WO2026006542A2

WO2026006542A2 - Compositions and methods for crispr/cas9 based reactivation of human angelman syndrome

Info

Publication number: WO2026006542A2
Application number: PCT/US2025/035407
Authority: WO
Inventors: Yong-hui JIANG; Xiaona LU; Jiangbing Zhou
Original assignee: Yale University
Current assignee: Yale University
Priority date: 2024-06-26
Filing date: 2025-06-26
Publication date: 2026-01-02
Anticipated expiration: 2026-12-26

Abstract

Systems and methods for highly-effective CRISPR-Cas based genomic editing within human chromosome 15q11-q13 have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNAs induce activity of non-pathogenic, paternal UBE3A alleles to reduce, reverse and/or prevent the causative neurodevelopmental defects of AS in a subject in need thereof. Compositions and methods of engineered crRNAs, sgRNAs thereof and ribonucleoprotein (RNP) complexes thereof are provided for enhanced genomic engineering with increased on-off target specificity and on-target editing efficacy for treatment of AS.

Description

COMPOSITIONS AND METHODS FOR CRISPR/CAS9 BASED REACTIVATION OF HUMAN ANGELMAN SYNDROME

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of and priority to U.S. Provisional Application No. 63/664,314 filed June 26, 2024, which is hereby incorporated by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under UG3 TR004713 awarded by National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO THE SEQUENCE LISTING

The Sequence Listing XML submitted as a file named “YU_8933_PCT_ST26.xml”, created on April 28, 2025, and having a size of 53,378 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.834(c)(1 ).

FIELD OF THE INVENTION

The invention is generally in the field of genetic engineering for amelioration of neurodevelopmental diseases and specifically in the area of CRISPR/Cas based genome editing for the treatment of Angelman Syndrome.

BACKGROUND OF THE INVENTION

Angelman syndrome (AS) is a severe neurodevelopmental disorder, caused by maternal deficiency of the UBE3A in the 15q 11 -ql3. The expression of UBE3A gene is subject to brain specific genomic imprinting. UBE3A is exclusively expressed from the maternal chromosome in normal individuals, while the silence of paternal UBE3A expression in neurons is mediated by the paternally expressed non-coding transcript that is antisense to UBE3A (UBE3A-AAS) (Jiang, el al., 1999; Meng, Person, et al. 2012; Meng, el al., 2013). Patients with AS typically have at least one structural intact copy of UBE3A but repressed expression in the paternal chromosome.

It is an object of the invention to provide reagents and methods thereof for enhanced treatment of genetic disorders within the brain and/or central nervous system (CNS).

It is also an object of the invention to provide compositions and methods for genome editing within the brain and/or CNS following systemic administration in vivo.

It is a further object of the invention to provide CRISPR-Cas9 compositions for therapeutic genomic engineering of mutations associated with Angelman syndrome (AS) that enhance therapeutic outcomes. SUMMARY OF INVENTION

Systems and methods for highly-effective CRISPR-Cas based genomic editing within the brain and/or central nervous system (CNS) have been developed as therapeutic interventions for Angelman Syndrome (AS). Selective single guide RNA molecules (sgRNAs) that impart enhanced CRISPR-Cas editing of genomic mutations associated with Angelman Syndrome in human cells are described. The engineered sgRNA scaffolds deactivate pathogenic maternal UBE3A alleles and induce reactivity of paternal UBE3A alleles to reduce, reverse and/or prevent the causative neurodevelopmental defects of AS in a subject in need thereof.

Engineered CRISPR RNA (crRNA) molecules that selectively target the UBE3A-anti- sense RNA (ATS) gene in or near the human genome in the region of chromosome 15ql 1— 13 (that expresses the long non-coding ATS RNA comprising a nucleic acid sequence set forth in SEQ ID NO:36), is provided. In some forms, the engineered crRNA molecule includes a nucleic acid sequence selected from CAGCUCAGUGCAGGAGACCA (SEQ ID NO:1), GGACCACCGUCACCCCUGCA (SEQ ID NO:2), GAGCCUGGGCUGCCUCACGG (SEQ ID NO:3), GAGCUGUGGUGAGCACAUCC (SEQ ID NOT), AGAGCUCACUGAAAGACACA (SEQ ID NO:5), UGCUCACCACAGCUCAGUGC (SEQ ID NO:6), or GAGCCUGGGCUGCCUCACAG (SEQ ID NO:7), UCUCAUCAUCGACCCAACCC (SEQ ID NO:8), AUUACGCUGAGGCCCAACCU (SEQ ID NO:9), UGUGUGGGAGGUGUUGUGUG (SEQ ID NOTO), UAGGUGAGUGGAUCCUGCUG (SEQ ID NO: 11), ACAGCUCAGUGCAGGAGACC (SEQ ID NO: 12), GGCUCACCACAGCUCAGUGC (SEQ ID NO: 13), GGAGACCUGGAGGCCCUGAA (SEQ ID NO: 14), CUCAUCAUCGACCCAACCCA (SEQ ID NO: 15), AGCUCACUGAAAGACACAAG (SEQ ID NO:16), and GCAGCCCAGGCUCCCUGUGU (SEQ ID NO:17). In some forms, the crRNA includes a nucleic acid sequence of any one of SEQ ID NOs:l-17, or a variant thereof having at least 75% identity to any one of SEQ ID NOs: 1-17. In some forms, the crRNA includes a nucleic acid sequence CAGCUCAGUGCAGGAGACCA (SEQ ID NO:1 ), or a variant thereof having at least 75% identity to SEQ ID NO: 1. In other forms, the crRNA includes a nucleic acid sequence GGACCACCGUCACCCCUGCA (SEQ ID NO:2), or a variant thereof having at least 75% identity to SEQ ID NO:2. In other forms, the crRNA includes a nucleic acid sequence GAGCCUGGGCUGCCUCACGG (SEQ ID NO:3), or a variant thereof having at least 75% identity to SEQ ID NO:3. In other forms, the crRNA includes a nucleic acid sequence

GAGCUGUGGUGAGCACAUCC (SEQ ID NOT), or a variant thereof having at least 75% identity to SEQ ID NO:4. Engineered single guide RNA (sgRNA) molecules, including the crRNA of any one of SEQ ID NOsl-17, are also provided. Typically, the sgRNAs include any one of SEQ ID NOs:l-17, conjugated with a Traer RNA. Exemplary sgRNAs include a nucleic acid sequence selected from any one of SEQ ID NOs: 19-22. In some forms, the Traer RNA includes a nucleic acid sequence set forth in SEQ ID NO: 18. In some forms, the sgRNA includes a nucleic acid sequence set forth in any one of SEQ ID NOs: 19-34. In other forms, the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO: 19. In other forms, the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:20. In other forms, the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:21. In other forms, the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:22.

Ribonucleoprotein complexes are also provided. Exemplary ribonucleoprotein complexes include: (a) a Cas enzyme; and (b) the variant sgRNA of any one of SEQ ID NOs: 19-35. In some forms, the Cas enzyme is a Cas9 enzyme. In some forms, the Cas9 enzyme is derived from Streptococcus pyogenes (spCas9).

In some forms, the described ribonucleoprotein complex functions to selectively repress the expression of a human UBE3A-ATS RNA in a subject in vivo. In some forms, the human UBE3A-ATS RNA includes a nucleic acid sequence of SEQ ID NO: 19. Nucleic acid vectors or a transposons encoding or expressing the engineered crRNA of any one of SEQ ID NOs:l-17 are also provided. In some forms, the vector is a viral vector, such as a lentiviral vector, an Adeno- associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV). In other forms, the vector is a non- viral vector. In some forms, the vector is selected from the group including a plasmid, a cosmid, and a replicon. Molecular delivery vehicles including the engineered crRNA of any one of SEQ ID NOs:l-17, and/or an sgRNA including a nucleic acid as set forth in any one of SEQ ID NOs: 19-35, and/or a vector expressing or encoding the crRNA or sgRNA, and/or a ribonucleoprotein complex including a Cas enzyme and an sgRNA any one of SEQ ID NOs: 19-35 are also provided. In some forms, the delivery vehicle includes a nanoparticle, or a microparticle. In an exemplary form, the crRNA, and/or sgRNA, and/or a vector expressing or encoding the crRNA or sgRNA, and/or a ribonucleoprotein complex including a Cas enzyme and the sgRNA is/are encapsulated within or conjugated to the nanoparticle or microparticle. Exemplary delivery vehicles include a polymeric particle, a viral particle, a liposome, a nucleic acid conjugate, and a metallic particle, or a combination thereof. In some forms, a delivery vehicle further includes a targeting motif, An exemplary targeting motif targets the delivery vehicle to the brain and/or the central nervous system (CNS) in vivo. In some forms, the targeting motif includes a peptide that facilitates passage across the blood-brain barrier (BBB).

A cell including the described crRNA, or sgRNA, or vector, or ribonucleoprotein complex, or the described delivery vehicle is also provided.

A pharmaceutical composition including (a) the crRNA of any one of SEQ ID NOs: 1- 17, or the sgRNA of any one of SEQ ID NOs: 19-35, or a ribonucleoprotein complex thereof including a Cas enzyme, or a vector expressing or encoding the crRNA of any one of SEQ ID NOs: 1-17, or the sgRNA of any one of SEQ ID NOs: 19-35, or a delivery vehicle encapsulating the crRNA of any one of SEQ ID NOs: 1-17, or the sgRNA of any one of SEQ ID NOs: 19-35, or a ribonucleoprotein complex thereof or a vector expressing or encoding the same; and (b)a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo is also provided.

Methods for CRISPR editing of one or more target genes to selectively repress the expression of a human UBE3A-ATS RNA in a cell are also described. Typically, the methods include administering into and/or expressing within the cell the crRNA of any one of SEQ ID NOs: 1-17, or the sgRNA of any one of SEQ ID NOs:19-35, or a ribonucleoprotein complex thereof including a Cas enzyme, or a vector expressing or encoding the crRNA of any one of SEQ ID NOs: 1-17, or the sgRNA of any one of SEQ ID NOs: 19-35, or a delivery vehicle encapsulating the crRNA of any one of SEQ ID NOs: 1- 17, or the sgRNA of any one of SEQ ID NOs: 19-35, or a ribonucleoprotein complex thereof or a vector expressing or encoding the same. In some forms the administering is in vivo. For example, in some forms, the methods administer the described pharmaceutical formulations to a subject in vivo.

Methods for treating or preventing Angelman Syndrome (AS) in a subject in need thereof, are also provided. Typically, the methods include administering to the subject the described pharmaceutical compositions. In some forms, the methods administer the pharmaceutical formulation to a subject in an amount effective to prevent or reduce expression of a UBE3A-ATS RNA in the subject. In some forms the subject is an infant or a child. In some forms, the administration is via a route selected from intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration. In some forms, the administration is via intracerebroventricular injection. In some forms, the administration is via intrathecal injection.

Kits including the described compositions of crRNAs and/or sgRNAs are also provided. Typically, the kits include: (i) a crRNA having a nucleotide sequence as set forth in any one of SEQ ID NOs:l-17, or the sgRNA of any one of SEQ ID NOs:19-35, or a vector expressing or encoding the crRNA of any one of SEQ ID NOs:l-17, or the sgRNA of any one of SEQ ID NOs: 19-35; and optionally (ii) a Cas9 enzyme, or vector encoding or expressing the Cas9 enzyme; and/or (iii) instructions for performing one of the described methods for CRISPR- based editing of one or more target genes expressed within the brain and/or CNS of a subject with AS.

BRIEF DESCRIPTION OF DRAWINGS

FIGs. 1A-1B are schematics depicting a CRISPR gene editing strategy as a therapeutic intervention for Angelman Syndrome. FIG. 1 A shows the molecular basis of Angelman Syndrome, based on the large deletion of maternal UBE3A and ongoing suppression of the paternal gene, due to expression of the paternal UBE3A antisense RNA (ATS). FIG. IB shows how knockout of paternally expressed antisense of UBE3A to remove the suppression effect will lead to the reactivation of expression of the paternal UBE3A.

FIGs. 2 -2C depict the in silica design and modeling of crRNAs that target the UBE3A- ATS RNA. respectively. FIGs. 2A-2C are graphs showing each of the top 20 putative sgRNA candidates, designated as hsgRNA-1-20, ranked according to the highest On-target score (FIG. 2A) and average On-target score (FIG. 2B), as well as number of repeats (FIG. 2C), respectively. Nos 5, 7 and 16 score highly across these parameters.

FIGs. 3A-3B are flowcharts, depicting the protocol used for generating neuronal cells expressing UBE3A antisense RNA (ATS) for use in screening the designed crRNAs. FIG. 3A depicts the protocol including first generating the NGN2 inducible iPSCs cells lines using lentivirus, then using double antibiotics select the NGN2 positive NPCs and overexpressing the NGN2 to get mature neurons. On the 14th day (Step 3, day 0), the neurons were treated with chemically modified RNP/Cas9 for 24 hours and collect the cells at the third day 7. FIG. 3B depicts the protocol to induce IPSC differentiate to mature neuron.

FIGs. 4A-4C are graphs showing, respectively. FIG. 4A is a graph of qRT-PCR data, showing Fold change (normalized to EIF4A2) for each of the twenty top candidate human sgRNAs (hsgRNAs) tested in AS patients IPSC induced neuron with minimal 6 repeats, and each culture has at least 3 batch of RT-qPCR. The hsgRNA 5,7 and 16 performed much better than others. FIGs. 4B-4C show a graphs of quantified immunofluorescence, showing the GFP positive (GFP+) neuron area, indicating the edited neuron number (FIG. 4B); and the GRP positive neuron integrity density (FIG. 4C), indicating the UBE3A expression level of each individual neuron, for all the sgRNA candidates. Consistent with the RT-qPCR results, the hsgRNA-5, 7 and 16 are the top 3.

Figures 5A-5F are micrograph images showing fluorescently labelled UBE3A expressed in a representative set of organoids following exposure to control (NT; Figure 5A), scrambled sgRNA (Figure 5B), or hsgRNA-5 at 15 pg (Figure 5C), 30 pg (Figure 5D), 60 pg (Figure 5E), or 120 pg (Figure 5F), respectively.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms "nucleic acid," "nucleic acid sequence," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" are used interchangeably and are intended to include, but not limited to, a polymeric form of nucleotides that may have various lengths, either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs or modified nucleotides thereof, including, but not limited to locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholines. An oligonucleotide is typically composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (uracil (U) for thymine (T) when the polynucleotide is RNA). Thus, the term "oligonucleotide sequence" is the alphabetical representation of a polynucleotide molecule; alternatively, the term may be applied to the polynucleotide molecule itself. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. Oligonucleotides may optionally include one or more nonstandard nucleotide(s), nucleotide analog(s) and/or modified nucleotides. In some cases nucleotide sequences are provided using character representations recommended by the International Union of Pure and Applied Chemistry (IUPAC) or a subset thereof. IUPAC nucleotide codes used herein include, A - Adenine, C = Cytosine, G = Guanine, T - Thymine, U = Uracil, R = A or G, Y = C or T, S = G or C, W = A or T, K = G or T, M = A or C, B = C or G or T, D = A or G or T, H = A or C or T, V = A or C or G, N = any base, or = gap. In some forms the set of characters is (A, C, G, T, U) for adenosine, cytidine, guanosine, thymidine, and uridine respectively. In some forms the set of characters is (A, C, G, T, U, I, X) for adenosine, cytidine, guanosine, thymidine, uridine, inosine, xanthosine, respectively. The modified sequences, non-natural sequences, or sequences with modified binding, may be in the genomic, the guide or the tracr sequences.

As used herein, the terms “percent (%) sequence identity,” or “% identical to (sequence)” are used interchangeably and are defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical with the nucleotides or amino acids in a reference nucleic acid sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software. Appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared can he determined by known methods.

The terms “protein” “polypeptide” or “peptide” refer to a natural or synthetic molecule including two or more amino acids linked by the carboxyl group of one amino acid to the alpha amino group of another.

The term “polynucleotide” or “nucleic acid” or “nucleic acid sequence” refers to a natural or synthetic molecule including two or more nucleotides linked by a phosphate group at the 3’ position of one nucleotide to the 5’ end of another nucleotide. The polynucleotide is not limited by length, and thus the polynucleotide can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

A cell can be in vitro. Alternatively, a cell can be in vivo and can be found in a subject. A “cell” can be a cell from any organism including, but not limited to, a bacterium.

The terms “editing fidelity” or “editing efficiency” or “targeting accuracy” or “on-target editing” or “on-off target specificity” or “on-target editing efficiency” are understood to mean the percentage of desired mutation achieved and are measured by the precision of the sgRNA variant in altering the DNA construct of the targeted gene with minimal off-target editing. A DNA editing efficiency of 1 (or 100%) indicates that the number of edited cells and/or edited alleles obtained when the sgRNA variant is used is approximately equal or equal to the number of edited cells and/or edited alleles obtained when the wild type or parent sgRNA variant is used. Conversely, a DNA editing efficiency greater than 1 indicates that the number of edited cells obtained when the sgRNA variant used is greater than the number of edited cells obtained when the parent sgRNA variant is used. In this case, the sgRNA variant has improved properties, for example improved editing efficiency when compared to the parent sgRNA.

The terms “single guide RNA” or “sgRNA” refer to the polynucleotide sequence including the guide sequence, tracr sequence and the tracr mate sequence. “Guide sequence” or “Protospacer” or “crRNA” refer to the around 20 base pair (bp) sequence within the guide RNA that specifies the target site and may be used interchangeably with the terms “guide” or “spacer.”

The term “stem-loop 2 region” refers to the polynucleotide sequence of the second hairpin structure of the sgRNA and the flanking sequence.”

The terms “genome editing,” “genome engineering” or “genome mutagenesis” refer to selective and specific changes to one or more targeted genes or DNA sequences within a recipient cell through programming of the CRISPR-Cas system within the cell. The editing or changing of a targeted gene or genome can include one or more of a deletion, knock-in, point mutation, substitution mutation or any combination thereof in one or more genes of the recipient cell.

The terms “vector” or “expression vector” refer to a system suitable for delivering and expressing a desired nucleotide or protein sequence. Some vectors may be expression vectors, cloning vectors, transfer vectors, etc.

The term “variant” or “mutant,” as used herein refer to an artificial outcome that has a pattern that deviates from what occurs in nature.

The terms “Protospacer adjacent motif’ or “PAM sequence” or “PAM interaction region” refer to short pieces of genetic code that flag editable sections of DNA and serve as a binding signal for specific CRISPR-Cas nucleases. The PAM interaction region in the wild-type SaCas9 or its variants contains amino acid residues 910-1053 (Nishimasu, et al. Cell, 162, 1113-1126, doi: 10.1016/j.cell.2015.08.007 (2016)) and includes a conserved 13-amino acid region spanning positions 982 to 994 which plays a role in binding to the 4th and Sth bases of the PAM (Ma, et al. Nature Communications, 10, 560, doi: 10.1038/s41467-019-08395-8 (2019)).

The terms “Cas9,” “Cas9 protein,” or “Cas9 nuclease” refer to a RNA-guided endonuclease that is a Cas9 protein that catalyzes the site-specific cleavage of double stranded DNA. Also, referred to as “Cas nuclease” or “CRISPR-associated nuclease.”

The term “mutation” refers to a substitution of a residue within a sequence, e.g., a nucleic acid or amino acid sequence, with another residue, or a deletion or insertion of one or more residues within a sequence. Mutations are described by identifying the original residue followed by the position of the residue within the sequence and by the identity of the change in residue. For the purposes of this disclosure, amino acid positions are identified using the amino acid positions shown in SpCas9 sequence UniProtKB/Swiss-Prot No. Q99ZW2 (PDB ID NO:6O0Y), with the numbering beginning at the initial methionine residue. Various methods for making the mutations in the amino acids provided herein are well known in the art, and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual 4th Edition, Cold Spring Harbor Laboratory Press, (2012).

The term “identity,” as used herein, can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, A. M., Ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., Ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., Eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., Eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J Applied Math., 48: 1073 (1988). Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. The percent identity between two sequences can be determined by using analysis software (i.e., Sequence Analysis Software Package of the Genetics Computer Group, Madison Wis.) that incorporates the Needelman and Wunsch, (J. Mol. Biol., 48: 443-453, 1970) algorithm (e.g., NBLAST, and XBLAST). In some forms, the default parameters can be used to determine the identity for the polynucleotides of the present disclosure. In some forms, the % sequence identity of a given nucleic acid sequence “C” to, with, or against a given nucleic acid or amino acid sequence “D” (which can alternatively be phrased as a given sequence C that has or includes a certain % sequence identity to, with, or against a given sequence D) is calculated as follows:

100 times the fraction W/Z, where W is the number of nucleotides or amino acids scored as identical matches by the sequence alignment program in that program’s alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of sequence C is not equal to the length of sequence D, the % sequence identity of C to D will not equal the % sequence identity of D to C. The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the present disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/- 10%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 5%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 2%; in other forms the values may range in value either above or below the stated value in a range of approx. +/- 1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as important to the practice of the disclosure.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed method and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a ligand is disclosed and discussed and a number of modifications that can be made to a number of molecules including the ligand are discussed, each and every combination and permutation of ligand and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited, each is individually and collectively contemplated. Thus, in this example, each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Likewise, any subset or combination of these is also specifically contemplated and disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E are specifically contemplated and should be considered disclosed from disclosure of A, B, and C; D, E, and F; and the example combination A-D. Further, each of the materials, compositions, components, etc. contemplated and disclosed as above can also be specifically and independently included or excluded from any group, subgroup, list, set, etc. of such materials.

These concepts apply to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific form or combination of forms of the disclosed methods, and that each such combination is specifically contemplated and should be considered disclosed.

All methods described herein can be performed in any suitable order unless otherwise indicated or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the forms and does not pose a limitation on the scope of the forms unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

IL CRISPR/Cas Genomic Editing Compositions

Engineered single guide RNA molecules (sgRNA) that impart enhanced editing activity and high targeting specificity for genomic engineering of genes associated with neurodevelopmental diseases in human cells have been developed. The engineered sgRNAs implement therapeutic mutations within the brain and/or CNS following system delivery in vivo. In some forms, the engineered sgRNAs selectively target and induce mutagenesis on one or more target genes associated with Angelman Syndrome (AS) pathology in human cells within the brain and/or CNS. Pharmaceutical formulations including the compositions are also provided.

The term “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats) is an acronym for DNA loci that contain multiple, short, direct repetitions of base sequences. The prokaryotic CRISPR/Cas system has been adapted for use as gene editing (silencing, enhancing or changing specific genes) tools in eukaryotes (see, for example, Cong, Science, 15:339(6121):819-823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). Methods of preparing compositions for use in genome editing using the CRISPR/Cas systems are described in detail in WO 2013/176772 and WO 2014/018423, which are specifically incorporated by reference herein in their entireties.

In general, the term “CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g., tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. One or more tracr mate sequences operably linked to a guide sequence (e.g., direct repeat-spacer-direct repeat) can also be referred to as pre-crRNA (pre-CRISPR RNA) before processing or crRNA after processing by a nuclease. Typically, a CRISPR-Cas9 system includes a guide RNA (gRNA) and Cas9 nuclease, which together form a ribonucleoprotein (RNP) complex. The presence of a specific protospacer adjacent motif (PAM) in the genomic DNA is required for the gRNA to bind to the target sequence. The Cas9 nuclease then makes a double-strand break in the DNA. Endogenous repair mechanisms triggered by the double-strand break may result in gene knockout via a frameshift mutation or knock-in of a desired sequence if a DNA template is present.

