Chemically modified poly(a) tail sequences

Abstract

The present disclosure provides a library of chemically modified poly(A) tail sequences that can be operably linked to an RNA sequence of interest as well as the RNA sequences comprising the chemically modified poly(A) tail sequences.

Description

CRISPR-44538.601CHEMICALLY MODIFIED POLY(A) TAIL SEQUENCESCross Reference to Related Applications

[0001] The present application claims priority to U.S. Provisional Application No.63 / 770,687, filed March 12, 2025, which is incorporated herein by reference in its entirety.Sequence Listing

[0002] The text of the computer readable sequence listing filed herewith, titled “CRISP_44538_601_SequenceListing.xml”, created March 12, 2026, having a file size of 146,587 bytes, is hereby incorporated by reference in its entirety.Field

[0003] The present disclosure provides a library of chemically modified poly(A) tail sequences that can be operably linked to an RNA sequence of interest as well as the RNA sequences comprising the chemically modified poly(A) tail sequences.Background

[0004] Therapeutic RNA drugs are a class of medications that utilize RNA molecules to treat diseases by targeting and modulating specific biological pathways. These drugs primarily work by interfering with the genetic information flow within cells. They can be categorized into several types based on their mechanism of action and therapeutic targets.

[0005] RNA Interference (RNAi): RNAi therapies use small RNA molecules, such as small interfering RNA (siRNA) or microRNA (miRNA), to silence the expression of specific genes. By targeting messenger RNA (mRNA), they prevent the production of harmful proteins. This approach has been used to treat conditions like genetic disorders (e.g., amyloidosis) and viral infections (e.g., hepatitis).

[0006] Antisense Oligonucleotides (ASOs): ASOs are synthetic strands of nucleotides designed to bind to specific mRNA molecules. This binding can block the translation of the mRNA into protein, correct genetic mutations, or modulate gene expression. ASOs are used to treat genetic diseases such as spinal muscular atrophy (SMA) and Duchenne muscular dystrophy (DMD).

[0007] mRNA Vaccines and Therapies: mRNA vaccines, like those developed for COVID- 19, instruct cells to produce a protein that triggers an immune response. ThisCRISPR-44538.601platform is being expanded to treat cancer, infectious diseases, and genetic disorders by encoding therapeutic proteins or antigens.

[0008] Gene Editing (CRISPR / Cas9 and Related Technologies): Although not strictly RNA drugs, these approaches involve RNA molecules (like guide RNA) to direct the Cas9 protein to specific DNA sequences to edit or correct genetic defects. CRISPR-based therapies are being explored for conditions like sickle cell anemia and certain types of cancer.

[0009] RNA-Based Enzyme Replacement: RNA drugs can also be used to produce therapeutic enzymes, which may be defective or lacking in certain diseases. These therapies help restore normal biological functions by delivering RNA that encodes the missing enzyme.

[0010] A major challenge for RNA drugs is ensuring stability of the RNA molecules. The present disclosure addresses this problem.Summary

[0011] The present disclosure provides a library of chemically modified poly(A) tail sequences that can be operably linked to an RNA sequence of interest as well as the RNA sequences comprising the chemically modified poly(A) tail sequences.

[0012] In some embodiments, the present disclosure provides a synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’, wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0, preferably from 1 to 4; Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides, and wherein at least one of Ri, R2, R3, R4, Rs, and Re is an adenosine ribonucleotide comprising a modified sugar moiety.

[0013] In some embodiments, at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.

[0014] In some embodiments, the modified sugar moiety is selected from the group consisting of a locked nucleic acid (LNA) subunit, a 2'0,4'C-ethylene-bridged nucleic acid (ENA) subunit, a tricyclo-DNA (tc-DNA) subunit, a 2' O-methyl subunit, a 2' O-methoxyethyl subunit, a 2'-fluoro subunit, and a 2'-O-[2-(N-methylcarbamoyl)ethyl] subunit, and combinations thereof when at least two of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.

