ 3 3 3 3 3 3 3 3 3 4 4 4 3 4 4 4 5 4 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 6 1 7 1 3 1 2 2 1 2 4 2 9 3 4 2 4 2 1 2 1 2 9 3 1 1 1 2 1 2 MO c e a g n r r i a t 5 t i 3 d e 0 0 . - 1 7 2 0- 3 7 4 0- 9 2 2 0- 3 9 4 0- 1 8 2 0- 9 9 0- 5 1 0- 0 2 0- 0 6 0- 1 8 0- 3 6 0- 2 3 0- 8 0 0- 5 0 0- 2 E . 1 E . 1 E . 1 E . 9 E . 7 E 3 . 2 E 7 . 2 E 1 . 1 E 5 . 4 E 0 . 7 E 7 . 3 8 E . 1 1 E . E 4 . E T N F 5 2 W n a n o i i c i 8 1 0 4 7 0 9 6 0 0 6 1 6 6 1 4 2 1 3 1 1 6 2 1 7 9 1 3 5 1 5 6 1 6 5 1 5 6 0 3 9 1 9 2 1 m t c fi c y t 4 0 7 0 4 0 1 0 - 0 0 - 9 0 7 0 7 0 3 0 8 0 1 0 3 0 6 0 0 0 9 0 u a H r e 1 F p s . + 0 1 E . + 0 1 E . + 4 1 E . 6 9 E . 2 9 E . - 5 9 E . - 6 9 E . - 2 - 5 - 8 - 4 - 0 + 1 - 7 - 9 E . 9 E . 9 E . 9 E . 9 E . 1 E . 9 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 4 4 4 4 5 5 5 5 5 5 5 5 8 5 9 5 0 6 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 0 1 5 1 8 1 9 1 9 1 1 2 6 2 1 2 7 3 9 2 4 2 7 2 2 1 7 2 5 2 MO c e a g n r r i a t 0 t i 6 d e 6 0 . - 8 8 6 0- 0 7 8 0- 0 2 4 0- 0 8 1 0- 2 0 3 0- 8 8 0- 6 8 0- 1 4 0- 4 9 0- 9 1 0- 2 6 0- 4 4 0- 3 8 0- 9 5 0- 1 E . 1 E . 1 E . 1 E . 1 E . 5 E 0 . 4 E 4 . 9 E 9 . 4 E 9 . 2 E 2 . 6 E 9 . 4 5 E . 4 1 E . E 5 . E T N F 5 4 W n a n o i i c i 6 7 1 8 7 1 1 0 0 7 8 1 3 6 1 5 5 1 6 2 1 5 5 1 4 8 1 4 8 1 0 8 1 9 3 1 3 6 1 7 0 1 6 0 1 m t c fi c y t 1 0 - 8 0 - 2 0 0 0 2 0 6 0 5 0 1 0 8 0 5 0 6 0 0 0 6 0 3 0 3 0 u a H r e 2 F p s . 7 9 E . 0 8 E . + 2 1 E . - 4 9 E . - 0 8 E . - 5 9 E . - 3 9 E . - 3 - 4 - 9 - 6 - 3 - 5 - 5 - 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 1 2 3 4 5 6 7 8 9 0 1 2 RQ 6 6 6 6 6 6 6 6 6 7 7 7 3 7 4 7 5 7 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 1 2 0 2 6 2 8 2 6 1 5 2 6 2 8 1 7 2 2 1 8 2 4 2 6 1 7 1 1 2 MO c e a g n r r i a t 0 t i 7 d e 5 0 . - 2 4 7 0- 6 1 1 0- 1 1 4 0- 1 1 1 0- 4 9 6 0- 9 0 0- 7 7 0- 1 8 0- 1 0 0- 7 4 0- 2 5 0- 9 5 0- 8 8 0- 7 7 0- 6 E . 8 E . 4 E . 3 E . 1 E . 8 E 3 . 9 E 4 . 1 E 6 . 6 E 3 . 1 E 4 . 4 E 5 . 6 3 E . 6 1 E . E 6 . E T N F 1 3 W n a n o i i c i 3 0 1 7 4 1 4 9 1 4 2 1 6 3 1 0 0 0 2 3 1 2 3 1 0 0 1 6 7 1 1 1 1 8 2 1 0 3 1 9 3 1 6 2 1 m t c fi c y t 9 0 - 4 0 - 5 0 4 0 1 0 1 0 7 0 3 0 7 0 2 0 0 0 9 0 3 0 4 0 4 0 u a H r e 1 F p s . 5 9 E . 2 9 E . - 0 9 E . - 6 9 E . - 0 9 E . + 9 1 E . - 4 8 E . - 4 - 5 - 4 - 0 - 3 - 5 - 5 - 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 7 7 7 7 8 8 8 8 8 8 8 8 8 8 9 8 0 9 E N 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 0 2 5 2 9 1 8 1 1 2 7 2 7 2 6 2 6 2 1 1 6 2 8 2 7 2 6 1 8 2 MO c e a g n r r i a t 5 t i 6 d e 4 0 . - 4 9 2 0- 8 7 2 0- 4 1 4 0- 3 0 0 0- 8 5 3 0- 7 9 0- 8 1 0- 9 5 0- 3 9 0- 6 6 0- 2 9 0- 6 2 0- 9 5 0- 5 8 0- 5 E . 8 E . 1 E . 1 E . 9 E . 3 E 2 . 7 E 9 . 2 E 6 . 2 E 6 . 1 E 5 . 7 E 6 . 8 6 E . 2 8 E . E 4 . E T N F 1 4 W n a n o i i c i 7 9 1 9 2 1 5 4 0 6 8 1 1 9 1 7 1 1 9 9 1 5 8 1 5 2 1 0 1 1 2 1 1 5 9 1 9 6 1 1 0 1 9 9 1 m t c fi c y t 7 0 - 9 0 - 1 0 0 0 0 0 9 0 4 0 0 0 2 0 8 0 8 0 8 0 5 0 0 0 1 0 u a H r e 0 F p s . 5 8 E . 0 9 E . + 6 1 E . - 4 8 E . - 2 9 E . - 8 9 E . - 4 9 E . - 1 - 4 - 8 - 5 - 7 - 1 - 1 - 9 E . 9 E . 9 E . 9 E . 9 E . 8 E . 9 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 1 2 3 4 5 6 7 8 9 0 1 2 RQ 9 9 9 9 9 9 9 9 9 0 0 0 3 0 4 0 5 0 E N 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 S