In some forms, a tracrRNA and crRNA are linked and form a chimeric crRNA-tracrRNA hybrid where a mature crRNA is fused to a partial tracrRNA via a synthetic stem loop to mimic the natural crRNA: tracrRNA duplex as described in Cong, Science, 15 :339(6121) : 819- 823 (2013) and Jinek, et al., Science, 337(6096):816-21 (2012)). A single fused crRNA-tracrRNA construct can also be referred to as a guide RNA or gRNA (or single-guide RNA (sgRNA)). Within an sgRNA, the crRNA portion can be identified as the ‘target sequence’ and the tracrRNA is often referred to as the ‘scaffold’.

CRSIPR systems having enhanced editing activity and high genome-wide targeting specificity typically include two components: (1) a single guide RNA configured for enhanced editing activity; and (2) a Cas enzyme.

CRISPR-Cas9 systems hold great promise for applying genome editing to biomedicine. CRISPR-Cas9 is a programmable gene-editing system that can be used to knock out genes and correct genetic mutations in human cells (Anzalone, et al., Nat Biotechnol 2020, 38, (7), 824- 844). This system utilizes a single guide RNA (sgRNA) that directs the Cas9 protein to the target genomic site for editing. Existing CRISPR/Cas9 toolkits exhibit varying efficiencies across loci, limiting their applicability for therapeutic genome editing. Optimization of such systems is in great need.

Applying genome editing technologies for applications in humans requires tools that are robust, reliable and specific, and a great deal of work has focused on enhancing the specificity of CRISPR/Cas9 by optimization of the sgRNA. The protospacer sequence of sgRNA is responsible for target site recognition, whereas its scaffold sequence binds to Cas9, which results in the conformational change of Cas9 for its activation. Many studies have been done on elucidating the determinants in the protospacer sequence for sgRNAs to exhibit high on-target and low off-target activities (Hanna, et al., Nat Biotechnol 2020, 38, (7), 813-823).

The scaffold sequence of sgRNA can be engineered to alter the overall editing activity by increasing its stability and assembly with the Cas9 protein. The “E+F” scaffold variant was engineered with a 5 -nucleotide-extended tetraloop that could strengthen the scaffold’s interaction with SpCas9 and an A-U base-pair flip in the lower stem that removes a putative polymerase-III terminator sequence (Chen, et al., Cell 2013, 155, (7), 1479-91). The E+F scaffold sequence was further mutated with different substitutions, and specific regions were identified to be more tolerant of mutations without compromising the sgRNA’s activity (Jost, et al., Nat Biotechnol 2020, 38, (3), 355-364). Six scaffold variants, three of them containing additional U61C + A66G mutations besides those in the E+F scaffold, were reported to generate more edits. Apart from these efforts, there has been limited success in enhancing SpCas9’s activity. Existing engineered guide RNA scaffolds that increase on-target editing of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease greatly compromise its on-to-off targeting specificity.

A. CRISPR/Cas Systems Targeting Genes Associated with Angelman Syndrome

Engineered single guide RNA molecules (sgRNA) that impart enhanced editing activity and high targeting specificity for genomic engineering of genes associated with Angelman Syndrome (AS) in human cells are provided.

In some forms, the engineered sgRNAs implement activity-enhancing sequences of nucleic acids that enhance their editing activities as compared with pre-existing strategies for silencing pathogenic maternal genes associated with AS RNA. An advantage of the described CRISPR-Cas systems is that a single Cas protein can be programmed by guide molecules to recognize a specific nucleic acid target within pathogenic maternal genes associated with AS. The described sgRNAs recruit CRISPR-Cas proteins to the specific nucleic acid target locus to achieve accurate and efficient genome editing at the defined target.

The single guide RNA is a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing. The gRNA is made up of two parts: CRISPR RNA (crRNA), a 17-20 nucleotide spacer sequence complementary to the target DNA and a conserved repeat fragment (“handle” or “tag”) region that pairs with the tracr RNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease. The crRNA component imparts specificity of CRISPR-directed nuclease activity and is the customizable component that directs specific editing.

1. Target Genes associated with AS

Angelman syndrome (AS) is a severe neurodevelopmental disorder characterized by severe developmental delay, speech impairment, a movement disorder, seizures and inappropriate “happy” disposition caused by mutation or deletion of the maternally inherited UBE3A allele. Human chromosome 15ql 1-ql 3, is known as the Prader-Willi syndrome (PWS)/Angelman syndrome (AS) region, since its deletion on the paternal chromosome leads to PWS (MIM ID: 176270) and deletion on the maternal chromosome leads to AS (MIM ID: 105830). AS is typically caused by large genomic deletions within 15ql 1- 13, affecting multiple genes, of which loss of the maternal copy of the ubiquitin protein ligase E3A (UBE3A) gene is causal for AS. UBE3A encodes an E3 ubiquitin ligase, named E6-AP, and is known to be the major disease gene for AS. Thus, UBE3A, is known to be “the Angelman syndrome gene”. UBE3A, is maternally expressed in neurons. Its antisense transcript, UBE3A-ATS, is also expressed only from the paternal chromosome and suppresses UBE3A in the paternal chromosome in cis. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding antisense RNA called UBE3A-ATS or Ube3A-ATS. UBE3A-ATS is an atypical RNAPII transcript that functions to suppress paternal UBE3A expression.

Human 15q 11-13 includes several imprinted genes together with regulatory elements named AS-IC (imprinting center) and PWS-IC. These concertedly confer allele specificity on 15ql 1— 13. It has been shown that DNA methylation status of 15ql 1— 13 and other autosomal imprinted differentially methylated regions (iDMRs) can influence the expression of UBE3A- ATS.

In Angelman syndrome patients, the UBE3A gene on the maternal allele has been either deleted or lost function, while the paternal Ube3a is structurally intact, but silenced by paternally expressed antisense UBE3A-AAS.

The described compositions to treat Angelman syndrome inactivate the UBE3A antisense on paternal chromosome, so as to remove the suppression effect, and enable expression of the paternal UBE3A in neurons and offer therapeutic benefit to Angelman patients.

Therefore, in some forms, the described compositions selectively inhibit the expression and/or presence of the long non-coding RNA called UBE3A-AAS in vivo. An exemplary nucleic acid sequence for the human UBE3A-AAS has a nucleic acid sequence of: GCCGACCTCGAATGCGGTCCTCCTGCTGCAGACCACGCCCACCAAGGGCTGGCCGCAGCCACTGTAGCTGAGCTCAG AGCCTTCTGTGTGGTTTGCGGGGGCAAGGTCAGCTGCCCCCTGCCCTCTCTCTGGGGCTATTTGAGGAATGCGGCCT TTTTGCATAAGAAAGGCTTTTCTCTACAGTAACTGTGGTCGCTGATCAATGACACCTTCCTGATGTAAGTGGGATCT GTGGATCTGAAAATTCACAGTGGCCAGCTCACCACCACCTGATGAAAGATACACCACAGGGTGAGAGCATCCTAACA GCAAACTGCAAATGAGATACTTTGACGAAGGTAATTGGGACTCCCATCAAGTCTCCACACAGCTAGCAGCCACGTGG GGCACTTCTCCAGCATATGTAAGTGGAACTCAGAAGGCAACATTCCCTGAACATACTCTTCCACCAGAATCTCCTCT ACAGATTTTTGCTGCCCCTTTTACCAGTGGCTGAATCTACTTTCTCTTATGGATCGCTTACACCTGAGACGAACTAC AGAACAGCACGTACCAGAGGTGGAAGTCCAAGTCAAACGCAGAAGGACTGCCTCACTGAGCAACCAAGAGTGTCAGT TGTACCCGAGGCGTTCTCAGCAGCAGCAAGTACCTGTGGTGGATTTCCAGGCTGAACTGAGGCAGGCATTCTTAGCT GAGACACCAAGAGGTGGTTAAAGCCATATTGGAGTAGCGAGGAATCTGATTCCAAGCAAAAACCAGGCTCCATCTAC TCTTTGAAGCTTCTGCCCAGCTTGCATTGTTTCTAGGAGAACCTGCGTCATACCTTTATCTATAGCCTTCCCCTAGG TCTTCAGAAGCATCAAGTTTTAACTGTGGACATTGGATTTGGTGGAACAGCAATCATGACTGTTGGCAAGAGTAGCA AGATGCTGCAGCACATTGACTATAGAATGAGATGTATCCTGCAAGATGGCCGAATCTTCATTGGCACCTTTAAGGCT TTTGACAAGCATATGAATTTGATCCTCTGTGATTGTGATGAGTTCAGAAAGATCAAGCCAAAGAATGCGAAGCAACC AGAGCGTGAAGAAAAGCGGGTTTTGGGTCTGGTGTTGCTGCGTGGGGAGAACTTGGTATCCATGACTGTGGAGGGGC CACCCCCCAAAGATACTGGCATTGCTCGGGTACCACTTGCTGGAGCTGCTGGAGGCCCTGGGGTTGGTAGGGCAGCT GGTAGAGGAGTACCAGCTGGTGTGCCAATTCCCCAGGCCCCTGCTGGATTGGCAGGCCCTGTCCGAGGAGTTGGGGG ACCATCCCAGCAGGTAATGACTCCACAGGGAAGAGGCACTGTAGCAGCTGCTGCTGTTGCTGCGACTGCCAGTATTG CTGGAGCCCCAACACAGTACCCACCAGGACGGGGCACTCCGCCCCCACCCGTCGGCAGAGCAACCCCACCTCCAGGC ATTATGGCTCCTCCACCTGGTATGAGACCACCCATGGGCCCACCAATTGGGCTTCCCCCTGCTCGAGGGACGCCAAT AGGCATGCCGCCTCCGGGAATGAGACCCCCTCCACCAGGCATTAGAGGTCCACCTCCCCCAGGAATGCGTCCACCAA GACCTTAGCATACTGTTGATCCATCTCAGTCACTTTTTCCCCTGCAATGCGTCTTGTGAAATTGTGTAGAGTGTTTG TGAGCTTTTTGTTCCCTCATTCTGCATTAATAATAGCTAATAATAAATGCATAGAGCAATTAAACTGTGAGGTACTG TTGTATATATTTTTTTGCCTGTTGATTTTGATGAGATCTTAAGTTACTGTGGATGAGGGTGATGCCTATTAAGCAGT TGATTCAAATCATATTCTCTTTAATTCTTAGGATAAAAAGGTTTTCTGCTATCTAACTTTCACTTTGTGCATTGACT GGTGTTGGTTAATAATGTTTTCATGACCCGGGGGTTTAAGTGGTTGTGCTGCTTCACACATGGCCTCCTGCTTCTCA

TGTTGCTATGTCACATTATCTGAAGGGAAGTGGGGATGGGATGAAAGGCCTTGTAAGGCCACCTATATACTGAAGGA

AGTTGTAGTGAATCACAGTCCATGCTCATTACATATCAAGGCATTTGAAATAGAAGTTACTCTTTTTCTTGGGATGT

TGACCTCAGTTGATCAGGTTTTACAGTGTCCTATGATCAGAATAGAGGATGTGATACTGGAATAATCATTTTCTACC

ATTGAGTAAGTATTTATTTATAAATTACTAAATGAGTGGAAGATCCTGGGTACTGGGAGGCAGGAGGTTGCATATTT

GAGTTGATAAGTTGTACTACTGATAATAAAGATAACTGACACACTAAAACATACTTGGTACACTGAAATACAAATTA

CGGTTATGTCCAGTGGTATCACCAAGTGACAAACTGGGAGCTAAATAGTTCAGAAATGCCCTAGTCTCTAGGTCCAG

TTTGAATTGGTTATGGAACAAAGAAGCCATTTATAAAAGGGAAGAGATTTTTAGGTTTTGCAGCTAAGGTTTATAGT

AGGCAGCGAGGGAATGATTCATAGTGATTGTAGCAGTTGGTTTACCAAGCACTAAAGAGATTGGTGTTAACTCCATA

ATACTGATAGACTGATTGGAATTACATTTTTTCTCTTGAATTTACCAGGAGTGGTTGTGAATGTCACCTTTTCCATC

TTTTAAATTACATCCTTGCAGGTGGTAATCACTCAAGGAAAAGGAATTATCAGAGTAACAATAGTTCCTGTTACTGA

CTTGATATGGTCTCTGCCAGAATAGCTTCTCAGATTAATGGCCACTCTCAGGGGAAGCATTTCTCCTTGCATTGGTT

TGCTGGTATGGATGGCCCTTAGATTTTGGCATGAGTGATTATGCAGCCTATTTGAAGAAAACCTGTTTTCAGGACCT

TTTTAATTAAATATGGCCTGTTACATGAGTTAAAGTGTCATACGATACCATTTTTTGACCACTTAGTTGATGCCTCT

GAAAGGAAGAGAGAATCAGATCGCTAAAATTTGATGTGTCGTCAGTGTGGATCCTACTCAGTAGTGTCACAACGAGG

CTGAAAATCATATTAAGTGCTTTTAGGACAGAAACGTAAACTGCTCAGGTAAAATAACCTTATAAAGAGAAATAAGC

ATATATTGCAGAGGCCTTGGCTAGGTTCATGATGACACAGGACCTTGTCTGAACATAATGATTTCAAAATTTGAGCT

TAAAAATGACACTCTGAAATCCAGTCAGTGTGCCTCACTAGACTTTTCGATTTCAAGATTTTCTGCAGAAAATGTTT

TGAAAACTTTGAATACTTAAAAATGGCAGGTGTAGTATTGCACTTTGCTAGTTGCTCAGATACCCTTTTTTATTTGT

ATAGATATTCTGAGTTCCTTTTTTTTTCTACATGTTGTACGTTGTCGAAAGCTAAAAGGAAACTTATCCTTGGATCA

CGGAAGGCAGAGGCATTTGGTGAGATGGAAACAAGGATGTGTAAAAATGAGACGACCACCTCTCGGATTAAAAAAAA

AAAGTGCCAGAGTTCTAGGGTTCTAAGTGATGTCCAGGAAGGAGGAGGAATAATATTTATGGAGCATATATTATGGA

ACACACCATTATGAATGGTTTTTGATTGATCTCACAAAGAGCATGTGCTTTAGACATTAATGCAGCGACTACCATGA

AACAGAGTATTGGATTACCTAAGGAAAGAAGCAGAGGATGGAATTTGAGGCAATCAGGATGAGTGAAAAATTGATTT

GCAGCTGACCTGCAAATGGAATCATCAGGAACATCCCTTTCTCATGGAGTCCCTTAATTTACAAGTTAACTGCAAAC

ATAGGAGATGATAGTTCCAAGAAGGAACATTTTATCGTCTTTGTTTTTAATCTCAAGAATGGTACCTACCATCAGTG

AATGACCTGTTGCAGTGCTTTCATTGAAGTGTTCTTCGTTCCCTCAGCAATATGATTGTGATGACTGAAAAAGGGAA

ACTGTGCCACTATTTGTACCATCATTTTCACCAAAATCTAAAAATGCTTTTTATGACGTATGGAGACATTCTTCATG

TTTGTTTCAGTGGACACTCCTTGCAGATGTAAAAAACTGAGAAAACTCACTTTTGGAAAGTGACCTAAAGAGTGTCA

TTGAAGTGAATTTTAAGTAGGCACGATGATTGTTTTCATGGTTGCTGTTGGATCATATCTCAGGAGCTGGAATGACA

GACATTATTGAACAAAGAAATCAGGATAGTGGAACTTAAAGGGCTTCATCTCAGTGCTTTCATAAGTATGAAGTGCA

TATATTTATAATTTTCACTAATCACAGGAATATTCCCTCTGTATACTTGCATTTTTTTCTATCAATTGACGCTTACA

ATTATTTTTGCAAGTTGGAAAAAAATGCAGTATTAATTTCATTTGCAACAAATTTAATAGATTAATTAGGAGAGAAT

AGATATCTTTAAAATTGAGTCTTCTGTCCAACTGCAAGTTATAGCTGAGGTGGTACCAGTTTAAGAAGTGAGAACTT

CTTCACTGACTGAAATTTGCATATCAATCCAAATCATTGTGCTTATTTACTATTTTTGTAGACGTCATTCTTCAAAA

GTGCTGCATCTTCAATAGTCAAGGAGGACAAAGACATCTTTCCTGCAGGTGGTGACCATGTGGATAATAGGATCATA

GACAAATGGTGGTTCCATCTACAGCATCACCGAAATGCTGTTGTTACTGCTTCTTTCTGGGATCTTCTTGCATCTTT

GCTTCTAAAGGGGCACACCTTTTATAGAAGTACGTCCAGGAATAGAAGTGACCTGCTTTTTCGCAATTGTCATGGGA GGCCAGCCGCCTGCTGGCAAGGAAGATGGTTGATTCTTCTGATCAATGGAGGCCCTGCAGAGTCCTGTAGTCACTGT

GGCCTGGCATCCATGGTGCACCCAATGCTGGTGGATGGCTTAGGACGAGGCAGTTGCTGTGGGAGGTGTGGTAACTG

CTGGAAGGAAGATGCTCAATTCTTCTGAGCAACGAAGGCCACACGGAGTCCTGGATTCTGTGTGGCCTGGCATCCAT

GGCGCACTGTGTGCTGGCAGGTGGCTCAGGACGGGGCAGTTATCATGGGACACCCGCCATATGCTGGCAAGGAAGAT

GGTCAGTTCTTCTGAGCCACAAAGGCCCCCAACATCCTGGAGTTGGTGTTGCCTAGCATCCACGGTGCGCCCCCTGC

TGGTGGATGGCTCAGGATGGGGCAGTTGCCGTGGGAGGCCCGCTGCCTGCTGGAAGGAAGATGGTCAGTTCTTCTGA

ACCACAAAGGCCCTGCAGAGTCCTGGAGTCTGTGTGGCCTGGCTTCCAGGGTTCACCTCATGCTGGTGGATTGCTCA

GGATGGGTGCCGAAGGTCTTTAAGGTCGTCAGCTCACCTACTGCCCAGCCTGTGGTGTCAAATGTCCTGCCTCTTCA

AATGTGTTTGGATCGATGATGAGTCCCCCCAAAAAAACATTCCTTGGAAAAGCTGAACAAAATGAGTGAAAACTCAT

ACCGTCGTTCTCAGCGGAACTGAGGTCCAGCGCGTTGCCTCCAGCAGGGACTGTTGGAGTGAGGGAGAGCTTCCACT

AGTCCCTATAGGTTCCACCACCCAGGGCATCCGCAGGTGCACCTCACCTAAGAGGGTGGCCAGAGACAGTGTCTTGT

CATGACATCAGAAGCCCCATGAGGAAGAGGAGGAGCCTGTGCTCCTGCCAGAGGTGGTGGCTGTTAGGGACAGTTGC

CGTGGGAGGTCCGCTGCCTGCTGTCAAGGAAGATGGTCGATGCTTCTGAGAAACAGGCCCTGCGGGTTCCTGGATTT

GGTGTGGCCTGGCATCCACGGTGCACCCCGTGCTGGCGGGTGGCCCAGGACGGGGCAGTTGCCTTGAGAGGCCCACC

GCCTGCTGGCATGACAGATGGTCAGTTCTTCCGAGCAGTGGAATCTCCATGGAGTCGTGGAGTCGGTGTGGCCTCGC

ATCCACAGTGCACCCTGTGCTGGTGGATGGCTCAGGACAGGGCAGTTGCCGTGGGAGGCCTGCCGTCTGCTGAAAGG

AAGATGGTTGATGCTTCTGAGCAATGAAGGCCCCATGGAGTCCTGGATTCGGTGTGGCCTTGCATCCGTGGTGCACC

CCGTGCTGGTGGATGGCTCAGGACGGGGCAGTTGCCATGGGAGGCGCAGGCCTGCTGGAAGGAAGATGGTCGATTCT

CCCAAGCAACAAAGGCCACGTAGAGTCCTGGTTTCAGTGTGGCCTGGTATCCACAGTGCACCCCGTGCTGGTGGGTG

GCTCAGGACGCACACTCATTTCCTCTGCACCTTCCTGGTGTCATTGGCATGGAAGGAAGGCCCTTGTCCAAGAAGGA

TGGTCTTCGTCTTGTGTGATCTTTGAGATGCCATGAGGCCCCTGGATACAAATGGTGTGGGCTCCTTTGGAGGCTTT

TGGATTTCTCCTGAATGTGGCGTTGGTATGGAAGGAAGGCCAGTGACCGCAAGGGATGACTTTGATCTTGGGAGATT

TTTGGAGATGGCAAAGTGCTTCTAAACATGTTTGGCATGGTCTCCTTCAGGCTGGTGGATACCTCAGGATGGTGGCG

TTGGCATGGAAGGAAGGACTGTGTCTGTGAGGGATGATCATCCTCTCATGTGATCCTTGGAGATGCCAGGAAGCCCC

TTGACACATGTGGCATGGGCTCCTTCAGAGGCTTTTGGATCCGTCGTGAATGTGGCATGGGCATGCAAAGAAAGACA

GAGGCTGCAAGGGATAAACTTCATCTTGGGAGATCCCTGGAGATGGCAGGGAACCCATGGACACATGTGGCATGGGC

TCCTTTGGGTGCCGGTGGATCCCTTCATACGGTAGTGTTGGTGTAAAAGGCAAACCCGTGTCATGAGGGATGGTCAT

CATCTTGTGTGATCCTTGGAGATGGCAGGAAGCCCTGGACATACATGGTGTGGGGGCTCCTCCAGAGGCTGTTGGGA

TCCTCCTGGATGTGGTGTGGGCATGGAAGGAAGGCCAGTGGAGACAATGGATGATCTTGTTCTTAGCAGATCACTGG

ATGTGGCAGGGAGTCCTAGGACATGTGTGGTGTGGGCTTCTTCAGGTGCTGGTGGTGTTGGCATGAGGAAAGGAGGT

ATCTTCGAGGGACAATCTTCTTCTTGTGCGATCCTTGGAGATGCCATGAGGCCCCTGGACACATGTGGTGTGGGCTC

CTTTGGAGGCTGTTGTATCCCTTCTGAATGTGGCGTGGGCATAGAAGGAAGGCCAGTGGCCACGAGGGACAATCTTG

GTCTTGGGAGATCCTGGAAATGATAGGGAGTCCCTTGATATGTGTGGCATGGGCTCCTTCAGGTGCTAGTGGATTCC

TTAGGATGGGACAAACACTGTGCGTGGATCGATGATGACTTCCATATATACATTCCTTGGAAAGCTGAACAAAATGA

GTGAAAACTCTATACCGTCATCCTCGTCGAACTGAGGTGGTGTTGGCATGAAAGGCAGGCTTGTATCATGAGGAATG

ATTGTCATCTTGTCTGATTCTTGGAGATGGCAGGAAGCCCCTGGAAACACATGGTGTGGACTCTTTCACAGGCTGTT

GAAACCCTCCTGAATGTGAGGCCAGAGACAGGCAGACAGCAGATGTATTGCAGGGAGCTGGATGACATGGCCCTTGG

AACCTGTGCACATGCCTGCCTTTCTGATGCACGTCCATGTTTTCTCTGCACCTCCCCGGTGGTGTTGGTATAAAAAG CAGGCTTACATCAGCAAGGGATGATTGTCGTCTCATGCGATCCTGGGAGATGGCAGAAGTCCCGGGACACATGGAGT