[0015] In some embodiments, the modified sugar moiety is selected from the group consisting of a 2' O-methyl subunit and a 2' O-methoxyethyl subunit, and combinations thereof when at least two of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.CRISPR-44538.601

[0016] In some embodiments, at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a 2' O-methyl subunit.

[0017] In some embodiments, at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a 2' O-methoxyethyl subunit.[0018| In some embodiments, at least one, two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides that comprise a modified internucleoside linkage.

[0019] In some embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

[0020] In some embodiments, X is an adenosine ribonucleotide.

[0021] In some embodiments, the RNA molecule of any of the foregoing embodiments has a 5’ terminal nucleotide, and the 5’ terminal nucleotide comprises a 5’ phosphorylation.

[0022] In some embodiments, the RNA molecule terminates at Re and Re is selected from the group consisting of an adenosine ribonucleotide comprising a 2' O-methyl subunit, an adenosine ribonucleotide comprising a 2' O-methyl subunit and a 3’ inverted T modification, a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification, and a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification and a 3’ inverted T modification.

[0023] In some embodiments, the present disclosure provides a synthetic RNA molecule of any one of SEQ ID NOs:l-48.

[0024] In some embodiments, the present disclosure provides a synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’, wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0; Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides, and wherein at least four of Ri, R2, R3, R4, Rs, and Re comprise a modified internucleoside linkage, preferably a phosphorothioate internucleoside linkage.

[0025] In some embodiments, the present disclosure provides a synthetic RNA molecule of SEQ ID NO:49 or 52.

[0026] In some embodiments, the present disclosure provides a synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-Rs-R6-3’, wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0; Ri, R2, R3, R4, and Rs are unmodified adenosine ribonucleotides and Re is a modified adenosine comprising a 3’ inverted T modification.

[0027] In some embodiments, the present disclosure provides a synthetic RNA molecule of SEQ ID NO:54 or 55.CRISPR-44538.601

[0028] In some embodiments, the present disclosure provides a RNA molecule comprising a RNA sequence of interest operably linked to a poly(A) tail comprising the synthetic RNA molecules as described in any of the foregoing embodiments.

[0029] In some embodiments, the RNA sequence of interest encodes a therapeutic protein.[0030| In some embodiments, the RNA sequence of interest encodes an enzyme.

[0031] In some embodiments, the enzyme is a therapeutic protein.

[0032] In some embodiments, the enzyme is a component of a gene-editing system.

[0033] In some embodiments, the present disclosure provides the enzyme is selected from the group consisting of a CRISPR / Cas nuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a nickase, a nickase-reverse transcriptase fusion enzyme, and a dCAS-deaminase fusion enzyme.

[0034] In some embodiments, the nuclease is a CRISPR / Cas nuclease.

[0035] In some embodiments, the present disclosure provides a lipid nanoparticle comprising the RNA molecule as described in any of the foregoing embodiments.

[0036] In some embodiments, the present disclosure provides a pharmaceutical composition comprising the RNA molecule as described in any of the foregoing embodiments and a pharmaceutically acceptable carrier.

[0037] In some embodiments, the present disclosure provides a gene editing system comprising: a nuclease as described above; and a guide sequence selected from the group consisting of a gRNA, sgRNA, and template armed guide RNA.

[0038] In some embodiments, the gene editing system further comprises a donor polynucleotide.

[0039] In some embodiments, the present disclosure provides a lipid nanoparticle comprising the gene editing system as described above.

[0040] In some embodiments, the lipid nanoparticle comprises an ionizable or cationic lipid and one or more additional lipids selected from the group consisting of helper lipids, cholesterol, and polymer conjugated lipids.

[0041] In some embodiments, the present disclosure provides methods of editing a genome comprising introducing a gene editing system or lipid nanoparticle as described above into a cell under conditions such that the genome of the cell is edited.

[0042] In some embodiments, the present disclosure provides the cell is in vivo.