1 0 6 9 Q n i t 6 7 . E 5 S 3 ( 2 2 9 9 P 1 C) 6 . E 4 o M : N XO t 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2P) C7 6 E 9 M: O T N W e s u o M Q ES ( 2P CE ) 8 M 6 : XO 86 N 1 D R I na mu H D I Q ES ( 2
P C) 7 n E 6 : o it t g 6 2 6 2 8 1 3 2 4 2 6 2 0 2 3 2 1 2 2 1 2 2 5 2 9 2 3 2 9 2 MO c e a g n r r i a t 3 t i 3 d e 2 0 . - 4 6 8 0- 5 3 3 0- 6 2 3 0- 7 8 9 0- 7 0 8 0- 4 0 0- 2 7 0- 2 0 0- 5 2 0- 9 3 0- 2 8 0- 1 0 0- 3 7 0- 9 9 0- 9 E . 5 E . 1 E . 5 E . 5 E . 2 E 7 . 6 E 4 . 1 E 7 . 5 E 1 . 1 E 5 . 8 E 3 . 4 2 E . 0 4 E . E 7 . E T N F 3 3 W n a n o i i c i 7 3 1 7 3 1 6 6 1 3 1 1 7 5 1 8 0 1 8 1 1 8 3 1 0 5 1 2 5 1 2 2 1 2 9 1 8 4 1 7 4 1 9 7 1 m t c fi c y t 6 0 - 5 0 - 1 0 6 0 6 0 6 0 1 0 5 0 9 0 5 0 7 0 6 0 2 0 1 0 3 0 u a H r e 0 F p s . 1 9 E . 2 9 E . - 9 8 E . - 4 9 E . - 7 9 E . - 3 9 E . - 2 9 E . - 5 - 8 - 1 - 3 - 3 - 1 - 1 - 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E . 9 E h 8 8 8 8 8 8 8 8 8 8 8 8 8 8 8 ct t a h c h t c h c h c h h h h h h h h h h h e B a t a t a t c a t c a t c a t c a t c a t c c c c c c a t a t a t a t a t t g B B B B B B B B B B B B B a B a B r a T AD I : N O 6 7 8 9 0 1 2 3 4 5 6 7 RQ 0 0 0 0 1 1 1 1 1 1 1 1 8 1 9 1 0 2 E N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 S
1 0 6 9 i t 6 Q n 7 . E 5 S 3 ( 2 2 9 9 P ) 1 C . E 6 4 o M : N O t X 8 N e k 6 1 D c I o R D e y s e u n o r M ott A D I QE S ( 2)
P C7 6 n E 9 oi t t c e g 9 g n 9 r it 8 3 8 0- 4 7 3 6 0- 3 0 2 9 0 2 8 2 0 M: O a r a t i d 5 . E 1 . E 6 . - E 1 . - E T N F e 2 1 1 1 W e s n u o i i c o t i 9 1 5 1 2 6 c fi c y 4 9 0 1 5 0 4 4 1 0 6 7 1 0 M a r e t F p 8 s . - 9 E 9 . - 9 E 9 . - 9 E 7 . - 9 E Q n E o l i c i t e g 8 2 4 4 5 S it ( c afi g r n a c i 3 o c a t ti 2 1 0- 7 9 3 9 0- 2 5 1 0 0- 0 3 1 4 0- 2 P r F l e p s y t d e . 7 E . 2 E . 1 E . 1 E 9 CE ) 8 2 M 6 : n 2 o t g 8 2 5 3 8 1 7 1 XO it e g n it 8 7 0 3 0 0 0 5 0 8 7 0 8 6 N c a r r F a t i d e 7 . - 7 E 9 . - 3 E 2 . - 1 E 7 . - 1 E 1 D R I na n o i m i c t i fi 7 4 7 u c a c e y 0 t 6 0 0 0 6 1 0 0 0 0 8 8 0 0 . + 7 9 . - 9 0 . + 1 1 . + H r F p s 1 E 9 E 1 E 1 E D I n o i i c t e t l a i f g g r n 4 7 6 i 0 3 5 3 1 2 3 4 2 Q E c c i c a t ti 7 8 0 - 2 0 - 6 0 - 0 0- S a r o l e ( F p s y t d e . 4 5 E . 1 E 9 . 2 E 6 . 1 E 2P C) 7 n o t g 8 3 6 3 4 9 E 6 : it e g n i 3 6 0 7 4 0 5 8 2 0 0 6 2 0 MO c a r r a t t i d e 5 . - 7 7 E . - 4 1 E . - 0 4 E . - 3 E T N F W n a n o i m i c t ifi 4 7 2 u c a c e y 3 t 3 1 7 0 6 9 1 0 4 1 1 0 0 1 0 . - E 7 . - 6 9 . - 2 7 . - H r F p s 9 9 E 9 E 9 E h 8 8 8 8 ct h h h h t a c t c c c e B a t a t a t a g B B B B r a T AD I : N 1 4 5 7 RQO 2 E N 5 3 5 4 S