GTGGGCTCTTTCGGAGGCTGTTGGATCCCTCCTGAATGTGTTGTGGCCATGGAAGTAAGGCCAGTGGCTACGAGGGA

CAGTCTTCATCTTGGGAGATCCCTAGAGAGGGCAGGGAGCCCCTTGACATGTGGAATGTGATCTCTGTTGGGTGCTG

GTGGATCCCACAGGTGGTCTGGCATGGAAGAAGGCCAGAGGCTGCGATGGATCATCTTTGTCTTGGGAGATTCCTGG

AGATGGTGGGGAGCCCCTGGGCACGTGCAGCATATGTGGCGTGGGCTTCTTCAGGTGCTGGTGGATCCCTCAGGATG

CTTTTCAAGGTTTTTCTATGAGTTCTGTTAAAGAGTCTGAAAATGGAGCGTAGAAGACATGCTTCGATTTCCAGGAT

GGTTGTGTTTTCCAGAGAGGCGGAGGTAAACCACTGGCGCAGCCCTGCCTCCCACAGATAATTCCGGGTGCAGTGTT

TCATATCTAGACTTGGACTTCTATCCTACCATACTTCTTTGTGTGCAGTTCATTGTGGAACCAGCACAGATTTTCAT

ACGAAAGTTATTTCTTTTTAACCTCAAAAAGTGACAGAATGGAGTGTATCATGAAAAAGTCTGACATCAAAACCTCA

AAATGTGATATTGATTGGGTGAGATAGAATTCCATGGAATTTCATTGCCAAACCAGTGAAGTGCTAATTTTGACATT

TGTACCAGCTAACGTAGCTGTGAAGAAACACACCAATACACTTCTAAGGACAGCATAGACCAAGATCTAGGTTTCAT

ATGGATGAATTTATTCTTCCCTTTGAGGCAAGACAAGAAGGAAAGAAAGTATTGGGAATAATGCTGCCCTCCCATCC

TCAAGGGCCAGTGGGAACTCTAATATTGATTGGTTTTCACAGAATTCCATGGACTTTAACTGCTGAACCAATGATGC

ACTGATTTTGTCACTTGTACCAGCTAACGCATCTGGGAGGCAACAAAACAATACTCTCCTGAGGTTAATGTGGACCA

AAAACTAGATTTCATATAAAAGAGTTTATTCCTTCTACTTGAGGTGTGACATGAAGGAAAGTAATGGAAATAATTCT

GCCCTCCCATTCTCAAGGGCCAGGGGGAATTCAAATATTGAACGGAAGTGATTTTGGAATTGCATGGAATTCTCACT

GCCATCCCAAAGAAATGCTGATCTTGTCACTCTTACCTGTTTCATTCCCTGGGGGCAGAACAAAAGGACGCACTCCA

AAGATCGGCATGAACCAAGAACTGGGTTTCATATAAAATATTTTATTCTGTCCTCTTGAGATGTGACATGAGAAAAG

TATTGAGAATACTTCTATCACCTCATCTTCAAGAATCCGTAGGAATTCTCCCATGGTGGACTTTACGGTTAACCTGA

ACAGGTCTTGGAAAAAGGACTTTAAACATTCTCCTGTTCTGTGGAATAAGTATACTTTTTTCTGGACATACACCTTG

TCAGTTCCATGTGATACTAGAGTTGCATAGATTTTTAACCTTTACGCCAATGAAGATCTGAGGCAAACATTGCCCCT

TTTTTAGAAGTTTGCATTTGTCACTGATAACTGTAATAATTGATCTTGTTGAAAAGCCTGGGAAATGAAGATCTGAT

ATGTCACTTTTACCGGCTTCCTCAACTGAAAAAGAATACAATGAAACACGCTGAGAATTTTTGTGCTTTCTCAGTCT

TGCTGATGATCTTTCTATGGGCCTGAAGCGTGGAGAGTGCCAGAGAGGCTGATCCTCAGCAGTGTGGCAGAGGAAAG

TTGGGTGCGACTAAGGGGTATGCGGTGCCCACATGCCACTGGGTCCTCAGCAGTACCACCTGATTGGAAGTGATATT

GGTGGAAGACAACATTTTGCCGCTTGGAATTCTCACCTCCACACCAAAGAAGTACTGTTTTTGTCATTTGCACCAGC

TTCTTTCTCCAGGAAAGATCAAAACGATGCACTGCAAGTGTGATTGGTCCAGATAGCTGCCTTATCCAACTGCCTCC

TTTGGACCACTTCATCATGGGACAGCTTGATGCAATCTACTTGACAAGACCCTGGAACCCCACACCCCTCATGGAAC

CAGTGTCCACCTCCCAGTCACAGTGTGACCCCAGGGAACTCTTGCCTGCTTGCTTTAAACCCACCACTTAAAAGTCT

CCACAGAAAACCTGTTTGAATAGTACCCTTGACCCAATAAGGGCATTGGCCCCTGGGTGTCTCTCTCTCTCTCTCTC

TCTCTCTCTCCCTCTCTCTCCCTCTCTCTCTCTCGCTCCCTGACCTCTGCGTGTGGACTCCAGGCATGCCATATACC

CCTCAGGACCTGAAGCAAAAGAATGAATTAAAGAAAACTCCAGAGAATGAGCTGACAACCTACTCCCTGGTCAGTGG

CTCTCCATGCCTACCTGTGGTCTCTTGGACACCCTTGCAGAAGATGACTTCCTGGGAACTCTTCTGGGAGTGAATGT

TATCAGCAAATTCCCAAAACAACACCCCTTAGACAAAAAGCCTCCTCACCTATGCATTGCCTAGACCACCCACTAAA

GATGGTGACCATGGAGGAAGACTTGTGTTGGGCCCAATGGCCCTCGGCCAGTGTCCGTCTGCCAGGTGCCAGCCCCC

GGTGCGCTGAAGCTCAGGCCATTCCTGATGCCCTGGCCTCCTGCACTGAGCTGTGTGAGCTCTTCTGCCCAGGCAGG

CCCCCTGGCATTGACCGGCATAGACAGTGAGCCTGGAGGAAGACTTGCCCTGGTCCCACTGTCCCTGGGCCAGTGTC

CATCAGCCAGGTGCCCAGACCCCAGTGCGCTGAAGCTCAGCCCTCCCTGGCACTCTGGTCTCCTGCACTGAGCTGTG TGAGCTCTTCCGCCCATGAGGGCCCCCTGGCCTTGAGCAGCATAGATGGTGACCCTGAAGGAGGACTTGTGTTGGGC

CCAATGGCCTGGGGCCAGTGTCTGTCAGCCAGGTGCCCAGCCCCTGGCGTGCTGAAGTTTGGGCCCTTCCTGGTGCC

CTGTTCTATCGGGGGAACCCGCCCCCAATAATTCAACGTCCCTCCGTTTGGGGTCCCTGACTTCCCTCAACACTGGT

CTCTTCCACTGAGCCTTGTGAGCTCTTCCACCAAGGAGGGCCCCCTGGCATTGACTGGCATAGTGAGCTCTTCCCCC

CAGCAGACCCCTGGGATTGACGAGCATAGATGGTGACTGTGGAAGAAGACTTGCTTTGGGACCGCTGGCCCCAGGCC

AGTGGTTGTCATCCAGGTGCCCAGCCCTTGGTGCACTTGAGCTCAAGCCCTTCCTGGTGCCCTGGCCTCCTGCCCTA

AGCTGTGCGAGCTCTTCTGCCCAGGGGGACCCCTGGCCTTAAGTGGGATAGATGGTGACCCCAGAGGAAGACGTGCA

TTGGGTCCGCTGGCCCCAGGCCAGTGTCTCTCAGCCATGTGCTCAACACCTGGTGAGCTGAAGCTCAGGCCCTTCCT

GGCATCCTGGTCTCCTGCACTGAGCTGTGGTCTGCAGCGGCATCTGGCATCCAGGCCAGCCTCCTTGGTGGGCCGTG

AACTGAATGGTGGCGCTGGCCTGTGCCCAGTATATCTGGGTTCGGTGGCTGAGAGAGCTTTTGTTGCCCTGTGTCCG

TGACGGGCCAGTGTTCTGCATGCATCCCATCACATGGATCCATGGAGGATGTTGAGCCCAGAGGAAGACTTGCACTG

GGCCCAATGGCCCCAGGTCAGCGTCCATCAGCCAGGTGTCCAGCCCCTGGTGCACTGAAGATCGGGCCCTTCCTGGT

GCCCTGGTCTCCTGCATTGAGCTGTGTGAGCTCTTCCACATGGAAGACCCCTGTCATTGACTGGCATAGGGTTCAGT

GGCGGAGAGAGCATCAGGTGGCCCACGTCTGTGATGGGCCAGTATTCCACATGGATCTCATCACATGGGTCCATGGA

AGGCGAGCATCCCTGAGCTGGCACACCCCACCTTCTGCCAGCATGGAAGGATGGTGAGCACAGAGGAAGAATTGCGT

TAGGCCCTTTGGCCCCGGACCAGTGTCTGCCAGCCACGTGCTCAGCCTCCCGTGCGCTAAAGCTCAGGTCCTTCCTG

TCATCCTGGTATCCTGCACTGAGGTGTGTGAGCTCTTCCCCCCAGCAGGCCCCCTGGGATTGACTGGCAAAGATGGT

GTCTGTGGAGGAAGACTTGCCATTGGGTTGCTGGCCCCAGGCCAGTGGTTGTCATCCAGGTGCCCAGCCTTGGTGCA

CTGAAGCTCAGGCCCTTCCTGGTGCTCTGGTCTCCTGCACTGAGCTGTGTGACCTCTTTTGCCCGGCAGACCCCCTG

GCATTGACCAGCATAGATAGTGAGACTGGAAGAAAACTTTCATTGGGCCCGCTGTCCCGGGGTGAGTGGTCATCAGT

CAGGTGCCCAGCCCTTGGTGTGGTGAAGCTCAGGCTCTTCCTGGCGCCCTGGTCTCCTGCACGGAGCTGCGTGAGCT

CTTCTACCCAGGCGGGCCCCTGCCCTTGAGCGGCATAGATGGTGACCCCAGAGGAAGACTTGCGTTGGGCCCACTGG

CTCTGGGCTCAGCCCCTGGTGAGCTGAAGCTCAGGCCCTTCCTGGCACCCTGGTCTGCTGCACTGAGCTGCGTGAGC

TCTTCCACCCAGGTGCAACCCCCTGGCATTGACCAGCATAGGTATTGAGCCTGGAGGAAGACTTGAATTGGTCCCAA

TGGTCCCAGGCCAGCATCCATCAGCTAGGTGCTGAGCCCCTGGTGCACTGAAGATTGGGCACTTCCTGGCACCCTGG

TGTGCTGCACCGAGCTGTGATGAGCACATCCGGGTCCTGCTGGATGCATGCGCCGGGAAGGACGTGCCCTGAGTTGG

GTCGATGATGAGAACCTTATATTATCCTGAAGAGAGGTGATGACTTAAAAATCATGCTCAATAGGATTACGCTGAGG

CCCAGTCTAGTGAGCTCTTCCCCACAGGTGGGCCCCCTGGCATTGACTAGCATCGTGAGCTCTTCTGCCCAGCAGCC

TCCCTGGCAGTGACCAGCATAGATGCTGACCCTGGAGGAAGAATTGCACTGGGCCCACTGGCCCCGGGAAAATGTCC

CTCAGCCAGGTGCTTAGCCCCTGGTAATCTGAAGCTCAGGCCCTTCCTGATGCCCTGGTCTCCTGCACTGAGCTGTG

TGAGCTCTTCAGCCCAGGTGGGCTCCCTGACACTGATGCGCATAGATGGTGAGCCTGGAGGAAGACTTGCATTGGGC

CCAATGGACCCAGGCCAGTGGCCATCAGCCAGGTGCCCACCCTGGTGTGCTGAAGCGCGGGACCTACCTGTTGTCCT

GGTTTCCTACACTGAGCTGAGTGAGCTCTTCAACCCAGGCGGGGCACCCTGGCATTCACCAGCATAGACTGTGAAAT

GGGAGGAAGAATTGTGTTGGGCCCAATGGCCCTGGGCCAGTATCCGTCAGCCTGGTGTCCAGCCCCTGGTGTGCTGA

AGCTCATGCCCTTCCTGGTGCCCTAGTTTCCTGCACTGAGCTTGTGAGCTCTTTTGCCCAGAAGACTCCCTGGCATT

GATCGGCATAGATGGCAGCGCTGGAGGAGGACTTGTACTGGGGCCGCTGGCCCCACATCAGTGTCCGTCATCCCGGC

GTCCAGCCCCTGGTGTGCTGAAGCTCAGGCCCTTCCTTGTGCCCTAGTCTCCTGCACTGAGCTTGTGAGCTCTTCTG

CTCAGGCGGGTCCCCTGGCATTGACCAGCATAGATGGTGGCCCTGGAGGAGGACTTGTATTGGGGCCACTGGCCCCA CGTCAGTGTTCGTCAGCCTGGCATCCAGCCCCTGGTGTGCTGAAGCTCAGGCCCTTCCTGTTGCCCTAGTCTCCTGC

ACTGAGCTTGTGAGCTCTTCTGCCCAGGTGGTCCCCCTGGTCTTGATGGGCATAGGGTGAGGCCCTGTTTGGACCCA

TCTTCCAGCTATCTCACCAGATGGTGAGCCCAGAGGAAGACCCCGTGGCTCTGGGCCAGTGTGCGTCAGCCAGGTCC

CCAGCCCCCAGTGTGCTGAAGCTCAGGCCCTTCCCAACGCCCTGGTCTCCTGCACTGAGTGAGCTCTTCCGCCCATT

TGGGCCCTCTGACATTGACCGGCATAGATGGTGAGCTCGGAGGAAGACTTGCATTGGGCCCAATGGAGCTGGGCCAG

TTTCTGTCAGCCAGGTCCCCAGCTCCTGATGCACTGAAGCTCAGGCCCTTCCTGGAGCCCTGGTCTCCTGCCCTGAG

CTGTGATGGTGAGCCTGAAGGAAGACTTGCATCAGGACCAATGGACCCTGGCAAGTGTCCGTCAGCCAGGTGCTCAG

CCCCTGGTGCACTGAAGCTCAGGCCCTTCCTGGCCCCTTGATCTCCTGCACTGAGCTGTGTGAGCTCTTCTGCCAAG

GGGGGGCCTCCTGGCATTGACCAACATAGATGGTGAGCCTGGAGGAAGATTTGCATTGGGCCCAATGGCCCTGGGCC

AGTGTCTGTCAGCCAGTTTCCCAGCCCCTGGTGCACTGAAGCTCAGGCCTTTCCTGGCATCCTGGCCTCCTGCACTG

AGCTGTGTGAGTGCTTCTGCCCAGGCAGGCCACTTGGCATTGACCGGTATAGATGGTGAGCCTGGAGGAAGATGTGT

GGGACCCGCAGGCCGAGGGCCAGTGTCTCTCAGCCAGGTGCTCAGCCCCCAGTGGGCTGAAACTCTGACCCTTCCTG

GCACCCTGCTCTCCTGCACTGTGCTGTGATGGTGAGACTTCTGTTGGGCTTGCTTTTCCTGGGCCAGTGTCCATCAG

CCAGGTGCTCAGCCATCGGTGCGCTGAAGCTCCAGCCCTTCCTGGTGCCCTGATCTCCTGCACTGAGCTGTGATGAT

GAGCTCTTTCACCTAGGAGGGCCCCTTGGCATTGACTGGCATAGATGGTGACCCTGAAGAAAGACTTCAGTTGGGCC

TGAAGGCCCCGGGCCAGTGTCCATCATCCAGGTGCTCAGCTCCCAGTGCACTGAAGCTCAGACCCTTCCTGGTTCCC

TGGTCTCTTGCACTGAGCTCTGATGGTGAGACTTGAGGAAGACTTCCACTGGGCCTGCTTTTCCTGAGCCAGTGCCC

ATCACCCAGGTGCTCAGTCCCTGGTGCACTGAAGCTCCAGCCCTTCCTGGCGCCCTGGTCTCCTGAACTGAGCTGTG

TGAGCTCTTCTGCCAAGCGGTTCCCCTGGCATTGACCAACATAGATGGTGAGCCTGGAGGAAGACTTGCGTTGGGCC

CAAAGGCCCTAGACCAGTGTCTGTCAGCCAGGTTCCCAACCCCCGATGCGCTGAAGCTCAAGCCTTTCCTGGCACCC

TGGTCTCCTGCACTGAGCTGTGTGAGCTCTTCTGCCCAGGCGGGCCCCCTGGAATTGATGGGTATAGATGGTGACCC

CGGAGGAAAATTTGTGTTGGGCCCACTATCCCTGGGCCAATGTCAGTCAGCTAGGCACCCAGCCCCCAGTGCGCTGA

AGCTCAGGACCTTCCTAGTGCCCTCGGCTCCTGCACTGAGCTCTGATGGTAAGCCTGGAGGAAGACTTGTGTTGGGC

CCACAGGCCCCAGGCCAATGTCTGTCAGCCAGGTGCTCAGCCTCTGGTGCGCTGAAACTCTGACCCTTCCTGGCACA

CTGGTCTCCTGCACTGAGCTGGGATTTTCCACCTAGGAGGTCCACCTGGCATTGACTGACATAGATGGTGAGACTTC

TGTCGGCCTCGCTTTTCCTGGGCCAGTGTCCATCAGCCAGGTGCTCAGCCCCAGTGCACTGAAGCTCCAGCCCTTCC

TGGCGCCCTGGACTGAGCTGTGTGAGCTCTTCTGCCAAGGGGGTCCTCCTGGCATTGACCAACATAGGTGGTGAGCC

TGGAGGAAGACTTGCATTGGGCCCAATGGCCCTGGACCAGTGTCTGTCAGCCAGGTTCCCAGCCCCCGATGCGCTGA

AGCTCAAGCCTTTCCTGGCACCCTGGTCTCCTGCACTGAGCTGTGTGAGCTCTTCGGCCCAAGTGGTCCCCCCGGCC

TTAAGCGGCATAGATGGTGAGCCTGGAGGAAGACTCGTGTTGGGCCCACAGGCCCCAGGCCAGTGTACGTCAGCCAG

GTGCTCAGCCCATGGTGTGCTGAAGCTCTGACCCTTCCTGACTCCGTGGTCTCCTGCACTGAGCTGTGATGACCATA

TCCAGTGAGCTCTTCCAAACAGGTGGGCTCCCTAGCATTGACCGACAGAGTGAGCTCTGCCACCCAGGACATCCCCC

TGGCATTGACTGGCATAGATGCTGACCCTGGAGGAAGACTTGTGTCGGGCCCCCAGGCCCCAGGCCAGTGTCCATCA

GCCAGGTGCTCAGCTCCCAGTGCGCTGAAGCTCAGACCCTTCCTGGCACCCTGGTGTCCAGCACTGAGCTGTGTGAG

CTTTTCCACCCAGGTGGGCCCCCTGGCATTGACCGACATAGATGATGAGACTGGAGGAAGACTTCCATTGGGCCCAC

TTTTCCTGGGCCAGTGTCCATCAGCCAGGTGCTCAGCCCCTGGTGTACTGAAGCTCCAGCCTTTCCTGGTGCCCCAG

TCTTCTGCACTAAGCTGTGTGAGCTCTTCCACCCAGGAGGGCCCCTTGGCATTGACTGGCATAGATGGTGAGACGGG

AGGAAGACTTGTGTTGGGCCTGCTGTTCCTGGGCCAGTGGCCATCAGCCTGTTGCCCAGCCCCTGAGGCGCTGAAGC TCAGGCCCTTCCTCACACCCTGGTCTCCTGCACTAGCTGTGTGAGCTCTTCTGCCCAGGTGGGGCCCATGGCCTTAC

ACGACATAGGAGCACGCATTTCTTGAAGTCCTGCAAAGGTGATGGTGGTCCAGACAGGAGAGGGTTCCTTCAGCCCA

ACATAGCGCAGCTCCCAGCTGAAACTCGGAAGGCTGGTCAGGCCTCGAGGACTGCCCAGGCCACATGAGGGTTTGGT GGCCGGGAGGGCCAATGATGCTCTGCGTCTGTGTTGGATCAGTGTTCTTCATGGATTCCATCACATGGTGAGCCTTG AGGAAGACTTGTATTGGGCCCCCACAGGCCCCAGGCCAGTGTCCGTCAGCCTGGTGCTCACCCCCAGTGTGCTGAAG CTCAGACCCTCCGTGGCACCCTGGTCTCCTGCACTGAGCTGTGTGAACTTTTCCTCCCAGATGGGCCCCCTGGCATT GACTGGCATAGATGGTGAGACCGGAGGAAGACTTGCATTGGGCCTGTTGTTCCTGGGCCAGTGGCCATCAGCCTGTT GCCCAGCCCCTGATGCGCTGAAGCTCGGGCCCTTCCTCGCGCCCTGGTCTCCTGCACTGAGCTGTGTGAGCTCTTCT GCCCAGGTGAGGCCCATGGCCTTAAGCGGCATAGATGGTGAGCCTGGAGGAAGACTTGTATTGGGACCCTACAGGCC CCAGGCCAGTGTCTGTCAGCCTGGTGCTCATCCCCCAGTGCGCTGAAGCTCAGACCCTCCCTGGCACCCTGGTCTCC TGCACTGAGCTGTGATGGTGAGTCTGGAGGAAGACTTGTGTTGGACCCCCACAGGCCCCAGGCCAGTGTCCATCAGC CTGGTGCTAATCCCCCAGTGCTCTGGAGTTTGGACCCTCCCTGGCACCCTGGTCTCCTGCACTGAGCTTGAGCTCTT CCACCCGGGCGGGCCCCCGGCATTGACTGGCATAGGTGGTGAGCCCGGAGGAGGACTTGCGTTCGGCCCGCTGGTCC CAGTGTGTCCGGAATTGGTGGGTTCTTGGTCTCACTGACTTCAAGAATGAAGCTGCGGACCCTCGCTATGATGATAT GGAAGAAAAGCACTCTTTGGCCTGTTGTGACTGGGACAGTTGACAGCACCCAGGTGTCCTTTAATGAAAATGCTCTT GACACCAATGCATCCTAGCATCACAGCTTCAGGAAGCCTTCTCAAGTGTGCATGGGGAGTACTATGTCTTTCATCAA TAATGAAATCTTCTGATTTGTAAGACATGCTGCCAAGAGATGTGCCATTCTATTATAAAAGATCAGTAGCTTCCTTT ACCGACGTGTATATTCTATCTAGAACATTGAGCTATGGAAGACTCCCACCTAAGGGAATTAGTTTTACACCTTCAGA TAAAGACTGCTGAGAAGAGCACCCTCTGGTGTTGTCACAGAGGCAAGTGCTACCGCACAGGCATGCTGCAGTGAATT TAACTGATCCTCTGTCCCTGCAACCGTTGTTTAAGGATGCTATTCTGAAAAGACTGTGGAGGAAGAAAACCCTTTAC CCTGTTGTTCAGGGAGAAACTGACACCACTCAACTGCCTGGCACTGAAAATGTGGCATCCAGTCCACTTTACCATCA GTGTTTAAGGAAACCATCTCTGATAAGGATGACTGAGGAAGAGTACTCTTTGGCTTGTTGACACCAGCACAGCTGAC ACACCCAGATATCTGTTTGGTCTCCTGTGAACTTTCAACCAGGATTTAAGGATGCCACTCTGGTTAAAAGCTGAAAC AACTTCAAGGAAACTTCAGGGAAAAGAGAAGGCCTGGAATCTGATCCTCCACTTCAGAGAACAGGGAGAAAAAAGTG CATGTGAAATACTACTACTTATCCATCTCCTGGCTCTGAGCAGTACATGTCAGGAACAAGAGGGAATTGTAGCTGGA AGACAGTAGAAGAGCCATAGGACAAGTCTCTAAGAGAAGGAAGAAAAGGCGCAATGAAAGAAAACAAGTTTAAGATA GTAGAAAAGCATTCAAAGACGGCAACCTGAGGTGGTGGAGTCTATGGCCCTTCTCCTTGAATCTGAGTGAAATTAGT GAGTCTTTCAATCCAAAGACTACAGTCATACCTCAACTATCTACAGAGGACTGGTTCCAGGACTCCCATGGATCCCA AAATCCAAGGATGCTTCGAATCCCTTATATAAAATGGCATGGTATTTGCATATAACCTACATGCATCTTCAAATATG TTAAACCATCTCTAGATTACTTATAATACCAAATACAATGTAATGCTATGTAAACAGTTGTTACAATGTATTGTCAA AGACAAGCTCTCCTTGCCAATCCCTGACCAAATGAGAGATTCATGAGCAAAATAAATAGTTGCTGATATTTAAGGCA CTAAGTTTGGGGCAATGTATTACACAGCAATAAATAACTGGAACAGCATAGTAAATTCTAGTTAAAAATACTAAACT ACTAAGTTTTAGTTAACATACATGCTCAAGTTAAGGGTGTGGTATACTAACAACTGCAACTTACTTTGAAAAGCATA AAAAAAGACACTAGAAGAAGACAGGATGGATAGATGGACAAGATAGAAGACAATAACAAATAGAACAAAATGTTGTG AAATTATATTCTACAATTATTTTAAAGTCTGTGTGGTTTGAAAAATTGTACGATAAAATGAAGAGTGAAATAAAGAC ATTTCCATACAAA (SEQ ID NO:36).