[0043] In some embodiments, the present disclosure provides methods of making a RNA molecule comprising ligating a poly(A) tail comprising a synthetic RNA as described above to an RNA sequence of interest.CRISPR-44538.601

[0044] In some embodiments, the present disclosure provides methods of treating a subject, comprising administering to the subject an RNA molecule, LNP, or pharmaceutical composition as described above.Brief Description of The Drawings

[0045] FIGs. 1 A-D provides data showing the ligation efficiency of selected oligos. 1A. OMe4_InvdT modified luciferase mRNA. IB. MOE2_InvdT modified luciferase mRNA. 1C. MOE4_modified luciferase mRNA. ID. InvdT_modified luciferase mRNA.

[0046] FIG. 2. provides live images of expression of luciferase at 4, 24, 48, 72, 96, 120 and 168 hours after administration of LNPs containing the mRNA sequences to mice.

[0047] FIG. 3 provides a graph showing measured luciferase activity over time.

[0048] FIG. 4 presents the results as total luciferase activity integrated as area under the curve.

[0049] FIG. 5 provides a bar graph showing the log fold change in luciferase activity at 4 hours.

[0050] FIGs. 6A and 6B provide the results of a pharmacokinetics analysis of luciferase activity for selected chemically modified poly (A) sequences.Detailed Description

[0051] mRNA instability is a major barrier to its use in therapeutic applications, often necessitating high dosages and frequent administration. Current strategies improve stability only to a limited extent and do not account for specific requirements in diverse biological or therapeutic contexts. This disclosure provides reagents and methods for enhancing the stability and translatability of messenger RNA (mRNA) through chemically modified poly(A) tail using a ligation-based approach. By incorporating diverse chemical modifications, particularly on the 2’ ribose on the distal end of poly(A) tail, the RNA sequences described herein allow for precise tunability of mRNA half-life in vivo. The technology described herein enables the adjustable modulation of mRNA attributes such as expression level and durability in vivo which are the two critical features determining potency, and hence provides a strategy to customize mRNA translatability to specific therapeutic applications. These tailored poly(A) modifications offer a robust solution to challenges related to mRNA degradation and limited translatability, optimizing performance for various biological and therapeutic applications such as vaccines, protein replacement therapies, and gene editing technologies.CRISPR-44538.601

[0052] Section headings as used in this section and the entire disclosure herein are merely for organizational purposes and are not intended to be limiting.1. Definitions

[0053] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0054] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0055] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.Genome Editing Definitions

[0056] As used in the specification and appended claims, unless specified to the contrary, the following genomic editing terms have the meaning indicated:

[0057] ‘ ‘Adenosine deaminase” refers to a deaminase enzyme that acts on deoxy adenosine, on adenosine, or both on deoxyadenosine and on adenosine to convert adenine to hypoxanthine or, alternatively, adenosine to inosine. Due to the structural similarity of inosine to guanosine (inosine does not include an exocyclic amino group of guanosine), inosine tends to behave as guanosine. Inosine is eventually replaced by guanosine through subsequent cellular processing. As such, an adenosine deaminase effects an A^G (or T— >C) substitution. Adenosine deaminase and adenine deaminase may be used interchangeably herein.

[0058] A “bulge” is an unpaired region of nucleotides within the duplex.

[0059] As used herein, the term “CRISPRs” or “Clustered Regularly Interspaced Short Palindromic Repeats” refers to an acronym for DNA loci that contain multiple, short, directCRISPR-44538.601repetitions of base sequences. Each repetition contains a series of bases followed by the same series in reverse and then by 30 or so base pairs known as “spacer DNA”. The spacers are short segments of DNA from a virus and may serve as a 'memory' of past exposures to facilitate an adaptive defense against future invasions (PMID 25430774). These sequences are transcribed and processed in CRISPR RNAs (crRNA).10060] ‘ ‘Cas” or “CRISPR-associated (cas)” refers to genes often associated with CRISPR repeat-spacer arrays (PMID 25430774). “Cas9” refers to a nuclease from Type II CRISPR systems, an enzyme specialized for generating double-strand breaks in DNA, with two active cutting sites (the HNH and RuvC domains), one for each strand of the double helix. Jinek combined tracrRNA and spacer RNA (or crRNA) into a “single-guide RNA” (sgRNA) molecule that, mixed with Cas9, could find and cleave DNA targets through Watson-Crick pairing between the guide sequence within the sgRNA and the target DNA sequence (PMID 22745249).