Attorney Docket No.199235.769601 EXAMPLE 17: In vivo editing, guide quantification and viral genome of exemplary MECP2- targeting engineered guide RNAs in mice [00365] This example describes the in vivo testing of the engineered guide RNAs in mutant (Mecp2R168X) and wild type (Mecp2WT) mice. Engineered guide RNAs (SEQ ID NO: 89 – SEQ ID NO: 521) are packaged into scAAV-PHP.eB (“vector”). Prior to packaging into scAAV-PHP.eB, the guides are designed for ITR-to-ITR configuration. The AAV vectors are delivered by retro-orbital administration in juvenile Mecp2R168X and Mecp2WT mice. A vehicle control is included to benchmark editing. Two to three weeks post-dosing with the AAV vectors or vehicle control, mice are euthanized and the brains are harvested. Mecp2 editing, Mecp2 protein restoration, engineered guide RNA expression, and vg/dg are analyzed to quantify delivery and functionality of the Mecp2 engineered guide RNAs. Individual brain regions (e.g., frontal brain, brainstem, and midbrain) from mice are also examined for editing and/or protein restoration of MECP2. RNA sequencing is performed to measure engineered guide RNA-induced changes in gene expression in vivo. Body weight measurements are recorded throughout the study as male Mecp2R168X mice are known to have a decreased body weight. Additionally, lifespan measurements are recorded throughout the study. EXAMPLE 18: In vivo Mecp2 protein restoration using exemplary MECP2-targeting engineered guide RNAs in mice [00366] This example describes in vivo assessment of protein restoration in Mecp2R168X mutant and Mecp2WT mice after treatment with ITR-to-ITR constructs that contain sequences encoding engineered guide RNAs (SEQ ID NO: 89 – SEQ ID NO: 521). Briefly, the AAV vectors are delivered by retro-orbital administration at a dose of either 5E11 vg/mouse or 1E12 vg/mouse in adult Mecp2R168X and Mecp2WT mice. Next, fluorescent detection immunofluorescence (IF) is performed using C-terminal MECP2 mAB and AF-647 secondary nanobody on sagittal tissue sections from mice two to three weeks after treatment. Fluorescent in situ hybridization (FISH) with probe sets specific for the engineered guide RNAs (SEQ ID NO: 89 – SEQ ID NO: 521) encoded by ITR-to-ITR constructs. Tissue sections are imaged and tiled on the Zeiss Axio Observer using the same settings for all samples. For each sample for IF, 4 separate fields of view (FOVs) of the same size, within brainstem, midbrain, frontal brain, are analyzed for quantification and the data was
Attorney Docket No.199235.769601 combined. For FISH, the entire hemisphere is quantified. Zeiss image analysis software are used to quantify MECP2 or gRNA+ nuclei and MFI of each stain. [00367] While preferred embodiments of the present disclosure have been shown and described herein, such embodiments are provided by way of example only. Numerous variations, changes, and substitutions are within the scope of the disclosure. Various alternatives to the embodiments of the disclosure described herein can be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.