Typically, the described molecules include crRNAs that selectively target and edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3 A- ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO:35. UBE3A-ATS is the distal portion of the long non-coding RNA of SNHG14 (set forth in NCBI Reference Sequence: NR_146177.1).

2. Engineered Single Guide RNA (sgRNA) specific for AS Genes

The single guide RNA is a specific RNA sequence that recognizes the target DNA region of interest and directs the Cas nuclease there for editing. The gRNA is made up of two parts: CRISPR RNA (crRNA), a 17-20 nucleotide spacer sequence complementary to the target DNA and a conserved repeat fragment (“handle” or “tag”) region that pairs with the tracr RNA, and a tracr RNA, which serves as a binding scaffold for the Cas nuclease. The crRNA component imparts specificity of CRISPR-directed nuclease activity and is the customizable component that directs specific editing. sgRNA is an abbreviation for “single guide RNA.” sgRNA is a single RNA molecule that contains both the custom-designed short crRNA sequence fused to the scaffold tracrRNA sequence. sgRNA is synthetically generated or made in vitro or in vivo from a DNA template.

While crRNAs and tracrRNAs exist as two separate RNA molecules in nature, sgRNAs include both a crRNA component and a scaffold component fused as a single molecule. i. crRNA/Protospacer Sequences

Engineered crRNA molecules including sequences targeting a human UBE3A-AES gene are provided.

In a first form, the crRNA “protospacer” targeting the UBE3A-AAS gene includes the 20 nucleotide sequence: CAGCUCAGUGCAGGAGACCA (“hsgRNA-5”; SEQ ID NO:1). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 1 targets the UBE3A-AES, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof.

In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 1. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 1). For example in some forms, the variant sequence has at least about 70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO:1. Typically, the variant crRNA has a nucleic acid sequence that has one or more amino acids different to SEQ ID NO: 1, such as one or more substitutions, deletions or additions at any one of the nucleotide positions of SEQ ID NO:1, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A- T , for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO:36.

In a second form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20- nucleotide sequence: GGACCACCGUCACCCCUGCA (“hsgRNA-16”; SEQ ID NO:2). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:2 targets the UBE3A-AAS. having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof.

In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:2. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:2. but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015. 10, for example, to repress the expression of a human UBE3A-AAS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO:36.

In a third form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20- nucleotide sequence: GAGCCUGGGCUGCCUCACGG (“hsgRNA-7”; SEQ ID NO:3).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:3 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:3. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:3, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a fourth form, the crRNA “protospacer” targeting the UBE3A- ATS includes the 20- nucleotide sequence: GAGCUGUGGUGAGCACAUCC (“hsgRNA-4”; SEQ ID NO:4).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:4 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:4. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:4, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-AAS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a fifth form, the crRNA “protospacer” targeting the UBE3A-AAS includes the 20- nucleotide sequence: AGAGCUCACUGAAAGACACA (“hsgRNA-6”; SEQ ID NO:5).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:5 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:5. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:5, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-AAS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a sixth form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20- nucleotide sequence: UGCUCACCACAGCUCAGUGC (“hsgRNA-8”; SEQ ID NO:6).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:6 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:6. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:6, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A- TS. for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a seventh form, the crRNA “protospacer” targeting the UBE3A- ATS includes the 20- nucleotide sequence: GAGCCUGGGCUGCCUCACAG (“hsgRNA-9”; SEQ ID NO:7). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:7 targets the UBE3A- TS. having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:7. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:7, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A- ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In an eighth form, the crRNA “protospacer” targeting the UBE3A- TS includes the 20- nucleotide sequence: UCUCAUCAUCGACCCAACCC (“hsgRNA-10”; SEQ ID NO:8).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:8 targets the UBE3A- ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:8. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:8, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO:36.

In a ninth form, the crRNA “protospacer” targeting the UBE3 A- ATS includes the 20- nucleotide sequence: AUUACGCUGAGGCCCAACCU (“hsgRNA-11”; SEQ ID NO:9). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO:9 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:9. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:9, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36. In a tenth form, the crRNA “protospacer” targeting the UBE3A-AAS includes the 20- nucleotide sequence: UGUGUGGGAGGUGUUGUGUG (“hsgRNA-12”; SEQ ID NO: 10). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 10 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 10. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 10, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In an eleventh form, the crRNA “protospacer” targeting the UBE3A- Al'S includes the 20-nucleotide sequence: UAGGUGAGUGGAUCCUGCUG (“hsgRNA-13”; SEQ ID NO: 11). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 11 targets the UBE3A-A1S, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 11. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 11, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a twelfth form, the crRNA “protospacer” targeting the UBE3A-A1S includes the 20- nucleotide sequence: ACAGCUCAGUGCAGGAGACC (“hsgRNA-14”; SEQ ID NO: 12). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 12 targets the UBE3A- AIS. having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 12. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 12, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A- ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a thirteenth form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20- nucleotide sequence: GGCUCACCACAGCUCAGUGC (“hsgRNA-15”; SEQ ID NO: 13). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 13 targets the UBE3A- ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO:13. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 13, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a fourteenth form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20-nucleotide sequence: GGAGACCUGGAGGCCCUGAA (“hsgRNA-17”; SEQ ID NO:14). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 14 targets the UBE3A- ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 14. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 14, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a fifteenth form, the crRNA “protospacer” targeting the UBE3A-ATS includes the 20- nucleotide sequence: CUCAUCAUCGACCCAACCCA (“hsgRNA-18”; SEQ ID NO:15). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 15 targets the UBE3A-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 15. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 15, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015. 10, for example, to repress the expression of a human C/BE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a sixteenth form, the crRNA “protospacer” targeting the UBE3A- ATS includes the 20- nucleotide sequence: AGCUCACUGAAAGACACAAG (“hsgRNA-19”; SEQ ID NO: 16).

In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 16 targets the I/BEJA-ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 16. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO:16, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human CBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36.

In a seventeenth form, the crRNA “protospacer” targeting the UBE3A-AVS includes the 20-nucleotide sequence: GCAGCCCAGGCUCCCUGUGU (“hsgRNA-20”; SEQ ID NO: 17). In some forms, the disclosed crRNA “protospacer” of SEQ ID NO: 17 targets the UBE3A- ATS, having a nucleic acid sequence set forth in NCBI Reference Sequence: NC_000015.10, or a homologue, paralogue, or ortholog thereof. In some forms, the engineered sgRNA includes a sequence having one, two, three, four, or five residues that are substituted, deleted, or added relative to the 20 nucleotide sequence of SEQ ID NO: 17. In some forms, the engineered sgRNA includes a variant having at least 75%, up to 99% identity to SEQ ID NO: 17, but retains the ability to specifically edit the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 36. ii. Traer Sequences

In some forms, the nucleic acid sequence of a Tracr/scaffold sequence of an sgRNA is or includes:

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCA CCGAGUCGGUGCU (SEQ ID NO:18). In the complete sgRNA, the guide sequence immediately precedes the first nucleotide of the tracr sequence. In some forms, the different regions of an sgRNA scaffold sequence are defined by the secondary structural elements formed within the sequence of scaffold RNA. For example, in some forms, the sgRNA scaffold sequence includes 77 nucleic acid residues, whereby nucleotides in positions 13-16 represent the “tetraloop” region; nucleotides in positions 31-43 represent the “nexus” region; 18 nucleotides in positions 44-61 represent the “stem-loop 2” region; and nucleotides in positions 62-77 represent the “stem-loop 3” region.

As described herein, the sgRNA scaffold stem-loop 2 region includes a hairpin region, as well as flanking regions. iii. Exemplary sgRNAs

Exemplary sgRNA molecules, including the described crRNA sequences fused to tracr sequences are provided.

An exemplary sgRNA molecule including the described hsgRNA-5crRNA of SEQ ID NO:1 includes a nucleic acid sequence of: ACCAGAGGACGUGACUCGACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO: 19).

An exemplary sgRNA molecule including the described hsgRNA-16crRNA of SEQ ID NO: 2 includes a nucleic acid sequence of: ACGUCCCCACUGCCACCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:20).

An exemplary sgRNA molecule including the described hsgRNA-7 crRNA of SEQ ID NO: 3 includes a nucleic acid sequence of: GGCACUCCGUCGGGUCCGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:21).

An exemplary sgRNA molecule including the described hsgRNA 4 crRNA of SEQ ID NON includes a nucleic acid sequence of: GAGCUGUGGUGAGCACAUCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:22).

An exemplary sgRNA molecule including the described hsgRNA 6crRNA of SEQ ID NO:5 includes a nucleic acid sequence of: AGAGCUCACUGAAAGACACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:23).

An exemplary sgRNA molecule including the described hsgRNA 8crRNA of SEQ ID NO:6 includes a nucleic acid sequence of:

UGCUCACCACAGCUCAGUGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:24).

An exemplary sgRNA molecule including the described hsgRNA 9crRNA of SEQ ID NO:7 includes a nucleic acid sequence of:

GAGCCUGGGCUGCCUCACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:25).

An exemplary sgRNA molecule including the described hsgRNA lOcrRNA of SEQ ID NO: 8 includes a nucleic acid sequence of:

UCUCAUCAUCGACCCAACCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:26).

An exemplary sgRNA molecule including the described hsgRNA 1 IcrRNA of SEQ ID NO:9 includes a nucleic acid sequence of:

AUUACGCUGAGGCCCAACCUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:27).

An exemplary sgRNA molecule including the described hsgRNA 12crRNA of SEQ ID NO: 10 includes a nucleic acid sequence of:

UGUGUGGGAGGUGUUGUGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:28).

An exemplary sgRNA molecule including the described hsgRNA 13crRNA of SEQ ID NO:11 includes a nucleic acid sequence of:

UAGGUGAGUGGAUCCUGCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:29).

An exemplary sgRNA molecule including the described hsgRNA 14crRNA of SEQ ID NO: 12 includes a nucleic acid sequence of:

ACAGCUCAGUGCAGGAGACCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU

AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NQ:30). An exemplary sgRNA molecule including the described hsgRNA 15crRNA of SEQ ID NO: 13 includes a nucleic acid sequence of: GGCUCACCACAGCUCAGUGCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:31).

An exemplary sgRNA molecule including the described hsgRNA 17crRNA of SEQ ID NO: 14 includes a nucleic acid sequence of: GGAGACCUGGAGGCCCUGAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:32).

An exemplary sgRNA molecule including the described hsgRNA 18crRNA of SEQ ID NO: 15 includes a nucleic acid sequence of: CUCAUCAUCGACCCAACCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:33).

An exemplary sgRNA molecule including the described hsgRNA 19crRNA of SEQ ID NO: 16 includes a nucleic acid sequence of: AGCUCACUGAAAGACACAAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:34).

An exemplary sgRNA molecule including the described hsgRNA 20crRNA of SEQ ID NO: 17 includes a nucleic acid sequence of: GCAGCCCAGGCUCCCUGUGUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:35).

B. Cas Enzymes

Systems including Cas enzymes are provided. Cas enzymes are RNA-guided DNA endonuclease enzymes. The CRISPR-associated Cas nuclease protein is a non-specific endonuclease. It is directed to the specific DNA locus by a gRNA, where it makes a doublestrand break. There are several versions of Cas nucleases isolated from different bacteria. The most commonly used one is the Cas9 nuclease from Streptococcus pyogenes (SpCas9).

As used herein, the term “Cas” generally refers to an effector protein of a CRISPR Cas system or complex. The term “Cas” may be used interchangeably with the terms “CRISPR” protein, “CRISPR Cas protein,” “CRISPR effector,” CRISPR Cas effector,” “CRISPR enzyme,” “CRISPR Cas enzyme” and the like, unless otherwise apparent. The Crispr-Cas effector protein may be without limitation a type II, type V, or type VI Cas effector protein. Non- limiting examples of Crispr-Cas effector proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some forms, the CRISPR enzyme has DNA cleavage activity.

1. Cas9

In some forms, the compositions include a Type II CRISPR enzyme that is a Cas9 enzyme. The signature Cas9 effector proteins are large multi-domain RNA-dependent endonucleases that locate, bind, and cleave the double- stranded DNA (dsDNA) targets which are complementary to their guide RNAs. For recognition and binding to target DNA, Cas9 requires the protospacer adjacent motif (PAM), as a short, conserved sequence located just downstream of the non-complementary strand of the target dsDNA. Recognition of the PAM (5'NGG3') triggers dsDNA melting, enabling crRNA strand invasion and base pairing. The dsDNA cleavage mediation happens via the activity of separate HNH and RuvC nuclease domains. Also, Cas9 is a member of a small subset of Cas effectors that need a second trans-acting crRNA (tracrRNA) for gRNA processing and DNA cleavage.

Exemplary Cas9 enzymes are disclosed in International Patent Application Publication No. WO/2014/093595. In some forms, the Cas9 enzyme is .S’, pneumoniae, S. pyogenes or 5. thermophilus Cas9, and may include mutated Cas9 derived from these organisms. The enzyme may be a Cas9 homolog or ortholog. Additional orthologs include, for example, Cas9 enzymes from Corynebacter diptheriae, Eubacterium ventriosum, Streptococcus pasteurianus, Lactobacillus farciminis, Sphaeroachaeta globus, Azospirillum B510, Gluconacetobacter diazotrophicus, Neisseria cinereal, Roseburia intestinalis, Parvibaculum lavamentivorans, Staphylococcus aureus, Nitratifractor salsuginis DSM 16511, Camplyobacter lari CF89 12, and Streptococcus thermophilus LMD 9.

In some forms, the Cas9 effector protein and orthologs thereof may be modified for enhanced function. For example, improved target specificity of a CRISPR Cas9 system may be accomplished by approaches that include, but are not limited to, designing and preparing guide RNAs having optimal activity, selecting Cas9 enzymes of a specific length, truncating the Cas9 enzyme making it smaller in length than the corresponding wild-type Cas9 enzyme by truncating the nucleic acid molecules coding therefor and generating chimeric Cas9 enzymes wherein different parts of the enzyme are swapped or exchanged between different orthologs to arrive at chimeric enzymes having tailored specificity.

A Cas9 enzyme may include one or more mutations and may be used as a generic DNA binding protein with or without fusion to or being operably linked to a functional domain. The mutations may be artificially introduced mutations and may include but are not limited to one or more mutations in a catalytic domain. Examples of catalytic domains with reference to a Cas9 enzyme may include but are not limited to RuvC I, RuvC II, RuvC III and HNH domains. Preferred examples of suitable mutations are the catalytic residue(s) in the N term RuvC I domain of Cas9 or the catalytic residue(s) in the internal HNH domain.

Generally, the Cas9 is (or is derived from) the Streptococcus pyogenes Cas9 (SpCas9). In such forms, preferred mutations are at any or all of positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 or corresponding positions in other Cas9 orthologs with reference to the position numbering of SpCas9 (which may be ascertained for instance by standard sequence comparison tools, e.g. ClustalW or MegAlign by Lasergene 10 suite). In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. The same mutations (or conservative substitutions of these mutations) at corresponding positions with reference to the position numbering of SpCas9 in other Cas9 orthologs are also preferred. Particularly preferred are DIO and H840 in SpCas9. However, in other Cas9s, residues corresponding to SpCas9 DIO and H840 are also preferred. These are advantageous as when singly mutated they provide nickase activity and when both mutations are present the Cas9 is converted into a catalytically null mutant which is useful for generic DNA binding.

In some forms, chimeric Cas9 proteins are used. Chimeric Cas9 proteins are proteins that include fragments that originate from different Cas9 orthologs. For instance, the N terminal of a first Cas9 ortholog may be fused with the C terminal of a second Cas9 ortholog to generate a resultant Cas9 chimeric protein. These chimeric Cas9 proteins may have a higher specificity or a higher efficiency than the original specificity or efficiency of either of the individual Cas9 enzymes from which the chimeric protein was generated. These chimeric proteins may also include one or more mutations or may be linked to one or more functional domains. Also suitable are Cas9 proteins that have different PAM specificities. Typically, Cas9 proteins, such as Cas9 from S. pyogenes (spCas9), require a canonical NGG PAM sequence to bind a particular nucleic acid region.

Cas9 nuclease sequences and structures are known to those of skill in the art (Ferretti, et al. Proc Natl Acad Sci U.S.A, 98, 4658-4863, doi: 10.1073/pnas.071559398 (2001); Deltcheva, et al. Nature, 471, 602-607, doi: 10.1038/nature09886 (2011)). Cas9 orthologs have been described in several species of bacteria, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus, Campylobacter jejuni and Neisseria meningitidis. (Slaymaker, et al. Science, 351, 84-88 doi: 10.1126/science.aad5227 (2016); Kleinstiver, et al. Nature, 529, 490- 495, doi: 10.1038/nature 16526 (2016); Chen, et al. Nature, 550, 407-410, doi: 10.1038/nature24268 (2017); Casini, et al. Nat Biotechnol, 6, 265-271, doi: 10.1038/nbt.4066 (2018); Lee, et al. Nat Commun,9, 3048, doi: 10.1038/s41467-018-05477-x (2018); Vakulskas, et al. Nat Med, 24, 1216-1224, doi: 1.1038/s41591-018-0137-0 (2018); Choi, et al. Nat Methods, 16, 722-730, doi: 10.1038/S41592-019-0473-0 (2019); Kim, et al. Nat Commun, 8, 14500, doi: 10.1038/ncommsl4500 (2017); (Edraki, et al. Mol Cell, 73, 714-726, doi: (2019)).

C. Ribonucleoprotein Complexes

Enhanced ribonucleoprotein complexes including a Cas enzyme and one of the described engineered sgRNAs are also provided.

Typically, the enhanced ribonucleoprotein complexes have high specificity and activity of on-target editing activity of CRISPR/Cas. In some forms, an enhanced ribonucleoprotein complex includes:

(i) a Cas enzyme; and

(ii) an engineered sgRNA with enhanced specificity and activity of on-target editing activity of CRISPR/Cas for one or more genes associated with AS.

Typically, the Cas9 enzyme is derived from S. pyogenes (spCas9).

In some forms, the ribonucleoprotein complex includes an engineered sgRNA including a crRNA targeting a nucleic acid sequence in the human genome in the region of chromosome 15ql 1— 13. Therefore, in some forms, the ribonucleoprotein complex with enhanced specificity and activity of on-target editing activity of CRISPR/Cas for one or more genes associated with AS includes a Cas9 enzyme and include a crRNA having a sequence of nucleic acids of any one of SEQ ID NOs:l-17, or an sgRNA having a sequence of nucleic acids of any one of SEQ ID NOs: 19-35. In some forms, the ribonucleoprotein complex includes an engineered sgRNA including a nucleic acid sequence of: ACCAGAGGACGUGACUCGACGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO: 19), together with a Cas enzyme, such as a Cas9 enzyme, such as a spCas9 enzyme.

In some forms, the ribonucleoprotein complex includes an engineered sgRNA including a nucleic acid sequence of: An exemplary sgRNA molecule including the described hsgRNA- 16crRNA of SEQ ID NO:2 includes a nucleic acid sequence of: ACGUCCCCACUGCCACCAGGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:20), together with a Cas enzyme, such as a Cas9 enzyme, such as a spCas9 enzyme.

An exemplary sgRNA molecule including the described hsgRNA-7crRNA of SEQ ID NO: 3 includes a nucleic acid sequence of: GGCACUCCGUCGGGUCCGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:21), together with a Cas enzyme, such as a Cas9 enzyme, such as a spCas9 enzyme.

An exemplary sgRNA molecule including the described hsgRNA-4crRNA of SEQ ID NO: 4 includes a nucleic acid sequence of: GAGCUGUGGUGAGCACATCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUU AUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU (SEQ ID NO:22), together with a Cas enzyme, such as a Cas9 enzyme, such as a spCas9 enzyme.

D. Vectors

In some forms, nucleic acids encoding the described engineered crRNAs, or sgRNAs thereof are present within vectors. In some forms, the vectors encode or express the described engineered crRNAs, or sgRNAs thereof. In some forms, a vector encodes or expresses the described engineered crRNAs, or sgRNAs thereof set forth by any one or more of SEQ ID NOS:1-17, or 19-22, or fragments or variants thereof.

Vectors including an isolated polynucleotide encoding a nucleic acid as set forth in SEQ ID NOS:1 -17, or 19-22, or fragments and variants thereof for the expression of the described engineered crRNAs, or sgRNAs thereof within a host cell are described. The term “vector” is a nucleic acid molecule used to carry genetic material into another cell, where it can be replicated and/or expressed. Any vector known to those skilled in the art in view of the present disclosure can be used. Examples of vectors include, but are not limited to, plasmids, viral vectors (bacteriophage, animal viruses, and plant viruses), cosmids, and artificial chromosomes (e.g., YACs). A vector can be a DNA vector or an RNA vector. In some embodiments, a vector is a DNA plasmid. One of ordinary skill in the art can construct a vector of the application through standard recombinant techniques in view of the present disclosure.