[0061] ‘ ‘Conversion” refers to any manipulation of a nucleic acid sequence that changes a mutated sequence into a wild type sequence, or a wild type sequence into a mutated sequence. For example, a converted sequence includes, but is not limited to, a base pair conversion, a nucleic acid sequence insertion or a nucleic acid sequence deletion.

[0062] “Correcting” a mutation means restoring a wildtype sequence at the place of the mutation in the double stranded target DNA, e.g., target gene, for example, by gene editing, base editing, or RT editing.

[0063] “Cytidine deaminase” refers to a deaminase enzyme that acts on deoxycytidine, on cytidine, or both on deoxycytidine and on cytidine to convert cytosine to uridine. Cytidine deaminase and cytosine deaminase may be used interchangeably herein.

[0064] ‘ ‘Deaminase domain” and “domain having deaminase activity” are used interchangeably to refer to one or more domains of a site-directed polypeptide that exhibits adenosine deaminase activity and / or cytidine deaminase activity.

[0065] ‘ ‘DNA-binding domain “ and “domain having DNA binding activity” are used interchangeably to refer to one or more domains of a site-directed polypeptide that have affinity for, and are capable of binding to, a target nucleic acid (e.g., DNA).

[0066] ‘ ‘DNA polymerase domain” and “domain having DNA polymerase activity” are used interchangeably to refer to one or more domains of a site-directed polypeptide that catalyze the synthesis of DNA.

[0067] ‘ ‘Donor polynucleotide,” “donor sequence,” “donor template,” or “donor” are used interchangeably to refer to an exogenous polynucleotide sequence that can be inserted into aCRISPR-44538.601target nucleic acid cleavage site. The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site.

[0068] “Editing,” “genome editing,” and “genetic editing,” are used interchangeably to refer to the process of modifying the nucleotide sequence of a genome.

[0069] An “editing template” of a RT editor guide RNA (tagRNA; template armed guide RNA)) is a single-stranded portion of the tagRNA that is 5' of the PBS and encodes a single strand of DNA. The editing template may comprise a region of complementarity to the PAM strand (i.e., the non-target strand or the edit strand), and comprises one or more intended nucleotide edits compared to the endogenous sequence of the double stranded target nucleic acid (e.g., DNA). The editing template and the PBS can be immediately adjacent to each other.

[0070] “Engineered reverse transcriptase” refers to a polypeptide that converts RNA into DNA and contains specific mutations that effect its activity efficiency. An exemplary reverse transcriptase used in RT editing is a Moloney murine leukemia virus reverse transcriptase (M-MLV RT).

[0071] “Enzymatically impaired” refers to a site-directed polypeptide that has been modified (e.g., mutated) to cleave only a single strand of a deoxyribonucleic acid backbone. Depending on the position of the mutation within the site-directed polypeptide (e.g., Cas9) sequence either the target or non-target strand is cleaved. A site-directed RT editor polypeptide can selectively cleave the non-target strand.

[0072] “Enzymatically inactive” refers to a site-directed polypeptide that has been modified to have no substantial nucleic acid-cleaving activity.

[0073] “Fragment” or “variant” refers to any functional fragment, variant, derivative or analog of a polynucleotide, polypeptide or biomolecule that possesses an in vivo or in vitro activity that is characteristic of the polynucleotide, polypeptide, or therapeutic agent. In some embodiments, the fragment, variant or analog has a length equal to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or greater of the length of the polynucleotide, polypeptide or biomolecule. Functional expression of the fragment or variant can be easily assayed by the person of ordinary skill in the art by testing activity (e.g., enzymatic activity) and the ability to manufacture products as described herein.