In some forms, the vector including nucleic acids encoding the described engineered crRNAs, or sgRNAs thereof is an expression vector. The term “expression vector” refers to any type of genetic construct including a nucleic acid coding for an RNA capable of being transcribed. Expression vectors include, but are not limited to, vectors for recombinant protein expression, such as a DNA plasmid or a viral vector, and vectors for delivery of nucleic acid into a subject for expression in a tissue of the subject, such as a DNA plasmid or a viral vector. It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc.

In some embodiments, vectors contain one or more regulatory sequences. The term “regulatory sequence” refers to any sequence that allows, contributes or modulates the functional regulation of the nucleic acid molecule, including replication, duplication, transcription, splicing, translation, stability and/or transport of the nucleic acid or one of its derivative (i.e. mRNA) into the host cell or organism. In the context of the disclosure, this term encompasses promoters, enhancers and other expression control elements (e.g., polyadenylation signals and elements that affect mRNA stability).

In some embodiments, the vector is a non-viral vector. Examples of non-viral vectors include, but are not limited to, DNA plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, bacteriophages, etc. Examples of non-viral vectors include, but are not limited to, RNA replicon, mRNA replicon, modified mRNA replicon or self-amplifying mRNA, closed linear deoxyribonucleic acid, e.g., a linear covalently closed DNA, e.g., a linear covalently closed double stranded DNA molecule. Preferably, a non-viral vector is a DNA plasmid. A “DNA plasmid”, which is used interchangeably with “DNA plasmid vector,” “plasmid DNA” or “plasmid DNA vector,” refers to a double- stranded and generally circular DNA sequence that is capable of autonomous replication in a suitable host cell. DNA plasmids used for expression of an encoded polynucleotide typically include an origin of replication, a multiple cloning site, and a selectable marker, which for example, can be an antibiotic resistance gene.

In some forms the vector is a viral vector. In general, viral vectors are genetically engineered viruses carrying modified viral DNA or RNA that has been rendered non-infectious, but still contains viral promoters and transgenes, thus allowing for translation of the transgene through a viral promoter. Because viral vectors are frequently lacking infectious sequences, they require helper viruses or packaging lines for large-scale transfection. Examples of viral vectors that can be used include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, pox virus vectors, enteric virus vectors, Venezuelan Equine Encephalitis virus vectors, Semliki Forest Virus vectors, Tobacco Mosaic Virus vectors, lentiviral vectors, arenavirus viral vectors, replication-deficient arenavirus viral vectors or replication-competent arenavirus viral vectors, bi-segmented or tri-segmented arenavirus, infectious arenavirus viral vectors, nucleic acids which include an arenavirus genomic segment wherein one open reading frame of the genomic segment is deleted or functionally inactivated (and replaced by a nucleic acid encoding a PC1-CTT polypeptide or another therapeutic polypeptide as described herein), arenavirus such as lymphocytic chori omeningitidis virus (LCMV), e.g., clone 13 strain or MP strain, and arenavirus such as Junin virus e.g., Candid #1 strain, etc.

In some embodiments, the viral vector is an adenovirus vector, e.g., a recombinant adenovirus vector. A recombinant adenovirus vector can for instance be derived from a human adenovirus (HAdV, or AdHu), or a simian adenovirus such as chimpanzee or gorilla adenovirus (ChAd, AdCh, or S AdV) or rhesus adenovirus (rhAd). Preferably, an adenovirus vector is a recombinant human adenovirus vector, for instance a recombinant human adenovirus serotype 26, or any one of recombinant human adenovirus serotype 5, 4, 35, 7, 48, etc. In other embodiments, an adenovirus vector is a rhAd vector, e.g. rhAd51, rhAd52 or rhAd53. In some forms, a recombinant viral vector is prepared using methods known in the art in view of the present disclosure. In some forms, the described engineered crRNAs, or sgRNAs is codon- optimized to ensure proper expression in the host cell (e.g., bacterial or mammalian cells). Codon-optimization is a technology widely applied in the art, and methods for obtaining codon- optimized polynucleotides will be well known to those skilled in the art in view of the present disclosure.

In some embodiments, the vectors, e.g., a DNA plasmid or a viral vector (particularly an adenoviral vector), include any regulatory elements to establish conventional function(s) of the vector, including but not limited to replication and expression of the described engineered crRNAs, or sgRNAs.

E. Molecular Deliver Vehicles

Any of the disclosed compositions including, but not limited the described engineered crRNAs, or sgRNAs and/or nucleic acid vectors the described engineered crRNAs, or sgRNAs, can be delivered to target cells using a delivery vehicle.

The delivery vehicles can be, for example, polymeric particles, inorganic particles, silica particles, liposomes, micelles, multilamellar vesicles, etc.

Delivery vehicles may be microparticles or nanoparticles. Nanoparticles are often utilized for inter-tissue application, penetration of cells, and certain routes of administration. The nanoparticles may have any desired size for the intended use. The nanoparticles may have any diameter from 10 nm up to about 1,000 nm. The nanoparticle can have a diameter from 10 nm to 900 nm, from 10 nm to 800 nm, from 10 nm to 700 nm, from 10 nm to 600 nm, from 10 nm to 500 nm, from 20 nm from 500 nm, from 30 nm to 500 nm, from 40 nm to 500 nm, from 50 nm to 500 nm, from 50 nm to 400 nm, from 50 nm to 350 nm, from 50 nm to 300 nm, or from 50 nm to 200 nm. In some embodiments the nanoparticles can have a diameter less than 400 nm, less than 300 nm, or less than 200 nm. The range can be between 50 nm and 300 nm.

Thus, in some embodiments, the delivery vehicles are nanoscale compositions, for example, 10 nm up to, but not including, about 1 micron. However, it will be appreciated that in some embodiments, and for some uses, the particles can be smaller, or larger (e.g., microparticles, etc.). Although many of the compositions disclosed herein are referred to as nanoparticle or nanocarrier compositions, it will be appreciated that in some embodiments and for some uses the carrier can be somewhat larger than nanoparticles. Such compositions can be referred to as microparticulate compositions. For example, a nanocarriers according to the present disclosure may be a microparticle. Microparticles can a diameter between, for example, 0.1 and 100 pm in size.

1. Viral Capsid Proteins

In some forms, the delivery vehicle is a viral capsid, or a virus-like particle formed from partly or entirely of a multiplicity of viral capsid proteins. Generally, virus capsids are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH. Exemplary viral-like particles that are stable toward thermal denaturation at temperatures up to 80-100°C, chaotropic agents, and to extremes of pH include bacteriophage capsids and phage particles.

In some forms, the delivery vehicle includes a viral-like particle (VLP), or vesicle, composed of a bacteriophage capsid protein.

The stability of a virus-like particle (VLP) is an important consideration for its use in nanobiotechnology. In some forms, the icosahedral capsid of a bacteriophage is cross-linked by disulfide bonds between coat protein dimers at its 5-fold and quasi-6-fold symmetry axes, providing enhanced stability to VLPs formed from capsid proteins. In some forms, the capsid is a modified capsid, for example, modified by attachment of a peptide, carbohydrate, small molecule or nucleic acid to the viral capsid.

2. Polymeric Particles

In some forms, the delivery vehicle is or includes one or more polymers, such as polymeric nanoparticles or microparticles. Exemplary polymers include biocompatible polymers. In some forms, the biocompatible polymer(s) is biodegradable or bioabsorbable. In other forms, the polymer is non-degradable. In some forms, the particles are a mixture of degradable and non- degradable particles.

In some forms, the delivery vehicle is a particles that includes one or more biocompatible polymer(s) including, but not limited to, polyamino acids; cyclodextrin-containing polymers, in particular cationic cyclodextrin-containing polymers, such as those described in U.S. Patent No. 6,509,323; polymers prepared from lactones such as poly (caprolactone) (PCL); polyhydroxy acids and copolymers thereof such as poly (lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co- glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), and blends thereof, polyalkyl cyanoacralate, polyurethanes, poly(valeric acid), and poly-L-glutamic acid; hydroxypropyl methacrylate (HPMA); poly anhydrides; other polyesters; poly orthoesters; poly(ester amides); polyamides; poly(ester ethers); polycarbonates; polyalkylenes such as polyethylene and polypropylene; polyalkylene glycols such as poly(ethylene glycol) (PEG) and polyalkylene oxides (PEO), and block copolymers thereof such as polyoxyalkylene oxide (“PLURONICS®” or block copolymers containing PEG where PEG has a molecular weight of any values within the range of 300 Daltons to 1 MDa); polyalkylene terephthalates such as poly (ethylene terephthalate); ethylene vinyl acetate polymer (EVA); polyvinyl alcohols (PVA); polyvinyl ethers; polyvinyl esters such as poly(vinyl acetate); polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone; poly siloxanes; polystyrene (PS); and celluloses including alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, and carboxymethylcellulose; polymers of acrylic acids including poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly (hexyl(meth)acry late), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as "polyacrylic acids"); polydioxanone and its copolymers; polyhydroxyalkanoates; polypropylene fumarate; polyoxymethylene; poloxamers; poly(butyric acid); trimethylene carbonate; and polyphosphazenes .

Examples of preferred natural polymers include proteins such as albumin, collagen, gelatin and prolamines, for example, zein, and polysaccharides such as alginate. Copolymers of the above, such as random, block, or graft copolymers, or blends of the polymers listed above can also be used.

Functional groups on the polymer can be capped to alter the properties of the polymer and/or modify (e.g., decrease or increase) the reactivity of the functional group. For example, the carboxyl termini of carboxylic acid contain polymers, such as lactide- and glycolide-containing polymers, may optionally be capped, e.g., by esterification, and the hydroxyl termini may optionally be capped, e.g. by etherification or esterification.

The weight average molecular weight can vary for a given polymer but is generally from about 1000 Daltons to 1,000,000 Daltons, 1000 Daltons to 500,000 Dalton, 1000 Daltons to 250,000 Daltons, 1000 Daltons to 100,000 Daltons, 5,000 Daltons to 100,000 Daltons, 5,000 Daltons to 75,000 Daltons, 5,000 Daltons to 50,000 Daltons, or 5,000 Daltons to 25,000 Daltons.

In some forms, the delivery vehicles are particles modified with one or more surfactants. Examples of surfactants include, but are not limited to, L-a-phosphatidylcholine (PC), 1 ,2- dipalmitoylphosphatidy choline (DPPC), oleic acid, sorbitan trioleate, sorbitan mono-oleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, natural lecithin, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, block copolymers of oxyethylene and oxypropylene, synthetic lecithin, diethylene glycol dioleate, tetrahydrofurfuryl oleate, ethyl oleate, isopropyl myristate, glyceryl monooleate, glyceryl monostearate, glyceryl monoricinoleate, cetyl alcohol, stearyl alcohol, polyethylene glycol 400, cetyl pyridinium chloride, benzalkonium chloride, olive oil, glyceryl monolaurate, corn oil, cotton seed oil, and sunflower seed oil, lecithin, oleic acid, and sorbitan trioleate.

In some forms where polyalkylene glycol (e.g. , PEG) is used in a composition of polymers to modify the particles, PEG surface density may be controlled by varying the amount of PEG in the polymer composition or by mixing a blend of pegylated polymer component and non-pegylated polymer component. The density of PEG or polyalkylene glycol on the surface of formed particles may be evaluated using several techniques.

In some forms, the delivery vehicles are modified by the addition of one or more polymers to possess a specific ^-potential. For example, in some forms, the delivery vehicles are modified by the attachment of PEG and/or other polymers to the surface to possess a ^-potential of between about 20 mV and about -20 mV, preferably between about 10 mV and about -10 mV, more preferably between about 2 mV and about -2 mV.

3. Liposomes, Micelles and Lipidic Particles

In some forms, the delivery vehicle is a lipidic particle, such as liposomes, or micelles. Lipidic particles include unilamellar phospholipid vesicles, liposomes, or lipoprotein particles. Liposomal encapsulation may be used and the liposomes may be derivatized with various polymers (e.g., U.S. Pat. No. 5,013,556). See also Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C. T. Rhodes, Chapter 10, 1979.

Formulations of liposomes and methods of making such formulations are well known to one of ordinary skill in the art. Liposomes are formed from commercially available phospholipids supplied by a variety of vendors including Avanti Polar Lipids, Inc. (Birmingham, Ala.).

Suitable methods, materials and lipids for making liposomes are known in the art. Liposome delivery vehicles are commercially available from multiple sources. The liposome may be formed from a single lipid; however, in some embodiments, the liposome is formed from a combination of more than one lipid. The lipids can be neutral, anionic or cationic at physiologic pH. In some forms, the liposomes incorporate PEG, or PEGylated lipid derivatives. Incorporation of one or more PEGylated lipid derivatives can result in a liposome which displays polyethylene glycol chains on its surface. The resulting liposomes may possess increased stability and circulation time in vivo as compared to liposomes lacking PEG chains on their surfaces. Liposomes are formed from one or more lipids, which can be neutral, anionic, or cationic at physiologic pH. Suitable neutral and anionic lipids include, but are not limited to, sterols and lipids such as cholesterol, phospholipids, lysolipids, lysophospholipids, sphingolipids or pegylated lipids. Neutral and anionic lipids include, but are not limited to, phosphatidylcholine (PC) (such as egg PC, soy PC), including, but limited to, 1 ,2-diacyl- glycero-3-phosphocholines; phosphatidylserine (PS), phosphatidylglycerol, phosphatidylinositol (PI); glycolipids; sphingophospholipids such as sphingomyelin and sphingoglycolipids (also known as 1-ceramidyl glucosides) such as ceramide galactopyranoside, gangliosides and cerebrosides; fatty acids, sterols, containing a carboxylic acid group for example, cholesterol; 1 ,2-diacyl-sn-glycero-3-phosphoethanolamine, including, but not limited to, 1 ,2- dioleylphosphoethanolamine (DOPE), 1 ,2-dihexadecylphosphoethanolamine (DHPE), 1 ,2- distearoylphosphatidylcholine (DSPC), 1 ,2-dipalmitoyl phosphatidylcholine (DPPC), and 1 ,2- dimyristoylphosphatidylcholine (DMPC). The lipids can also include various natural (e.g., tissue derived L-a-phosphatidyl: egg yolk, heart, brain, liver, soybean) and/or synthetic (e.g., saturated and unsaturated 1 ,2-diacyl-.s7?-glycero-3-phosphocholines, l -acyl-2-acyl-.s/?-glycero-3- phosphocholines, l,2-diheptanoyl-SN-glycero-3-phosphocholine) derivatives of the lipids. In some forms, the liposomes contain a phosphaditylcholine (PC) head group, and preferably sphingomyelin. In another form, the liposomes contain DPPC. In a further form, the liposomes contain a neutral lipid, preferably 1 ,2-dioleoylphosphatidylcholine (DOPC).

In certain forms, the liposomes are generated from a single type of phospholipid. In such forms, preferably the phospholipid has a phosphaditylcholine head group, and, most preferably is sphingomyelin. The liposomes may include a sphingomyelin metabolite. Sphingomyelin metabolites used to formulate the liposomes include, without limitation, ceramide, sphingosine, or sphingosine 1 -phosphate. The concentration of the sphingomyelin metabolites included in the lipids used to formulate the liposomes can range from about 0.1 mol % to about 10 mol %. Preferably from about 2.0 mol % to about 5.0 mol %, and more preferably can be in a concentration of about 1.0 mol %.

Suitable cationic lipids in the liposomes include, but are not limited to, N-[l-(2,3- dioleoyloxy)propyl]-N,N,N-trimethyl ammonium salts, also references as TAP lipids, for example methylsulfate salt. Suitable TAP lipids include, but are not limited to, DOTAP (dioleoyl-), DMTAP (dimyristoyl-), DPTAP (dipalmitoyl-), and DSTAP (distearoyl-). Suitable cationic lipids in the liposomes include, but are not limited to, dimethyldioctadecyl ammonium bromide (DDAB), 1 ,2-diacyloxy-3-trimethylammonium propanes, N-[l-(2,3- dioloyloxy)propyl]-N,N-dimethyl amine (DODAP), 1 ,2-diacyloxy-3-dimethylammonium propanes, N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2- dialkyloxy-3 -dimethylammonium propanes, dioctadecylamidoglycylspermine (DOGS), 3 -[N- (N',N'-dimethylamino-ethane)carbamoyl]cholesterol (DC-Chol); 2,3-dioleoyloxy-N-(2- (sperminecarboxamido)-ethyl)-N,N-dimethyl-l-propanaminium trifluoro- acetate (DOSPA), P- alanyl cholesterol, cetyl trimethyl ammonium bromide (CTAB), diCi4-amidine, N-ferf-butyl-N'- tetradecyl- 3 -tetradecyl amino-propionamidine, N-(alpha-trimethylammonioacetyl)didodecyl-D- glutamate chloride (TMAG), ditetradecanoyl-N-(trimethylammonio-acetyl)diethanolamine chloride, 1 ,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), and N , N , N' , N'- tetramethyl- , N'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-l ,4-butanediammonium iodide. In one form, the cationic lipids can be l-[2-(acyloxy)ethyl]2-alkyl(alkenyl)-3-(2-hydroxyethyl)- imidazolinium chloride derivatives, for example, l-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)- heptadecenyl-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), and l-[2- (hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazolinium chloride (DPTIM). In one embodiment, the cationic lipids can be 2,3-dialkyloxypropyl quaternary ammonium compound derivatives containing a hydroxyalkyl moiety on the quaternary amine, for example, 1 ,2-dioleoyl-3-dimethyl-hydroxyethyl ammonium bromide (DORI), 1 ,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), 1 ,2-dioleyloxypropyl-3-dimetyl- hydroxypropyl ammonium bromide (DORIE-HP), 1 ,2-dioleyl-oxy-propyl-3-dimethyl- hydroxybutyl ammonium bromide (DORIE-HB), 1 ,2-dioleyloxypropyl-3 -dimethylhydroxypentyl ammonium bromide (DORIE- Hpe), 1 ,2-dimyristyloxypropyl-3 -dimethylhydroxylethyl ammonium bromide (DMRIE), 1 ,2-dipalmityloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DPRIE), and 1 ,2-disteryloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DSRIE).

The lipids may be formed from a combination of more than one lipid, for example, a charged lipid may be combined with a lipid that is non-ionic or uncharged at physiological pH. Non-ionic lipids include, but are not limited to, cholesterol and DOPE (1,2-dioleolylgly ceryl phosphatidylethanolamine), with cholesterol being most preferred. The molar ratio of a first phospholipid, such as sphingomyelin, to second lipid can range from about 5 : 1 to about 1 : 1 or 3:1 to about 1:1, more preferably from about 1.5:1 to about 1:1, and most preferably, the molar ratio is about 1:1. 4. Intracellular Transport Systems

Compositions configured for the intracellular transportation of therapeutic sgRNA agents are provided.

It may be that direct delivery of CRISPR/Cas9 system as a ribonucleoprotein (RNP) complex, e.g., composed of a Cas9 protein and one of the described engineered single guide RNA (sgRNA) molecules to the brain and/or central nervous system (CNS) in vivo is impeded by the blood-brain barrier (BBB). The BBB is a dense protective biological barrier that limits passage of large molecules and thereby can represent a hurdle to treatment of the brain/CNS with systemically delivered reagents. While adeno-associated viruses (AAVs) navigate some of these hurdles, their immunogenic nature, and the risk of off-target effects from persistent Cas9, along with the potential for genomic integration and cancer development, restrict their clinical application in vivo.

Contemporary developments in nanoparticle-based delivery is promising; however, the considerable size (40-200 pm) restricts its systemic application. Fortunately, stimuli-responsive cargo delivery system is emerging, which are able to control the biodistribution of therapeutic payloads in response to endogenous stimuli, to initiate the cleavage of the stimuli-responsive element, such as changes in pH, enzyme concentration or redox gradients. Meanwhile, chemical structures containing self-immolative chemical moieties that can be sued to generate suitable linkers.

Therefore, in some forms, compositions for delivery of the described engineered sgRNAs to target cells in the brain and/or CNS include a chemical linker and a cell membrane fusogenic/penetrating molecule. In some forms, the compositions are designed to exhibit one or more functions in one or more specific environments.

An exemplary composition configured for intracellular transportation of therapeutic sgRNA agents as described herein is a “Stimuli-Responsive Traceless Engineering Platform”, or “STEP” composition. An exemplary STEP composition designed for the intracellular transportation of therapeutic agents includes a linker that incorporates a stimuli-responsive chemical group, a self- immolative spacer, or both. In some forms, whereby the liker includes both a stimuli-responsive chemical group and a self-immolative spacer, the compositions are designed to release the therapeutic payload, such as an engineered sgRNA, directly into a target cell in a controlled and traceless manner. In an exemplary form, upon cellular entry the STEP encounters the intracellular microenvironment, and initiates a cleavage reaction, followed by self-immolation to release the therapeutic payload, such as an engineered sgRNA, directly into the target cell, ensuring precise delivery without residual effects.

In some forms, the a STEP composition is configured to initiate cellular internalization via one or more cellular pathways. In some forms, a STEP composition is configured for cellular internalization via the endocytosis pathway. The endocytosis pathway has been studies in the art (Dougherty, Sahni, and Pei 2019; Sousa de Almeida et al. 2021; Rennick, Johnston, and Parton 2021; Zhang, Gao, and Bao 2015).

In some forms, after cellular internalization of a STEP composition, for example, via the cellular endocytosis pathway, the linker of a STEP composition is configured to be cleaved to release the therapeutic payload, such as an sgRNA, within a target cell.

In some forms, release of the therapeutic payload is induced in response an intracellular stimulus such as a high concentration of glutathione (GSH), and/or a low pH. High levels of intracellular concentrations of GSH, and/or a low pH are known in the art, for example, as described in Montero et al. 2013; Pei and Buyanova 2019; Zhou et al. 2017. In some forms, release of a therapeutic payload including Cas9 enzyme induces an increase in intracellular positive charge. It has been shown that positive charge can cause problems in vivo, due to rapid clearance by the reticuloendothelial system (Zhang, Gao, et al. 2021). Therefore, in some forms, the STEP system is modified with biocompatible poly(ethylene glycol) (PEG) units (Vlassi, Papagiannopoulos, and Pispas 2017), to shield the positive charges of Cas9 protein and increase the complex stability in vivo (Schubert and Chanana 2018).

In some forms, the STEP system delivers a Ribonucleoprotein Complex. After released by STEP, the positive charged Cas9 leads the RNP bypass following lysosomal pathway through endocytosis escaping, and enter the nucleus following the guidance of nucleus localization signal (NLS) on the Cas9 protein (Figl.c-g). i. Linkers

In some forms, a STEP composition includes a linker. Exemplary linkers include one or more functional moieties, such as a stimuli-responsive chemical group, and/or a self-immolative group. a. Stimuli-responsive Chemical Group

In some forms, a STEP composition includes a linker including a stimuli-responsive chemical group. Exemplary stimuli-responsive chemical groups that can be included within a linker of a STEP composition include disulfide bond containing materials. Disulfide bond- containing materials are responsive to glutathione (GSH). Since the intracellular GSH concentration is much higher than the extracellular, the use of disulfide bond containing materials in STEP for intracellular delivery of biomacromolecules efficiently releases the bound cargoes after cell internalization. Therefore, in some forms, once delivered into cytosol, the disulfide linkage in the STEP is cleaved by GSH, degrading the STEP into segments, and releasing loaded payload (e.g., sgRNA and/or RNP molecules). After released by STEP, the positive charged Cas9 leads the payload to bypass following lysosomal pathway through endocytosis escaping, and to enter the nucleus following the guidance of nucleus localization signal (NLS) on the Cas9 protein. b. Sclf-Immolative Chemical Group

In some forms, a STEP composition includes a linker including a self-immolative chemical group. Macromolecules that are designed so as to self-immolate upon a pre-determined cleavage event are highly advantageous, in addition to their use in therapeutic and diagnostic application, in various industrial applications. Self-immolative linkers, or spacers, are covalent groups, which have the role of cleavaging two bonds between a protector group and a drug, in the case of drug delivery systems, after a stimuli. Typically, following a stimuli, a cascade of reactions allows to control the release of an attached sgRNA or ribonucleoprotein complex. In some forms, a linker includes one or more self-immolative groups selected from the group including a photo-labile self-immolative group, a chemically removable self-immolative group, a hydrolizable self-immolative group and a bio-cleavable self-immolative group. In some forms, one or more of the releasable self-immolative group is selected from the group including a detectable agent, a therapeutically active agent, a second self-immolative compound, a chemosensitizing agent, a targeting moiety, an agrochemical, a chemical moiety and a chemical reagent. Exemplary self-immolative chemical groups that can be included within a linker of a STEP composition include any self-immolative chemical groups that are known in the art. Self- immolative chemical groups are reviewed in Gavriel, et al., Polym. Chem., 2022, 13, 3188-3269, the content of which is incorporated herein in its entirety.

F. Pharmaceutical Formulations

Pharmaceutical formulations including the disclosed compositions, e.g., one of the disclosed crRNA “protospacers” targeting UBE3A-ATS of SEQ ID NO:1, or 2, or 3, or an sgRNA including these crRNAs, such as an sgRNA of SEQ ID NO: 19, or 20, or 21 or a ribonucleoprotein complex thereof, including a Cas9 enzyme. In some forms, the pharmaceutical compositions include one or more of a pharmaceutically acceptable buffer, carrier, diluent or excipients. The term “pharmaceutically acceptable carrier” describes a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, in some forms the carrier is a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in contact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

In some forms, pharmaceutical compositions include buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The pharmaceutical compositions can be formulated for delivery via any route of administration. The term “Route of administration” can refer to any administration pathway known in the art, including but not limited to aerosol, nasal, oral, intravenous, intramuscular, intraperitoneal, inhalation, transmucosal, transdermal, parenteral, implantable pump, continuous infusion, topical application, capsules and/or injections. The pharmaceutical compositions are preferably formulated for intravenous administration.

Typically, the disclosed pharmaceutical compositions are administered in a manner appropriate to a disease to be treated (or prevented). The quantity and frequency of administration is typically determined by such factors as the condition of the patient, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

The disclosed pharmaceutical compositions can be delivered in a therapeutically effective amount. The precise therapeutically effective amount is that amount of the composition that will yield the most effective results in terms of efficacy of treatment in a given subject. This amount will vary depending upon a variety of factors, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through routine experimentation, for instance, by monitoring a subject's response to administration of a compound and adjusting the dosage accordingly. For additional guidance, see Remington: The Science and Practice of Pharmacy (Gennaro ed. 20th edition, Williams & Wilkins PA, USA) (2000).

III. Methods of Use

Methods for using the described compositions for enhanced gene editing are described.

The described variant sgRNAs and ribonucleoprotein complexes thereof can be used for any suitable purpose and in any suitable method for CRISPR-based editing of DNA.

Generally, the disclosed compositions of sgRNAs can be used to cleave genomic DNA within the human genome in the region set forth in NCBI Reference Sequence: NC_000015.10, for example, to repress the expression of a human UBE3A-ATS, for example, an RNA having a nucleic acid sequence set forth in SEQ ID NO: 8 in vivo with high on-target and very low off- target activity.

Such cleavage is preferably used in a method of editing the target DNA to repress the expression of a human UBE3A-ATS in a subject having Angelman syndrome (AS), or who is at increased risk of developing one or more of the symptoms of AS. For example, in some forms, the disclosed sgRNAs can be used for and within any known methods of DNA editing, including in vitro and in vivo DNA editing for altering the genome of a cell.

The described sgRNAs that selectively repress the expression of a human UBE3A-ATS, can be used for various therapeutic methods, for example, for the treatment and/or prevention of one or more symptoms of AS in a subject in need thereof.

Various methods for selectively altering the genome of a cell using RNA-guided endonucleases are described in the following exemplary U.S. Patent documents: U.S. Patent Nos. 8,993,233, 9,023,649, and 8,697,359 and U.S. Patent Application Publication Nos. 20140186958, 20160024529, 20160024524, 20160024523, 20160024510, 20160017366, 20160017301, 20150376652, 20150356239, 20150315576, 20150291965, 20150252358, 20150247150, 20150232883, 20150232882, 20150203872, 20150191744, 20150184139, 20150176064, 20150167000, 20150166969, 20150159175, 20150159174, 20150093473, 20150079681, 20150067922, 20150056629, 20150044772, 20150024500, 20150024499, 20150020223, 20140356867, 20140295557, 20140273235, 20140273226, 20140273037, 20140189896, 20140113376, 20140093941, 20130330778, 20130288251, 20120088676, 20110300538, 20110236530, 20110217739, 20110002889, 20100076057, 20110189776, 20110223638, 20130130248, 20150050699, 20150071899, 20150050699, 20150045546, 20150031134, 20150024500, 20140377868, 20140357530, 20140349400, 20140335620, 20140335063, 20140315985, 20140310830, 20140310828, 20140309487, 20140304853, 20140298547, 20140295556, 20140294773, 20140287938, 20140273234, 20140273232, 20140273231, 20140273230, 20140271987, 20140256046, 20140248702, 20140242702, 20140242700, 20140242699, 20140242664, 20140234972, 20140227787, 20140212869, 20140201857, 20140199767, 20140189896, 20140186958, 20140186919, 20140186843, 20140179770, 20140179006, 20140170753, and 20150071899, each of which is incorporated by reference herein, and in particular for their description of the uses of RNA-guided endonucleases.

Various methods for selectively altering the genome of a cell using RNA-guided endonucleases are described in the following exemplary publications: WO 2014/099744; WO 2014/089290; WO 2014/144592; WO 2014/004288; WO 2014/204578; WO 2014/152432; WO 2015/099850; WO 2008/108989; WO 2010/054108; WO 2012/164565; WO 2013/098244; WO 2013/176772; Makarova et al., “Evolution and classification of the CRISPR-Cas systems” 9(6) Nature Reviews Microbiology 467-477 (1-23) (June 2011); Wiedenheft et al., “RNA-guided genetic silencing systems in bacteria and archaea” 482 Nature 331-338 (Feb. 16, 2012); Gasiunas et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria” 109(39) Proceedings of the National Academy of Sciences USA E2579-E2586 (Sep. 4, 2012); Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity” 337 Science 816-821 (Aug. 17, 2012); Carroll, “A CRISPR Approach to Gene Targeting” 20(9) Molecular Therapy 1658-1660 (September 2012); U.S. Appl. No. 61/652,086, filed May 25, 2012; Al-Attar et al., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs): The Hallmark of an Ingenious Antiviral Defense Mechanism in Prokaryotes, Biol Chem. (2011) vol. 392, Issue 4, pp. 277-289; Hale et al., Essential Features and Rational Design of CRISPR RNAs That Function With the Cas RAMP Module Complex to Cleave RNAs, Molecular Cell, (2012) vol. 45, Issue 3, 292-302.

Methods of CRISPR also provided. A method for CRISPR editing of one or more target genes to selectively repress the expression of a human UBE3A-ATS RNA in a cell, including administering into and/or expressing within the cell a crRNA of any one of SEQ ID NOs 1-3, or the sgRNA of any one of SEQ ID NOs 5-7, or a ribonucleoprotein complex thereof. In some forms, the administering is in vivo. Methods of treatment of neuro-developmental diseases are also provided.

A. Methods of Treatment

Methods of treating diseases and/or disorders in a subject in need thereof are provided. The subject to be treated can have a disease, disorder, or condition such as but not limited to, neurodevelopmental diseases or disorders.

An exemplary method involves treating a subject (e.g., a human) having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including the described engineered crRNAs. In some forms, the methods administer the described engineered crRNAs to a subject (e.g., a human) having a disease, disorder, or condition in an amount effective to treat the disease, disorder, or condition.

In some forms the methods include administering a therapeutic composition including or encoding the described engineered crRNAs to treat one or more disease or disorder in a subject in need thereof. For example, in some forms the methods treat one or more genetic disease or disorders in a subject, such as a hereditary genetic disease or disorder, or a somatic genetic disease or disorder in a subject.

Any of the methods can include treating a subject having an underlying disease or disorder. For example, in some forms, the methods treat a neurodevelopmental disease or disorder, such as Angelman syndrome (AS), in a patient having another disease or disorder, such as diabetes, a bacterial infection (e.g., Tuberculosis), viral infection (e.g., Hepatitis, HIV, HPV infection, etc.), or a drug-associated disease or disorder. In some forms, the methods treat an immunocompromised subject. In some forms, the methods treat a subject having a disease of the kidney, liver, heart, lung, brain, bladder, reproductive system, bowel/intestines, stomach, bones or skin.

The disease, disorder, or condition can be associated with inhibit the expression of a pathological maternal UBE3A gene in the brain and/or CNS. Disclosed are methods of editing human genomic DNA to repress the expression of a human UBE3A-ATS antisense RNA to treat a disease or disorder.

In some forms, the method includes administering to a subject a crRNA having the sequence of SEQ ID NO:1, or 2, or 3, or an sgRNA having the sequence of SEQ ID NO:5, or 6, or 7, optionally together with a Cas9 enzyme or a ribonucleoprotein thereof, in an amount to repress the expression of a UBE3A- AAS RNA within the subject. Typically the subject harbors a maternal UBE3A allele including one or more substitutions or deletions associated with a neurodevelopmental disease, such as Angelman syndrome (AS).

1. Treatment of Angelman syndrome (AS).

Methods of treating a subject having Angelman syndrome (AS) are provided.

AS is a rare genomic imprinting related, neurogenetic disorder, that affects approximately one in 15,000 people and results in severe intellectual deficiency, loss functional speech, gross and fine motor impairment, and/or frequent seizures. In normal, healthy humans, the UBE3A gene is exclusively expressed from the maternal chromosome in neurons in the brain, while the paternal copy of the UBE3A gene is silenced by an antisense RNA, due to a genetic imprinting mechanism. In Angelman syndrome patients, the maternal UBE3A gene is either deleted or functionally impaired, while the paternal UBE3A gene is structurally intact, but silenced by paternally expressed antisense RNA (UBE3A-AE RNA). A schematic depicting the molecular basis for AS is set forth in Figure 1A. A strategy to treat AS, is to inactivate the UBE3 A- AES RNA antisense on the paternal chromosome of a subject with AS, so as to remove the suppression effect, such that the paternal UBE3A can be expressed in the subject, as depicted in Figure IB.

Methods of using the described engineered crRNAs, to treat a disease or disorder are provided. Typically the methods edit the genome to repress the expression of a UBE3A-AAS RNA in the CNS and/or brain of the subject, for example, halting or limiting the suppression of a non-pathological paternal UBE3A gene. Therefore, in some forms, the methods reduce or prevent one or more of the symptoms of AS in a subject in need thereof. For example, in some forms, the methods treat a subject having a disease, disorder, or condition by administering to the subject an effective amount of a pharmaceutical composition including the described engineered crRNAs, such as an sgRNA or a RNP thereof.

In some forms, methods of treating a subject having (AS) include administering to the subject an effective amount of a crRNA having the sequence of SEQ ID NO:1, or 2, or 3, or an sgRNA having the sequence of SEQ ID NO:5, or 6, or 7, optionally together with a Cas9 enzyme or a ribonucleoprotein thereof, in an amount to repress the expression of a UBE3A-AAS RNA within the subject.

As set forth in the Examples below, therapeutic composition including or encoding the described engineered crRNAs substantially inhibit the expression of a pathological maternal UBE3A gene. Therefore, the described therapeutic composition including or encoding the described engineered crRNAs can be applied to a broad range of therapy and can be used in combination with other therapeutic approaches.

Typically, the methods prevent suppression of a non-pathological UBE3A gene in the cells of a subject, and/or reduce or halt the progression of a neurodevelopmental disorder such as AS in the subject. Generally, the methods reduce, ablate or prevent one or more symptoms associated with expression of a pathological UBE3A gene and/or suppression of a non-pathological UBE3A gene in the cells of a subject. Thus, the methods can not only stop and prevent the development of neurodevelopmental diseases and disorders resulting expression of a pathological UBE3A gene, but in some forms the methods may also reverse the pathological effects associated with expression of a pathological UBE3A gene. For example, in some forms, the methods can reverse one or more symptoms associated with neurodevelopmental diseases and disorders, such as AS, in a subject. Tn some forms, when a subject has suffered a neurological deficit associated with a disease or disorder resulting from the expression of a pathological UBE3A gene, the methods can restore some or all of the neurological ability of the subject to a level similar to that on a normal control subject.

It has been shown that Restoring UBE3A function during the embryonic and early postnatal period is more efficacious at treating AS than restoring UBE3A in adulthood. Therefore, in some forms, the methods treat AS in a subject during the embryonic and/or immediate post-natal period. In some forms, the subject is an infant. In some forms, the subject is less than one day old, less than one week old, less than one month old, less than one year old, or less than 2 years old. In some forms, the subject has been diagnosed as having, or as likely to have AS as an infant, or prior to birth. In some forms, the subject is treated in utero. In some forms, the subject is treated prior to adulthood. In other forms, the subject is treated during adulthood.

An exemplary method for treating or preventing Angelman Syndrome (AS) in a subject in need thereof, includes administering to the subject the pharmaceutical composition of a crRNA having the sequence of SEQ ID NO:1, or 2, or 3, or an sgRNA having the sequence of SEQ ID NO:5, or 6, or 7, optionally together with a Cas9 enzyme or a ribonucleoprotein thereof in an amount effective to prevent or reduce expression of a t/BEJA-ATS RNA in the subject. In some forms, the subject is an infant or a child. i. Symptoms to be Treated

In some forms, the methods treat or prevent one or more symptoms of Angelman syndrome (AS) in a subject. Methods for ameliorating, preventing, reducing or limiting the progression of, or reversing one or more symptoms of AS in a subject in need thereof include administering to the subject an effective amount of the described engineered crRNAs. AS is a genetic nervous system disease that affects nerve cells in the brain and spinal cord. AS causes delayed development, problems with speech and balance, difficulty with feeding and/or suckling, mental disability, and seizures. Delays in maturing, called developmental delays, begin between about 6 and 12 months of age. The delays often are the first signs of Angelman syndrome. Seizures may begin between the ages of 2 and 3 years old.

In some forms, the methods treat or prevent one or more neurological or psychological symptoms of Angelman syndrome (AS) in a subject including smiling and laughing often and at inappropriate times undesirable excitability, stiff or jerky movements, tongue thrusting/protruding tongue, uncontrolled movement of the hands (“hand flapping”) and/or raising arms while walking, seizures/fits, gross and/or fine motor-neuron impairment, loss of functional speech and severe intellectual deficiency, for example, an intelligence quotient (IQ) score of around 20. In some forms, the methods treat or prevent one or more physically evident symptoms of Angelman syndrome (AS) in a subject, including microcephaly (a reduced head size) by age 2, strabismus, scoliosis, and/or obesity. In some forms, subjects with Angelman have sleep disorders and /or hyperactivity. Symptoms often start/are noticed during infancy and are lifelong. In some forms, the methods include administering to a subject an effective amount of the described engineered crRNAs to ameliorate, prevent or reverse one or more symptoms of AS in the subject.

B. Effective Amounts

In some forms the methods administer the described the described engineered crRNAs, such as an sgRNA or a RNP thereof in an effective amount. The effective amount or therapeutically effective amount of a pharmaceutical compositions including the described the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same can be a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease or disorder, such as AS, or to otherwise provide a desired pharmacologic and/or physiologic effect, for example, reducing, inhibiting, or reversing one or more of the underlying pathophysiological mechanisms underlying AS a subject in need thereof. In some forms, when administration of the pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same elicits an effective therapeutic response, the amount administered can be expressed as the amount effective to achieve a desired effect in the recipient. For example, in some forms, the amount of the pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same, is effective to reduce the distribution and/or expression of a pathological UBE3A gene product in the brain and/or CNS of the recipient. In some forms, the amount of the pharmaceutical composition including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same is effective to reduce or prevent the expression of a L/SE3A-ATS RNA in the CNS and/or brain of the subject. In some forms, the amount of the pharmaceutical composition including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same is effective to reduce the symptoms of a disease in the recipient, or prevent or reduce a neurodevel opmental deficit or decline, and combinations thereof. In other forms, the amount of the pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same is effective to reduce one or more symptoms or signs of AS in an AS patient. Signs of AS can include biochemical markers, such as levels of a biomarker detectable in the blood of a patient.

The effective amount of the pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same that is required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the disorder being treated, and its mode of administration. Thus, it is not possible to specify an exact amount for every pharmaceutical composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. For example, effective dosages and schedules for administering the pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same can be determined empirically, and making such determinations is within the skill in the art. In some forms, the dosage ranges for the administration of the compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same are those large enough to effect reduction in the expression of a UBE3A- TS RNA in the CNS and/or brain of the subject and/or a symptom thereof, for example. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, and sex of the patient, route of administration, whether other drugs are included in the regimen, and the type, stage, and location of the disease to be treated. The dosage can be adjusted by the individual physician in the event of any counter-indications. It will also be appreciated that the effective dosage of the composition including the described engineered crRNAs used for treatment can increase or decrease over the course of a particular treatment. Changes in dosage can result and become apparent from the results of diagnostic assays.

Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the subject or patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages can vary depending on the relative potency of individual pharmaceutical compositions, and can generally be estimated based on EC50S found to be effective in in vitro and in vivo animal models.

It can generally be stated that a pharmaceutical composition containing the described engineered crRNAs can be administered at a dosage of 10"⁴ mg/kg body weight to 100 mg/kg body weight, inclusive, preferably 0.1 to 10.0 mg/kg body weight, including all integer values within those ranges. In some forms, patients can be treated by infusing a disclosed pharmaceutical composition containing the described engineered crRNAs in the range of about 0.1 to 10.0 or more mg/kg body weight of the subject. As set forth in the Examples, it has been established that dosages of 15, 30, 60 or 120 pg sgRNA in a volume of 3 ml were sufficient to increase or induce expression of the paternal 3UBE gene in organoids. Therefore, corresponding amounts (e.g., 5, 10, 15 or 30 pg sgRNA/ml) are envisioned as effective dosages.

An infusion can be repeated as often and as many times as the patient can tolerate until the desired response is achieved. Compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same can also be administered once or multiple times at these dosages. The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly. In some forms, the unit dosage is in a unit dosage form for intravenous injection. In some forms, the unit dosage is in a unit dosage form for oral administration. In some forms, the unit dosage is in a unit dosage form for inhalation. In some forms, the unit dosage is in a unit dosage form for intra-cranial injection.

Treatment can be continued for an amount of time sufficient to achieve one or more desired therapeutic goals, for example, a reduction of the amount of/the expression of a UBE3A- ATS RNA in the CNS and/or brain of the subject relative to the start of treatment, or complete absence of UBE3A-NAS RNA in the brain and/or CNS in the recipient. Treatment can be continued for a desired period of time, and the progression of treatment can be monitored using any means known for monitoring the presence of UBE3A-AHS RNA in the brain and/or CNS in a patient. In some forms, administration is carried out every day of treatment, or every week, or every fraction of a week. In some forms, treatment regimens are carried out over the course of up to two, three, four or five days, weeks, or months, or for up to 6 months, or for more than 6 months, for example, up to one year, two years, three years, or up to five years.

The efficacy of administration of a particular dose of the pharmaceutical compositions, such as the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same, according to the methods described herein can be determined by evaluating the aspects of the medical history, signs, symptoms, and objective laboratory tests that are known to be useful in evaluating the status of a subject in need for the treatment of AS or other neurodevelopmental diseases and/or conditions. These signs, symptoms, and objective laboratory tests will vary, depending upon the particular disease or condition being treated or prevented, as will be known to any clinician who treats such patients or a researcher conducting experimentation in this field. For example, if, based on a comparison with an appropriate control group and/or knowledge of the normal progression of the disease in the general population or the particular individual: (1) a subject’s physical condition is shown to be improved (e.g., a diseases state has partially or fully regressed), (2) the progression of the disease or condition is shown to be stabilized, or slowed, or reversed, or (3) the need for other medications for treating the disease or condition is lessened or obviated, then a particular treatment regimen will be considered efficacious.

C. Modes of Administration

In some embodiments the methods the methods administer the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same via any route that enables access of the compositions to the site of action in vivo, i.e., to the brain and/or CNS. For example, in some forms, the route of administration is tailored depending upon the form of the composition, to provide the greatest and/or deepest penetration of agents into the brain parenchyma. Circumventing the BBB via cerebral spinal fluid (CSF) microcirculation is one strategy for the delivery of the described peptides into the brain, achieving deep penetration and distribution. For example, in some forms, the methods the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same directly to the CNS/brain via cerebral spinal fluid (CSF) administration. In some forms, when the methods administer the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same via the CSF, the amount of the compositions required to achieve a therapeutic effect is less than that required when using other routes of administration, such as intravenous (i.v.) administration.

In some forms, the methods include administering the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same directly into cerebral spinal fluid (CSF), for example, via lumbar puncture. Therefore, in some forms, the methods include administering the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same via injection into cerebral spinal fluid (CSF) in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF. In some forms, when the methods include administering the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same including a targeting and/or blood/brain barrier penetrating motif, the methods include administering the compositions into the bloodstream, to traverse the BBB, or via injection directly into CSF, e.g., in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF. In some forms, when the methods include administering the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same as nucleic acids, such as vectors encoding or expressing crRNA, the methods include administering the nucleic acids in an expression vector, for example, encapsulated within a nanoparticle. A nanoparticle can be delivered into the bloodstream to traverse the BBB, or via injection directly into CSF in combination with a pharmaceutically acceptable carrier suitable for administration to the CSF.

The compositions described herein can be conveniently formulated into pharmaceutical compositions composed of one or more of the compounds in association with a pharmaceutically acceptable carrier. See, e.g., Remington’s Pharmaceutical Sciences, latest edition, by E.W. Martin Mack Pub. Co., Easton, PA, which discloses typical carriers and conventional methods of preparing pharmaceutical compositions that can be used in conjunction with the preparation of formulations of the therapeutics described herein and which is incorporated by reference herein. These most typically would be standard carriers for administration of compositions to humans. In one aspect, for humans and non -humans, these include solutions such as sterile water, saline, and buffered solutions at physiological pH. Other therapeutics can be administered according to standard procedures used by those skilled in the art.

Another administration route that successfully reaches the brain is the intranasal route. Intranasal (i.n.) administration has recently been explored by researchers because it reaches the brain, bypassing the BBB through the olfactory bulb.

The pharmaceutical compositions the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same can include, but are not limited to, carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the therapeutic(s) of choice.

Pharmaceutical compositions containing the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same, and optionally one or more additional therapeutic agents can be administered to the subject in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, for example, a pharmaceutical composition including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same, can be administered as an intravenous infusion, or directly injected into a specific site, for example, into or surrounding the brain and/or CNS. Moreover, a pharmaceutical composition can be administered to a subject as an ophthalmic solution and/or ointment to the surface of the eye, vaginally, rectally, intranasally, orally, by inhalation, or parenterally, for example, by intradermal, subcutaneous, intramuscular, intraperitoneal, intrarectal, intraarterial, intralymphatic, intravenous, intrathecal and intratracheal routes. In some forms, the compositions are administered directly into a tumor or tissue, e.g., stereo-tactically.