[0074] ‘ ‘Genome-targeting nucleic acid” refers to a nucleic acid associated with a site-directed polypeptide to direct it to a specific sequence within a target nucleic acid.

[0075] A genome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein.CRISPR-44538.601

[0076] A “hairpin” refers to a secondary structure of DNA with intrastrand base pairing.

[0077] “Heterologous” means a non-native gene or protein component of, e.g., an engineered complex or fusion protein that does not naturally occur in the same organism, or in a naturally occurring fusion protein or complex, as other components of the complex or fusion protein, but which is engineered into the complex or fusion protein.

[0078] A “linker” can be any chemical group or a molecule linking two molecules or moieties. A linker can be an organic molecule, group, polymer, or chemical moiety.

[0079] A “minimum CRISPR repeat sequence” refers to a sequence that has substantial homolog to a reference CRISPR repeat sequence.

[0080] A “minimum tracrRNA sequence” refers to a sequence that has substantial homology to a reference tracrRNA sequence. A “3’ tracrRNA sequence” refers to a tracrRNA sequence that has substantial homology to a reference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

[0081] ‘ ‘Nickase,” “polypeptide having nickase activity,” “nickase domain,” and “domain having nickase activity” are used interchangeably to refer to a polypeptide or functional fragment thereof that cleaves a single strand of a target nucleic acid (e.g., DNA). A nuclease that has been enzymatically impaired to cleave only a single strand of a target nucleic acid may be referred to as a nickase. For example, introducing a H40A mutation into a Cas9 nuclease produces a variant Cas9 H840A nickase.

[0082] A “nick site” refers to a specific position in between two nucleotides or two base pairs of the double stranded target nucleic acid (e.g., DNA) where a nickase or nuclease will nick or cleave the target nucleic acid.

[0083] ‘ ‘Nuclease,” “polypeptide having nuclease activity,” “nuclease domain,” and “domain having nuclease activity” are used interchangeably to refer to a polypeptide or functional fragment thereof that cleaves a double- stranded target nucleic acid (e.g., DNA). A Cas nuclease is an exemplary nuclease of use herein.

[0084] ‘ ‘Nucleic acid” refers to a polymer containing at least two nucleotides (i.e., deoxyribonucleotides or ribonucleotides) in either single- or double-stranded form and includes DNA and RNA. “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, andCRISPR-44538.601alkylhalides. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages or modified sugar residues, or non-canonical / chemically-modified nucleobases and combinations thereof, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs and / or modified residues include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0085] ‘ ‘Nucleic acid” includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, pre-condensed DNA, a PCR product, vectors, expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.

[0086] A “nucleic acid” may include one or more nucleotide variants, including nonstandard nucleotide(s), non-natural nucleotide(s), nucleotide analog(s), and / or modified nucleotides. Polynucleotides introduced into cells can comprise one or more modifications that can be used individually or in combination, for example, to enhance activity, stability or specificity, alter delivery, reduce innate immune responses in host cells, or for other enhancements, as further described herein and known in the art.

[0087] Modified polynucleotides can be used in a CRISPR / Cas9 / Cpfl system, in which case the gRNAs (sgRNA or dsRNA) and / or a DNA or an RNA encoding a Cas or Cpfl endonuclease introduced into a cell can be modified. Such modified polynucleotides can be used in the CRISPR / Cas9 / Cpfl system to edit any one or more genomic loci. Using the CRISPR / Cas9 / Cpfl system for purposes of nonlimiting illustrations of such uses, modifications of gRNAs can be used to enhance the formation or stability of the CRISPR / Cas9 / Cpfl genome editing complex comprising gRNAs (sgRNAs or dgRNAs), and a Cas or Cpfl endonuclease. Modifications of gRNAs can also or alternatively be used to enhance the initiation, stability or kinetics of interactions between the genome editing complex with the target sequence in the genome, which can be used, for example, to enhance on-target activity. Modifications of gRNAs can also or alternatively be used to enhanceCRISPR-44538.601specificity, e.g., the relative rates of genome editing at the on-target site as compared to effects at other (off-target) sites.