Parenteral administration, if used, is generally characterized by injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. A more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein. Suitable parenteral administration routes include intravascular administration (e.g., intravenous bolus injection, intravenous infusion, intra-arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature); peri- and intratissue injection (e.g., intraocular injection, intra-retinal injection, or sub-retinal injection); subcutaneous injection or deposition including subcutaneous infusion (such as by osmotic pumps); direct application by a catheter or other placement device (e.g., an implant including a porous, non-porous, or gelatinous material).

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions which can also contain buffers, diluents and other suitable additives. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.

Administration of the pharmaceutical compositions containing the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same can be localized (i.e., to a particular region, physiological system, tissue, organ, or cell type) or systemic. In some forms, the described methods administer the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same via a route selected from intravenous (i.v.), intramuscular (i.m.), intracranial (i.c.), intraosseus (i.o.), intranasal (i.n.), intrathecal (i.th.), intraventricular (i.ven.), intraparenchymal (i.par.) and intracerebroventricular (i.c.v.) administration.

D. Combination therapy

In some embodiments the methods administer the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same in combination with other therapeutic agents or treatment modalities. Any of the disclosed pharmaceutical compositions including the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same, can be used alone, or in combination with other therapeutic agents or treatment modalities, for example, conventional medications. As used herein, “combination” or “combined” refer to either concomitant, simultaneous, or sequential administration of the therapeutics.

In some forms, the pharmaceutical compositions and other therapeutic agents are administered separately through the same route of administration. In other forms, the pharmaceutical compositions and other therapeutic agents are administered separately through different routes of administration. The combinations can be administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject; one agent is given orally while the other agent is given by infusion or injection, etc.,), or sequentially (e.g., one agent is given first followed by the second).

Examples of preferred additional therapeutic agents include other conventional therapies known in the art for treating the desired disease, disorder or condition. In some forms, the therapeutic agent is one or more other targeted therapies.

The compositions and methods described herein may be used as a first therapy, second therapy, third therapy, or combination therapy with other types of therapies known in the art, such as chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radio- frequency ablation or the like, in an adjuvant setting or a neoadjuvant setting.

The disclosed pharmaceutical compositions and/or other therapeutic agents, procedures or modalities can be administered during periods of active disease, or during a period of remission or less active disease. The pharmaceutical compositions can be administered before the additional treatment, concurrently with the treatment, post-treatment, or during remission of the disease or disorder. When administered in combination, the disclosed pharmaceutical compositions and the additional therapeutic agents e.g. , second or third agent), or all, can be administered in an amount or dose that is higher, lower or the same than the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain forms, the administered amount or dosage of the disclosed pharmaceutical composition, the additional therapeutic agent (e.g., second or third agent), or all, is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy (e.g., required to achieve the same therapeutic effect).

1. Additional Therapeutic agents

In some embodiments, the methods administer the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same together with one or more additional therapeutic agents to a subject in need thereof.

Treatment strategies for AS can include treatment with common medication used to manage seizures. Therefore, in some forms, the methods include treating a subject with symptoms of AS, or who is at risk of developing symptoms of AS, with the described engineered crRNAs, such as an sgRNA, or a delivery vehicle including the same in addition to one or more agents effective to treat or prevent seizures, such as an anticonvulsant medication. In some forms, additional treatment strategies for AS can include treatment with selective serotonin reuptake inhibitors (SSRIs) such as sertraline and citalopram, antipsychotic medications such as risperidone and quetiapine, cholinesterase inhibitors such as donepezil, rivastigmine, and galantamine, gene therapy, stem cell therapy, neurotrophic factors, occupational therapy, speech and language therapy, or physiotherapy. Exemplary additional therapeutic agents used to manage seizure include Depacon (sodium valproate), Klonopin (clonazepam), lamotrigine, Luminal (phenobarbital), Topamax (topiramate), and Keppra (levetiracetam). In some forms, the methods include performing one or more one or more additional procedures on the subject, such as physical therapy to assist joint mobility and/or movement of the subject.

The description will be further understood by reference to the following numbered paragraphs:

1. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15 q 11 — 13, wherein the crRNA includes a nucleic acid sequence CAGCUCAGUGCAGGAGACCA (SEQ ID NO:1), or a variant thereof having at least 75% identity to SEQ ID NO:1.

2. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence GGACCACCGUCACCCCUGCA (SEQ ID NO:2), or a variant thereof having at least 75% identity to SEQ ID NO:2.

3. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence GAGCCUGGGCUGCCUCACGG (SEQ ID NO:3), or a variant thereof having at least 75% identity to SEQ ID NO:3.

4. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence GAGCUGUGGUGAGCACAUCC (SEQ ID NO:4), or a variant thereof having at least 75% identity to SEQ ID NO:4.

5. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11- 13, wherein the crRNA includes a nucleic acid sequence AGAGCUCACUGAAAGACACA (SEQ ID NO:5), or a variant thereof having at least 75% identity to SEQ ID NO:5. 6. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence UGCUCACCACAGCUCAGUGC (SEQ ID NO:6), or a variant thereof having at least 75% identity to SEQ ID NO:6.

7. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence GAGCCUGGGCUGCCUCACAG (SEQ ID NO:7), or a variant thereof having at least 75% identity to SEQ ID NO:7.

8. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence UCUCAUCAUCGACCCAACCC (SEQ ID NO:8), or a variant thereof having at least 75% identity to SEQ ID NO:8.

9. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence AUUACGCUGAGGCCCAACCU (SEQ ID NO:9), or a variant thereof having at least 75% identity to SEQ ID NO:9.

10. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence UGUGUGGGAGGUGUUGUGUG (SEQ ID NO: 10), or a variant thereof having at least 75% identity to SEQ ID NO: 10.

11. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence UAGGUGAGUGGAUCCUGCUG (SEQ ID NO: 11), or a variant thereof having at least 75% identity to SEQ ID NO: 11.

12. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15 q 11 —

13. wherein the crRNA includes a nucleic acid sequence ACAGCUCAGUGCAGGAGACC (SEQ ID NO: 12), or a variant thereof having at least 75% identity to SEQ ID NO: 12.

13. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence GGCUCACCACAGCUCAGUGC (SEQ ID NO: 13), or a variant thereof having at least 75% identity to SEQ ID NO: 13. 14. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15q 11— 13, wherein the crRNA includes a nucleic acid sequence GGAGACCUGGAGGCCCUGAA (SEQ ID NO: 14), or a variant thereof having at least 75% identity to SEQ ID NO: 14.

15. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence CUCAUCAUCGACCCAACCCA (SEQ ID NO: 15), or a variant thereof having at least 75% identity to SEQ ID NO: 15.

16. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence AGCUCACUGAAAGACACAAG (SEQ ID NO: 16), or a variant thereof having at least 75% identity to SEQ ID NO: 16.

17. An engineered CRISPR RNA (crRNA) molecule that selectively targets putative regulatory regions and genes in or near the human genome in the region of chromosome 15ql 1— 13, wherein the crRNA includes a nucleic acid sequence GCAGCCCAGGCUCCCUGUGU (SEQ ID NO: 17), or a variant thereof having at least 75% identity to SEQ ID NO: 17.

18. An engineered single guide RNA (sgRNA) molecule, including the crRNA of any one of paragraphs 1-17, conjugated with a Traer RNA.

19. The engineered sgRNA of paragraph 18, wherein the Traer RNA includes a nucleic acid sequence set forth in SEQ ID NO: 19.

20. The engineered sgRNA of paragraph 18, wherein the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:20.

21. The engineered sgRNA of paragraph 18, wherein the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:21.

22. The engineered sgRNA of paragraph 18, wherein the sgRNA includes a nucleic acid sequence set forth in SEQ ID NO:22.

23. A ribonucleoprotein complex including:

(a) a Cas enzyme; and

(b) the crRNA of paragraph 18.

24. A ribonucleoprotein complex including:

(a) a Cas enzyme; and

(b) the variant sgRNA of paragraph 19. 25. A ribonucleoprotein complex including:

(a) a Cas enzyme; and

(b) the variant sgRNA of paragraph 20.

26. A ribonucleoprotein complex including:

(a) a Cas enzyme; and

(b) the variant sgRNA of paragraph 21.

27. A ribonucleoprotein complex including:

(a) a Cas enzyme; and

(b) the variant sgRNA of paragraph 22.

28. The ribonucleoprotein complex of any one of paragraphs 23-27, wherein the ribonucleoprotein complex functions to selectively repress the expression of a human UBE3A- ATS RNA in a subject in vivo.

29. The ribonucleoprotein complex of any one of paragraphs 23-28, wherein the Cas enzyme is a Cas9 enzyme.

30. The ribonucleoprotein complex of paragraph 29, wherein the Cas9 enzyme is derived from Streptococcus pyogenes (spCas9).

30. A nucleic acid vector or a transposon encoding or expressing the engineered crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22.

31. The nucleic acid vector of paragraph 30, wherein the vector is a viral vector.

32. The nucleic acid vector of paragraph 31, wherein the viral vector is selected from the group including a lentiviral vector, an Adeno-associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV), or a chimeric vector including a combination of any two or more of a Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV).

33. The nucleic acid vector of paragraph 30, wherein the vector is selected from the group including a plasmid, a cosmid, and a replicon.

34. A molecular delivery vehicle including the engineered crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22 or ribonucleoprotein complex of any one of paragraphs 23-30, or the vector of any one of paragraphs 31-33.

35. The delivery vehicle of paragraph 34, wherein the delivery vehicle includes a nanoparticle, or a microparticle. 36. The delivery vehicle of paragraph 35, wherein the engineered crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22 or ribonucleoprotein complex of any one of paragraphs 23-30, or the vector of any one of paragraphs 31-33 is encapsulated within or conjugated to the nanoparticle or microparticle.

37. The delivery vehicle of any one of paragraphs 34-36, wherein the delivery vehicle is selected from the group including a polymeric particle, a viral particle, a liposome, a nucleic acid conjugate, and a metallic particle, or a combination thereof.

38. The delivery vehicle of any one of paragraphs 34-37, further including a targeting motif that targets the delivery vehicle to the brain and/or the central nervous system (CNS) in vivo.

39. The delivery vehicle of paragraph 38, wherein the targeting motif includes a peptide that facilitates passage across the blood-brain barrier (BBB).

40. A cell including the crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22, or ribonucleoprotein complex of any one of paragraphs 23-30, or the vector of any one of paragraphs 31-33, or the delivery vehicle of any one of paragraphs 34-39.

41. A pharmaceutical composition including

(a) the crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22 or ribonucleoprotein complex of any one of paragraphs 23-30, or the vector of any one of paragraphs 31-33 or the delivery vehicle of any one of paragraphs 34-39, or the cell of paragraph 40; and

(b) a pharmaceutically acceptable buffer, carrier, diluent or excipient for administration in vivo.

42. A method for CRISPR editing of one or more target genes to selectively repress the expression of a human UBE3A-AAS RNA in a cell, including administering into and/or expressing within the cell the crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22, or the ribonucleoprotein complex of any one of paragraphs 23-30.

43. The method of paragraph 42, wherein the administering is in vivo.

44. A method for treating or preventing Angelman Syndrome (AS) in a subject in need thereof, including administering to the subject the pharmaceutical composition of paragraph 41, in an amount effective to prevent or reduce expression of a UBE3A-ATS RNA in the subject.

45. The method of paragraph 42, wherein the subject is an infant or a child.

46. The method of any one of paragraphs 42-45, wherein the administration is via a route selected from the group including intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration.

47. The method of paragraph 46, wherein the administration is via intracerebroventricular injection.

48. The method of paragraph 46, wherein the administration is via intrathecal injection.

49. A kit including

(i) the crRNA of any one of paragraphs 1-17, or the sgRNA of any one of paragraphs 18-22, or the ribonucleoprotein complex of any one of paragraphs 23-30, or the vector of any one of paragraphs 31-33, or the delivery vehicle of any one of paragraphs 34-39; and optionally

(ii) a Cas9 enzyme, or vector encoding or expressing the Cas9 enzyme; and/or

(iii) instructions for performing the method of any one of paragraphs 42-48.

The description will be further understood by reference to the following Examples.

EXAMPLES

Example 1: Design of CRISPR/Cas9 gene editing systems for amelioration of Angelman Syndrome

Methods crRNA Design and validation

Taking advantage of the highly repetitive sequence region of the target UBE3A antisense as the input for sgRNA design, a total of 20 sgRNA candidates were designed (hsgRNAs 1-20).

Firstly, sgRNA designer from Broad institute was used with an input genome region. The total number of sgRNAs output from the designer program was 17,800. The number of sgRNA which have at least 4 repeats was 115.

Secondly, the top 20 candidates were selected based on:

(i) high on-target scores; and

(ii) high number of repeats.

These top 20 candidates were tested (see Figures 2A-2C). The tested pool also included 3 hsgRNAs as positive controls. The sgRNA7 was also used for the Cas9 comparison.

Protocol for inducible iPSCs and development into neurons, and RNP testing

The protocol for inducible iPSCs and development into neurons, and RNP testing takes about 2 months (or longer) from the inducible iPluripotent Stem Cell (IPSC) to produce mature neurons. While the induced neurons are used for hsgRNA screening and Cas9 comparison, according to the schematic set forth in Figure 3A. The NGN2 were first generated as inducible iPSCs cells lines using lentivirus and then selected using double antibiotics to select the NGN2 positive NPCs and overexpress the NGN2 to get mature neurons.

On the 14th day, the neurons were treated with test ribonucleoproteins (RNP) including engineered for 24 hours and the cells were collected on around day 21. In a second/advanced protocol, RNP administration was advanced to the fourth day after differentiation is initiated. Using NeuN as the mature neuron marker, successful differentiation was observed according to this protocol. After 24 hours incubation with the STEP-RNP construct, the paternal Ube3a-EGFP protein was detectable in more than 90% of the neurons. This indicated the STEP-RNP effectively inactivated the UBE3A antisense and reactivated the expression of Ube3a protein from the silenced paternal chromosome.

Finally, a 7 day method was used for each batch of experiments to induce IPSC differentiate to mature neuron (as set forth in Figure 3B). Using this 7 day protocol, 24 conditions were tested, including negative control, scramble sgRNA, 20 hsgRNA candidates, and Cas9 comparison groups. Each of the conditions were carried out using at least 6 repeats for DNA and RNA examination, 3 repeats for IHC, and additional 3 repeats for the top 3 hsgRNA candidates, cross compared with 3 kinds of Cas9 protein.

Results

All the top 20 sgRNA candidates have been tested in AS patients’ IPSC induced neurons having a minimal 6 repeats, and each culture has at least 3 batches of RT-qPCR. Since the RT- qPCR is the most sensitive quantification method, the best three candidates according to this assay were identified and selected. These three candidates performed much better than others, including hsgRNA5, hsgRNA7 and hsgRNA16 (see Figure 4A).

A neuron cell-based immunofluorescence assay was also carried out for all hsgRNA candidates and the Cas9 comparison groups, to further validate the RT-qPCR data. The cellbased assay relied upon detection of a fluorescent labeled paternal UBEJA-EGFP protein. The data corresponded to the RT-qPCR data, showing that RNP effectively inactivated the UBE3A antisense and reactivated the expression of UBE3A from the silenced paternal chromosome; among the 20 sgRNA candidates, the sgRNA 5, 7, and 16 performed better than others. Quantification for the immunofluorescence is depicted in Figures 4B-4C. As depicted in FIG.4A, the GFP positive neuron area indicating the edited neuron number; and the GRP positive neuron integrity density in FIG.4A indicated the UBE3A expression level of each individual neuron, for all the sgRNA candidates and the different Cas9. Consistent with the RT- qPCR results, the hsgRNA-5, 7 and 16 are the top performing candidates.

An on-target event assessment was also carried out using the top three candidates and DNA. For hsgRNA5, among the 8 potential targeting repeats, the target 1, 2 and 5 are used in all sequenced 4 samples, while none used the target loci 3, 4, 6, 7 and 8. This is not too surprising since the DNA 3 -dimensional structure could restrict the binding. The situation in hsgRNA#16 and #7 are similar. Both have 4 potential targeting loci, and they are used alternatively in different samples.

Example 2: Non- viral delivery of CRISPR/Cas9 gene editing for amelioration of Angelman Syndrome

Methods

STEP sgRNA Construct Design

The Stimuli-Responsive Traceless Engineering Platform (STEP) constitutes a pharmaceutically conjugated composition designed for the intracellular transportation of therapeutic agents. STEP'S conjugates include a chemical linker and a cell membrane fusogenic/penetrating molecule. The linker harbors both a stimuli-responsive chemical group and a self-immolative spacer.

Upon cellular entry, these STEP conjugates encounter the intracellular microenvironment, initiate a cleavage reaction, followed by self-immolation, to facilitate the release of the therapeutic payload directly into the cell in a controlled and traceless manner, ensuring precise delivery without residual byproducts.

The cellular internalization of STEP-RNP occurs mainly through endocytosis pathway, which is similar to majority of nanoparticles and delivery platform (Dougherty, Sahni, and Pei 2019; Sousa de Almeida et al. 2021; Rennick, Johnston, and Parton 2021; Zhang, Gao, and Bao 2015).

Results

Construct Characterization

After internalization, the linker of STEP-RNP was cleaved to release the free RNP due to the responsive to the stimuli of intracellular high glutathione (GSH) and low pH(Montero et al. 2013; Pei and Buyanova 2019; Zhou et al. 2017). Positive charge usually cause problems when applied in vivo due to rapid clearance by the reticuloendothelial system (Zhang, Gao, et al. 2021). Therefore, the STEP-RNP modified with biocompatible poly(ethylene glycol) (PEG) units (Vlassi, Papagiannopoulos, and Pispas 2017), to shield the positive charges of Cas9 protein and increase the complex stability in vivo (Schubert and Chanana 2018). After released by STEP, the positive charged Cas9 leads the RNP bypass following lysosomal pathway through endocytosis escaping (Xie et al. 2022), and enter the nucleus following the guidance of nucleus localization signal (NLS) on the Cas9 protein.

STEP-RNP is highly efficient to edit Ai9 reporter line (Madisen et al. 2010) in vitro and in vivo.

Efficacy of STEP-RNP in paternal Ube3a-YFP fusion gene reporter mouse model (UBE3Am+/p-YFP)

STEP-RNP was tested in the paternal YFP fusion gene mouse model (Ube3am+/p-YFP) (JAX stock #017765) (Dindot et al. 2008). STEP-RNP was synthesized with 10 sgRNA candidates that targeted UBE3A- TS and assess the efficacy of these sgRNA individually. The STEP-RNP/sgRNAs were delivered to Pl newborn via intracerebroventricular (ICV) injection. Out of 10 sgRNA candidates, which were summarized and redesigned from published studies (Schmid et al. 2021; Wolter et al. 2020), sgRNA33 was distinguished by its effectiveness in suppressing UBE3A-AYS measured by primer pairs at multiple loci as well as in reactivating the paternal UBE3A- PP gene. The results were comparable among brain regions of the prefrontal cortex, striatum, hippocampus, and cerebellum at 30 days after Pl-2 delivery of STEP-RNP. Intrathecal (IT) injection at Pl-2 day yielded comparable results to the ICV injection, attributed to a larger volume administered (10 pl per mouse for IT versus 2pl per lateral ventricle for ICV) (Lu and Jiang 2023). Consequently, IT injection of sgRNA33 was the chosen method for delivering STEP-RNP in subsequent experiments described below.

In the UBE3Am+/p-'YFP mice, the STEP-RNP achieved extensive brain penetration. The widespread reactivation of paternal UBE3A was evident across the entire brain, in contrast to the untreated mice 30 days after Pl -2 delivery. In the prefrontal cortex, reactivated UBE3A expression coincided with 76% of NeuN positive neurons, indicating a high gene editing efficiency. Comparable editing efficiency was noted in the cerebellum and other brain regions. The sustained reactivation of UBE3A was also confirmed by western blot at 90-day post administration. Compared to the level of maternally expressed UBE3A, the level of reactivated UBE3A was -40% in prefrontal cortex and -60% in cerebellum.

STEP-RNP Delivery in AS UBE3A Maternal Aexon2 Deletion Mouse Model (UBE3Am-/p+).

Whether the STEP-RNP treatment could mitigate neurobehavioral deficits was examined in AS mouse model with maternal UBE3A deficiency (t/5E3Am-/p+) (JAX stock #016590) (Jiang et al. 1998). A single dose of STEP-RNP was administered at three distinct developmental stages: Pl, P21, and P42 day, indicating neonatal, juvenile and adult timeframe in rodent respectively, each as 3 single dose groups. A battery of behavioral tests were conducted in order at given time points to evaluate different functional behavioral domains across the treated groups, the non-treated (NT), and a group injected with STEP-RNP containing scrambled sgRNA.

The administration of STEP-RNP improved the proprioception and motor function of AS mice. Mice with AS that received STEP-RNP exhibited an increase in the total distance traveled during 1-hour open field test in all treatment mice, including those with delayed administration at P42. However, significantly decreased center time, indicating reduced anxiety, was only observed in the mice treated on day 1. Improvements in motor coordination were evident in the prolonged latency to fall in both accelerating and steady speed rotarod tests. Notably, with an advanced formulation of Cas9 (G5, described in a subsequent section) that includes dual NLS, mice treated on day 42 displayed a complete restoration of motor coordination. Additionally, STEP-RNP treatment resulted in a partial correction of marble burying behavior, reflecting an improvement in proprioceptive function in AS mice.

The administration of the STEP-RNP ameliorated cognitive deficits in AS mice, as showed improved performance in tests of short-term and long-term memory. Mice that received treatment on day 1 exhibited a complete recovery in short-term memory assessments, whereas those in the delayed treatment group showed only partial improvement. Furthermore, all groups, regardless of the timing of treatment, demonstrated moderate enhancements in long-term memory performance. The assessment of fear conditioning, encompassing both contextual and cued paradigms, revealed that STEP-RNP treatment nearly fully restored contextual learning in AS mice, while cue learning was only partially ameliorated.

Seizures constitute a principal symptom in patients with AS and are a primary cause of mortality (Sun et al. 2019; Suiting, Williams, and Horsthemke 2016). AS mice exhibit increased susceptibility to seizure induction. The administration of STEP-RNP at any age has been effective in suppressing both myoclonic and tonic seizures. The immunostaining of STEP-RNP treated mice shown compared to non-treated mice.

High gene editing efficiency of STEP-RNP in IPSC derived neuron from Angelman Syndrome patients

The imprinting mechanism of UBE3A is conserved but sequences of UBE3A-NTS are not significantly conserved between human and rodent. Therefore, human specific sgRNAs have to be identified from the experiments conducted in human neurons. To facilitate the screening of sgRNAs, a UBE3A-EGFP fusion gene was engineered in the paternal chromosome using an IPSC line-derived AS patient with a large maternal deletion of 15ql l-ql3. The enhanced green fluorescent protein (EGFP) was fused in-frame to the C-terminus of the paternal UBE3A gene after mutating the STOP codon. It was achieved using the CRISPR/Cas9 system via electroporation. Thereafter, the hIPSCs were induced to differentiate into neurons (hlPSCs- iNeurons) following NGN2 overexpression protocol (Zhang et al. 2013).