[0088] Modifications can also or alternatively be used to increase the stability of a gRNA, e.g., by increasing its resistance to degradation by ribonucleases (RNases) present in a cell, thereby causing its half-life in the cell to be increased. Modifications enhancing gRNA halflife can be particularly useful in aspects in which a Cas or Cpfl endonuclease is introduced into the cell to be edited via an RNA that needs to be translated in order to generate endonuclease, because increasing the half-life of gRNAs introduced at the same time as the RNA encoding the endonuclease can be used to increase the time that the gRNAs and the encoded Cas or Cpfl endonuclease co-exist in the cell.

[0089] Modifications can also or alternatively be used to decrease the likelihood or degree to which RNAs introduced into cells elicit innate immune responses. Such responses, which have been well characterized in the context of RNA interference (RNAi), including smallinterfering RNAs (siRNAs), as described below and in the art, tend to be associated with reduced half-life of the RNA and / or the elicitation of cytokines or other factors associated with immune responses.

[0090] One or more types of modifications can also be made to RNAs encoding an endonuclease that are introduced into a cell, including, without limitation, modifications that enhance the stability of the RNA (such as by increasing its degradation by RNAses present in the cell), modifications that enhance translation of the resulting product (i.e. the endonuclease), and / or modifications that decrease the likelihood or degree to which the RNAs introduced into cells elicit innate immune responses.

[0091] Combinations of modifications described herein can also be used. In the case of CRISPR / Cas9 / Cpf 1 , for example, one or more types of modifications can be made to gRNA, and / or one or more types of modifications can be made to RNAs encoding Cas endonuclease.

[0092] By way of illustration, guide RNAs used in the CRISPR / Cas9 / Cpf 1 system, or other smaller RNAs can be readily synthesized by chemical means, enabling a number of modifications to be readily incorporated. While chemical synthetic procedures are continually expanding, purifications of such RNAs by procedures such as high-performance liquid chromatography (HPLC, which avoids the use of gels such as PAGE) tends to become more challenging as polynucleotide lengths increase significantly beyond a hundred or so nucleotides. One approach that can be used for generating chemically-modified RNAs of greater length is to produce two or more molecules that are ligated together. Much longer RNAs, such as those encoding a Cas9 endonuclease, are more readily generatedCRISPR-44538.601enzymatically. While fewer types of modifications are available for use in enzymatically produced RNAs, there are still modifications that can be used to, e.g., enhance stability, reduce the likelihood or degree of innate immune response, and / or enhance other attributes, as described further below and in the art; and new types of modifications are regularly being developed.

[0093] By way of illustration of various types of modifications, especially those used frequently with smaller chemically synthesized RNAs, modifications can comprise one or more nucleotides modified at the 2' position of the sugar, in some aspects a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide. In some examples, RNA modifications can comprise 2'-fluoro, 2'-amino or 2' O-methyl modifications on the ribose of pyrimidines, abasic residues, or an inverted base at the 3' end of the RNA. Such modifications can be routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target.

[0094] Many nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligonucleotide; these modified oligos survive intact for a longer time than unmodified oligonucleotides. Examples of modified oligonucleotides include those comprising modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Some oligonucleotides are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH2 — NH — O — CH2, CH, ~N(CH3)~O~CH2 (known as a methylene(methylimino) or MMI backbone), CH2 — O — N(CH3)— CH2, CH2— N(CH3)— N(CH3)— CH2and O— N(CH3)— CH2— CH2backbones, wherein the native phosphodiester backbone is represented as O — P — O — CH); amide backbones [see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)]; morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a poly(A)mide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the poly(A)mide backbone, see Nielsen et al., Science 1991, 254, 1497). Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3 'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphor amidate andCRISPR-44538.601aminoalky Iphosphoramidates , thionophosphoramidates , thionoalkylphosphonates , thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0095] Morpholino-based oligomeric compounds are described in Braasch and David Corey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue 3, (2001);Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al., Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci., 97: 9591-9596 (2000); and U.S. Pat. No.5,034,506, issued Jul. 23, 1991.