The efficacy of STEP-RNP was initially assessed on above hIPSCs-iNeurons utilizing 3 hsgRNAs from a published study (Wolter et al. 2020), along with a commercially available CRISPR/Cas9 protein obtained from IDT (Alt-R® S.p. Cas9 Nuclease V3) equipped with a single NLS. Subsequently, STEP-RNP was administrated with 3 variants of CRISPR/Cas9 proteins (detailed in a subsequent section) conjugated with hsgRNA#3, to the hIPSCs-iNeurons. Among these, the G5-CRISPR/Cas9 protein variant exhibited the best performance. Hence, G5- CRISPR/Cas9 was selected for further screening of hsgRNA candidates.

Among the 20 hsgRNA candidates evaluated, hsgRNA#5 and hsgRNA#7 shown the highest efficacy. The paternal CBEdA-EGFP fusion protein was detectable in more than 90% of the iNeurons, at 24 hours of administering the STEP-RNP . This result suggests that the STEP- RNP effectively targeted the UBE3A- ATS genomic region, subsequently silenced the UBE3A antisense transcripts, and reactivated UBE3A expression from the silenced paternal allele.

Pharmacokinetics, Biodistribution and General Toxicity of STEP-RNP

To investigate the pharmacokinetics and biodistribution of STEP-RNP in vivo, wholebody specimens were collected from neonatal mice at 1-, 3-, 6-, 12-, and 24-hours following IT administration for immunofluorescence analysis. The STEP-RNP was rapidly distributed throughout the body and maintained a stable biodistribution up to 6 hours after IT administration, with a significant decrease observed by 12 hours and nearly complete clearance at 24 hours after IT administration. This rapid pharmacokinetic profile ensures an extremely low likelihood of off-target effects. General toxicity was evaluated by examining liver and kidney function, body weight, and survival rates after dayl administration, with no significant toxicity associated with the STEP-RNP treatment observed.

On-target and off-target detection

To assess the off-target events in vivo in mice, the top 10 predicted off-target sites were first assessed using amplicon sequence for the detection of variants (Wolter et al. 2020). 8 prefrontal cortex tissue collected from the mice treated with single dose STEP-RNP incorporating 3 variants of CRISPR/Cas9 protein were analyzed, and a double dose STEP-RNP incorporating with G221-CRISPR/Cas9 protein. No off-target effects were detected in any samples. Thereafter, Whole Genome Sequence (WGS) was performed at 300X depth on brain and liver tissue from STEP-RNP treated mice. Among the few off-target events identified across all samples, only 2 unique to the brain. Further RNA-seq for the same brain tissue revealed no significant gene expression changes related to these two events. Notably, the on-target effects were clearly and effectively identified in the WGS data.

Modification of STEP-RNP

To further enhance the efficiency and cell-type specificity or universality of genomic editing by STEP-RNP, variations in the number of NLS affiliated with the CRISPR/Cas9 protein component were engineered. While the current commercial CRISPR/Cas9 proteins contain a single NLS, in addition to the G5-CRISPR/Cas9 protein used in current study, a variant designated as “G221”, which is equipped with 3 NLS motifs was introduced. This synthesized G221-CRISPR/Cas9, in conjunction with sgRNA and STEP, was applied to embryonic fibroblasts from Ai9 reporter mice and demonstrated superior genomic editing efficiency compared to both the commercial Cas9 and G5 variant. Further comparative analysis between the G5 and G221 versions in Ai9 and UBE3A- YFP fusion mouse models revealed that STEP- RNP formulated with the G5-CRISPR/Cas9 protein exhibited the most robust neuron specificity relative to the other Cas9 variants). While the STEP-RNP formulated G221-CRISPR/Cas9 protein have stronger affinity to endothelial cell.

Discussion

As the first ex vivo gene therapy using CRISPR/Cas9 based technology for sickle cell disease has been approved recently (Frangoul et al. 2021), the next milestone will be to precisely deliver the CRISPR/Cas9 system to solid organs for in vivo gene therapy (Suzuki et al. 2016), as this is the only long-lasting treatment. Development of safe and effective delivery system is the primary issue for the applications of CRISPR/Cas9 system. Direct delivery of the CRISRP/Cas9 RNP takes advantages in transient function, higher genome-editing efficiency and lower off- target effect when compared with the delivery of Cas9 plasmid and mRNA. To overcome the potential immunogenic and tumori genic issue and the risk of off-target effects from persistent Cas9 associated with the AAV delivery system, and the large particle size and the instability in the circulatory system of nanoparticle, the non- viral, non-nanoparticle gene editing delivery system STEP was introduced. A very prerequisite for CRISPR/Cas9 complex to function is the efficient delivery of the complex into nucleus of target cells (Rui, Wilson, and Green 2019). STEP-RNP delivery enables the swiftest genome editing by reasons of eliminating the need for intracellular transcription and translation. Meanwhile, the transient genome editing not only permits high editing efficiency, but also reduces off-target effects, insertional mutagenesis, and immune responses (Lattanzi et al. 2019; Li et al. 2023).

Exploiting the extraordinary physical properties, STEP employed a biodegrade platform for RNP delivery. The Cas9 protein were fused with more than one NLSs at C-terminus to enhance its electrostatic interaction with STEP and improve the nuclear transportation of RNP after cellular internalization. Due to the enhanced electrostatic interaction provided by NLSs and the 2D puckered honeycomb structure of STEP, the STEP exhibited a remarkable RNP loading capacity and maintained small size at the same time. The outermost cholesterol further enhance the capacity and flexibility of the STEP to encapsulate RNP. It also provides an optimal balance between rigidity and fluidity, which is crucial for the functionality of targeting molecules and for the affinity with cell membranes during the delivery process. By stabilizing the STEP structure, cholesterol further help STEP to evade the immune system and extend the latency in circulation system, improving the bioavailability of the RNPs. Distinct from nucleic acids with high density of negative charges, Cas9 protein is highly cationic (theoretical net change: +22)(Zhang, Shen, et al. 2021). However, the RNPs delivered by STEP are negatively charged due to the appropriate mole ratio between Cas9 protein and sgRNA, and further shadowed by biocompatible PEG units and cholesterol to enable a long blood circulation and selective cell uptake.

The complexes of STEP-RNP enter cells through active endocytosis pathways. Precise spatiotemporal control which is important for in vivo genome editing applications. Materials responsive to endogenous triggers such as pH, redox potential, enzymes and ATP may promote intracellular release of RNP and increase editing efficiency. The acidic endosomal environment triggers the charge conversion of the STEP-RNP, facilitating the escape of RNP from endosomes. Finalization of STEP-RNP in alkalescent buffer (pH=7.4) facilitated the endosomal escape of RNP complexes via the pH-buffering mechanism. The endosomal escape of RNP complexes is an important step in the delivery of CRISPR/Cas9 systems into the cytoplasm of cells for genome editing. The pH-buffering mechanism is one of the strategies used to facilitate this process. It involves the use of substances or materials that can buffer the acidic environment within the endosomes. As the endosome matures, its interior becomes more acidic, which can trigger the buffering agents to accept protons (H+ ions). This protonation can lead to an influx of water into the endosome, resulting in increased osmotic pressure, swelling, and eventual rupture of the endosomal membrane. Once the endosome is disrupted, the RNP complexes can escape into the cytoplasm where they can reach their target DNA and perform the intended gene editing. This mechanism is crucial because without efficient endosomal escape, the RNPs would be degraded in the endolysosomal pathway, rendering the CRISPR/Cas9 system ineffective. The proton sponge hypothesis has been explored using validated endosome burst computational models (Freeman, Weiland, and Meng 2013; Freeman, Weiland, and Meng 2010), and experimental observations supportive of the proton sponge effect have been reported by Sonawane et al (Sonawane, Szoka, and Verkman 2003).

Disulfide bond containing materials are also responsive to glutathione (GSH). Since the intracellular GSH concentration is much higher than the extracellular, the use of disulfide bond containing materials in STEP for intracellular delivery of biomacromolecules could efficiently release the bound cargoes after cell internalization. Once delivered into cytosol, the disulfide linkage in the STEP could be cleaved by GSH, degrading the STEP into segments, and releasing loaded RNP molecules. After released by STEP, the positive charged Cas9 leads the RNP bypass following lysosomal pathway through endocytosis escaping (Xie et al. 2022), and enter the nucleus following the guidance of nucleus localization signal (NLS) on the Cas9 protein.

STEP-RNP offers a robust platform to deliver CRISPR/Cas9 to neuron which is relatively low transcription and translation activity (Lee and Fields 2021). Neurons possess receptors and transport proteins that can interact with cholesterol, as cholesterol is important for the proper functioning of nerve cells (Cantuti-Castelvetri et al. 2018). Cholesterol is also important in maintaining membrane fluidity and is involved in the formation of synapses and the maintenance of synaptic function (Yang et al. 2022). Although high affinity of STEP-RNP was observed in both brain and liver, the exact types of receptors on neurons that directly interact with cholesterol are less characterized compared to liver cells, but neurons rely on cholesterol for various cellular processes and obtain it through different mechanisms, such as from astrocytes that supply cholesterol through APOE-containing lipoproteins (Chen et al. 2023). The pH value of extracellular fluid is kept constant at 7.4 while the cellular cytosol is at 7.2. In addition, the pH values of brain and liver are lower than other organs, and the pH values of early endosome are maintained at a much lower level (pH=6.3) (Pei and Buyanova 2019). The pH-responsive STEP- RNP facilitate the targeting to neuronal cells, in addition to the endosomal escape and intracellular release.

Equally important, the natural cellular mechanisms of protein turnover ensure that the RNP can be degraded within a few hours after entering the cell and nucleus (Cooper, 2006) and this would certainly minimize the potential off-target associated with Cas9/gRNA. In addition, there is no concern for virus mediated immunogenicity. The general concern for the Cas9 protein related immunogenicity is also minimal due to the single IT delivery and shorter half-life of Cas9 protein inside of cells. For the translational application, the apparent benefit is that because the genome editing effect is expected permanent, it may only require a single administration to application lifetime.

Besides the ongoing ASO trial, there is no effective treatment targeting the molecular defect for AS. Patients with AS typically carry at least one intact copy of UBE3A gene on paternal chromosome that is silenced by an epigenetic mechanism mediated by paternally expression long noncoding and antisense to UBE3A transcripts, the UBE3A-ATS (Rougeulle et al. 1998). Studies have shown that interrupting the transcription of paternal UBE3A-ATS in rodent models by a small molecule of topotecan and ASOs could un-silence the UBE3A from the paternal chromosome, restore the UBE3A expression, and ameliorate AS phenotypes (Huang et al. 2011; Meng et al. 2015). However, both ASOs (a currently FDA approved clinical trial) and any small molecule compounds have a short half-life and require repeated injection by an invasive procedure. The potential of topotecan is also limited by its significant toxicity (Huang et al. 2011).

CRISPR/Cas9, a genome editing tool, has the potential to permanently disrupt the expression of UBE3A-ATS to achieve a long-lasting therapeutic efficacy. The efficiency of CRISPR/Cas9 gene disruption mechanism is much higher than other editing modalities (Cox, Platt, and Zhang 2015) and this makes Cas9 gene editing an attractive alternative to ASOs. Two studies have identified several gRNAs that are close to the UBE3A gene body and could reduce the expression of UBE3A-ATS and reactivate the expression of UBE3A in brain of AS mouse models via AAV or lenti virus delivery (Schmid et al. 2021; Wolter et al. 2020). However, the viral delivery of CRISPR/Cas9 to brain is a challenge for clinical usage, due to undesired effects such as variable efficiency, cytotoxicity and immunogenicity, and higher potential of off target events due to prolonged expression of Cas9 DNA vector in vivo (Yin et al. 2017). It is noted the in vivo delivery of using lentivirus is not feasible due to its high frequency of genome integration that will produce situations hazardous to human health (Osten, 2006). To develop alternative gene editing delivery platform, NIH SCGE launched the phase 1 project in 2017 (Saha et al. 2021). The goals are to develop and improve genome editors and new delivery tools.

The STEP technique was applied to deliver CRISPR/Cas9-sgRNA RNP to brain for the treatment of Angelman syndrome. The mice with AS treated on postnatal dayl had the comprehensive improvement in all neurobehavioral domains including locomotion, motor coordination, cognitive function, and seizure susceptibility, while the delayed treated groups have full recover in motor function but not the cognitive function. This is consistent with the findings in previous studies that reinstating UBE3A functionality during early postnatal phase is anticipated to be more effective in treating AS than restoring UBE3A in adulthood (Silva-Santos et al. 2015; Zylka 2020). This is also in line with those studies using virus based CRTSPR delivery and ASO to treat Angelman syndrome (Meng et al. 2015; Dindot et al. 2023; Milazzo et al. 2021). To be noted, in clinic, rare Angelman syndrome patients with mosaic defect have significant mild clinical phenotype, especially the seizures and speech.

Although the exact residual level of UBE3A in brain of mosaic patient is not known, it was estimated to have 10% expression of normal UBE3A. Thus, it is possible to predict that partial reactivation of UBE3A such as 40% observed in mic from paternal chromosome may offer significant clinical benefit for AS patients with complete deficiency of UBE3A.

The sgRNA screening data from mouse model suggested the selection of human hsgRNA taking advantage of the multiple repetitive sequences on the UBE3A-ATS to be tested on AS patient’s ISPC-iNeurons, which is clearly an advantage for hsgRNA targeting and increased the efficiency of gene editing. These repetitive sequences of the UBE3A-ATS is also the highly conservative region across species, and this phenomenon likely exist in other diseases related genomic regions.

The significant behavior rescue data prompted us to examine safety profile for the STEP- RNP delivery. The assessment of long-term safety certainly will take long time to complete. From observation of many cohorts of behavioral analysis, the treatment were well tolerated when given different time and different dosages. As mentioned earlier, CRISPR/Cas9 protein delivered through STEP-RNP transiently stay in vivo should significantly minimize the potential off-target event, although the assessment of off-target event also remains a challenge, technically and conceptually. Many groups are trying to apply the CRISPR/Cas9 technique to clinical gene therapy, however, the best practice to evaluate the off-target events is still in debating(Kleinstiver et al. 2016; Kim et al. 2015; Ran et al. 2013). Amplicon sequence is the major approach currently; however, it’s restricted by the prediction tools and parameters. Using Whole Genome Sequence (WGS) to detect the potential off-target events is an unbiased method, however, there has no good standard for the sufficient sequence depth, and acceptable off-target ratio. In this study, 300x WGS was applied, which is 10 times depth of routine WGS in clinic and followed by RNAseq to confirm the consequence in transcriptome level. There were also no deleterious off-target events.

In conclusion, the data demonstrate that applying the STEP technique to deliver CRISPR/Cas9 system paves the way for the development of rapid and cost-effective ways for in vivo genome editing for neurogenetic disorders.

Example 3: Neuron-organoid model of CRISPR/Cas9 gene editing

Methods

The hsgRNA#5 was tested for efficacy using a neuron-based “brain” organoid model system. The activity of the hsgRNA#5 was measured by detecting expression of the paternal UBE3A allele (i.e., to assess deactivation of the UBE3A antisense gene)). Organoid culture followed published protocols (Y. Xiang et al., Generation and Fusion of Human Cortical and Medial Ganglionic Eminence Brain Organoids. Curr Protoc Stem Cell Biol 47, (2018)).

Briefly, AS-iPSCs were dissociated into a single-cell suspension and seeded into an ultralow attachment 96-well plate at a density of 9,000 cells per well in 150 pL of induction medium supplemented with SB431542, LDN193189, and XAV939 on day 0 to initiate embryoid body (EB) formation. Induction medium was refreshed daily until day 10. On day 10, EBs were transferred to ultra-low-attachment 6-well plates for spinning culture at 80 rpm in neural differentiation medium supplemented with brain-derived neurotrophic factor (BDNF) and ascorbic acid, without vitamin A. Medium changes were performed every other day until day 18. From day 18 onward, organoids were cultured in neural differentiation medium containing BDNF and ascorbic acid to promote neural maturation, with medium refreshed every four days. A first batch of organoids was taken down for the assessment at day35 (the STEP-RNP were added on the day 28 for 24 hours, then washed out); a second batch of organoids were taken down at 70 days of culture (the STEP-RNP were added on the day 63 for 24 hours, then washed out).

Results

Results are depicted in Figs. 5A-5F, showing increasing expression of the paternal UBE3A allele with increasing dose of sgRNA across all organoids tested. Distribution of the paternally-expressed UBE3A within organoid neurons in response to different doses of the UBE3A sgRNAs (UBE-YFP) (with additional data for neuronal protein NeuN, SOX2 stem cell transcription factor, and DNA in the nuclei of cells (DAPI) on pages 2, 4, 5 and 7) at 2 time points (35 and 70 days) demonstrated that sgRNAs effectively deactivate the antisense UBE3A- ATS gene and thereby induce expression of the paternal UBE3A allele.

Based on the organoid data, the most efficacy dosage is 60 pg per 3 ml culture medium, using hsgRNA#5.

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Zylka, M. J. (2020). Autism Res 13(1): 11-17. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS We claim:

1. An engineered CRISPR RNA (crRNA) molecule that selectively targets a U E3A-MA\- sense RNA (ATS) gene, wherein the gene expresses a long non-coding ATS RNA comprising a nucleic acid sequence set forth in SEQ ID NO: 36.

2. The engineered crRNA molecule of claim 1, comprising a nucleic acid sequence selected from the group consisting of CAGCUCAGUGCAGGAGACCA (SEQ ID NO:1),

GGACCACCGUCACCCCUGCA (SEQ ID NO:2), GAGCCUGGGCUGCCUCACGG (SEQ ID NO:3), GAGCUGUGGUGAGCACAUCC (SEQ ID NO:4), AGAGCUCACUGAAAGACACA (SEQ ID NO:5), UGCUCACCACAGCUCAGUGC (SEQ ID NO:6), or

GAGCCUGGGCUGCCUCACAG (SEQ ID NO:7), UCUCAUCAUCGACCCAACCC (SEQ ID NO:8), AUUACGCUGAGGCCCAACCU (SEQ ID NO:9), UGUGUGGGAGGUGUUGUGUG (SEQ ID NO: 10), UAGGUGAGUGGAUCCUGCUG (SEQ ID NO: 11),

ACAGCUCAGUGCAGGAGACC (SEQ ID NO: 12), GGCUCACCACAGCUCAGUGC (SEQ ID NO:13), GGAGACCUGGAGGCCCUGAA (SEQ ID NO: 14),

CUCAUCAUCGACCCAACCCA (SEQ ID NO: 15), AGCUCACUGAAAGACACAAG (SEQ ID NO: 16), and GCAGCCCAGGCUCCCUGUGU (SEQ ID NO: 17).

3. An engineered single guide RNA (sgRNA) molecule, comprising the crRNA of any one of claims 1-2, conjugated with a Traer RNA.

4. The engineered sgRNA of claim 3, wherein the Traer RNA comprises a nucleic acid sequence set selected from the group consisting of SEQ ID NO: 19-21 and 22.

5. A ribonucleoprotein complex comprising:

(a) a Cas enzyme; and

(b) the crRNA of claim 3.

6. A ribonucleoprotein complex comprising:

(a) a Cas enzyme; and

(b) the sgRNA of claim 4.

7. The ribonucleoprotein complex of any one of claims 5-6, wherein the ribonucleoprotein complex functions to selectively repress the expression of a human UBE3A-ATS RNA in a subject in vivo.

8. The ribonucleoprotein complex of any one of claims 5-7, wherein the Cas enzyme is a Cas9 enzyme.

9. The ribonucleoprotein complex of claim 8, wherein the Cas9 enzyme is derived from Streptococcus pyogenes (spCas9).

10. A nucleic acid, vector or transposon encoding or expressing the engineered crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4.

11. The nucleic acid vector of claim 10, wherein the vector is a viral vector.

12. The nucleic acid vector of claim 11 , wherein the viral vector is selected from the group consisting of a lentiviral vector, an Adeno- associated virus (AAV) vector, or an adenovirus vector, or a Herpes Simplex virus (HSV) vector, or a vesicular stomatitis (VSV) vector, or a human Bocavirus vector (hBoV), or a chimeric vector comprising a combination of any two or more of a Adeno-associated virus (AAV) vector, Herpes Simplex virus (HSV) vector, vesicular stomatitis (VSV) vector, and a human Bocavirus vector (hBoV).

13. The nucleic acid vector of claim 10, wherein the vector is selected from the group consisting of a plasmid, a cosmid, and a replicon.

14. A molecular delivery vehicle comprising the engineered crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5- 9, or the vector of any one of claims 10-13.

15. The delivery vehicle of claim 14, wherein the delivery vehicle comprises a nanoparticle, or a microparticle.

16. The delivery vehicle of claim 15, wherein the engineered crRNA of any one of claims 1-

17. or the sgRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13 is encapsulated within or conjugated to the nanoparticle or microparticle.

17. The delivery vehicle of any one of claims 14-16, wherein the delivery vehicle is selected from the group consisting of a polymeric particle, a viral particle, a liposome, a nucleic acid conjugate, and a metallic particle, or a combination thereof.

18. The delivery vehicle of any one of claims 14-17, further comprising a targeting motif that targets the delivery vehicle to the brain and/or the central nervous system (CNS) in vivo.

19. The delivery vehicle of claim 18, wherein the targeting motif comprises a peptide that facilitates passage across the blood-brain barrier (BBB).

20. A molecular delivery vehicle comprising

(a) a payload, wherein the pay load comprises the engineered crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13; and (b) a stimuli-responsive, self-immolative chemical linker, and;

(c) a cell membrane fusogenic molecule, wherein the payload is conjugated with the cell membrane fusogenic linker via the stimuli-responsive, self-immolative chemical linker.

21. A cell comprising the crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13, or the delivery vehicle of any one of claims 14-20.

22. A pharmaceutical composition comprising

(a) the crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13 or the delivery vehicle of any one of claims 14-20, or the cell of claim 21; and

23. A method for CRISPR editing of one or more target genes to selectively repress the expression of a human UBE3 A- ATS RNA in a cell, comprising administering into and/or expressing within the cell the crRNA of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13.

24. The method of claim 23, wherein the administering is in vivo.

25. A method for treating or preventing Angelman Syndrome (AS) in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 22, in an amount effective to prevent or reduce expression of a UBE3A-ATS RNA in the subject.

26. The method of claim 24, wherein the subject is an infant or a child.

27. The method of claim any one of claims 23-26, wherein the administration is via a route selected from the group consisting of intravenous, intramuscular, intracranial, intraosseus, intranasal, intrathecal, intraventricular, intraparenchymal and intracerebroventricular administration.

28. The method of claim 27, wherein the administration is via intracerebroventricular injection.

29. The method of claim 27, wherein the administration is via intrathecal injection.30. A kit comprising

(i) the crRNA of any one of claims of any one of claims 1-2, or the sgRNA of any one of claims 3-4 or the ribonucleoprotein complex of any one of claims 5-9, or the vector of any one of claims 10-13 or the delivery vehicle of any one of claims 14-20, or the cell of claim 21; and optionally

(ii) a Cas9 enzyme, or vector encoding or expressing the Cas9 enzyme; and/or

(iii) instructions for performing the method of any one of claims 25-29.