[0096] Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

[0097] Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

[0098] One or more substituted sugar moieties can also be included, e.g., one of the following at the 2' position: OH, SH, SCH3, F, OCN, OCH3OCH3, OCH3O(CH2)n CH3, O(CH2)n NH2, or O(CH2)nCH3, where n is from 1 to about 10; Cl to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; poly(A)lkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic propertiesCRISPR-44538.601of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. In some aspects, a modification includes 2'-methoxyethoxy (2'-0 — CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl)) (Martin et al, Helv. Chim. Acta, 1995, 78, 486). Other modifications include 2'-methoxy (2'-0 — CH3), 2'-propoxy (2'-OCH2CH2CH3) and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics, such as cyclobutyls in place of the pentofuranosyl group.

[0099] In some examples, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units can be replaced with novel groups. The base units can be maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can be replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases can be retained and bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA compounds can be found in Nielsen et al, Science, 254: 1497-1500 (1991).

[0100] Guide RNAs (sgRNAs, dsRNAs, tagRNAs, etc.) can also include, additionally or alternatively, nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include adenine (A), guanine (G), thymine (T), cytosine (C), and uracil (U). Modified nucleobases include nucleobases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5 -methylcytosine (also referred to as 5-methyl-2' deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5 -bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine, and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H.Freeman & Co., San Francisco, pp 75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-CCRISPR-44538.601substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C.(Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions.

[0101] Modified nucleobases can comprise other synthetic and natural nucleobases, such as 5 -methylcytosine (5-me-C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.

[0102] Further, nucleobases can comprise those disclosed in U.S. Pat. No. 3,687,808, those disclosed in ‘The Concise Encyclopedia of Polymer Science And Engineering’, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandle Chemie, International Edition’, 1991, 30, page 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’, pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, comprising 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research and Applications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspects of base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications. Modified nucleobases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588; 5,830,653; 6,005,096; and US Patent Application Publication 2003 / 0158403.

[0103] ‘ ‘Modified” in the context of a nucleic acid refers to a non-natural sugar, phosphate, or base that is incorporated into a gRNA (e.g., sgRNA, dsRNA, tagRNA, etc.), an endonuclease, or both a gRNA (e.g., sgRNA, dsRNA, tagRNA, etc.) and an endonuclease. ItCRISPR-44538.601is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single oligonucleotide, or even in a single nucleoside within an oligonucleotide.

[0104] The gRNAs (e.g., sgRNA, dsRNA, tagRNA, etc.) and / or mRNA (or DNA) encoding a site-directed polypeptide or site-directed polypeptide fusion complex can be chemically linked to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties comprise, but are not limited to, lipid moieties such as a cholesterol moiety [Letsinger et al., Proc. Natl. Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al., Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g., hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660: 306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3: 2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. Acids Res., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol or undecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) and Svinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. Acids Res., 18: 3777-3783 (1990)]; a poly(A)mine or a polyethylene glycol chain [Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)]; adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36: 3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys. Acta, 1264: 229-237 (1995)]; or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J. Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

[0105] Sugars and other moieties can be used to target proteins and complexes comprising nucleotides, such as cationic polysomes and liposomes, to particular sites. For example, hepatic cell directed transfer can be mediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, et al., Protein Pept Lett. 21(10): 1025-30 (2014).CRISPR-44538.601

[0106] Other systems known in the art and regularly developed can be used to target biomolecules of use in the present case and / or complexes thereof to cells of interest.

[0107] These targeting moieties or conjugates can include conjugate groups covalently bound to functional groups, such as primary or secondary hydroxyl groups. Conjugate groups of the invention include intercalators, reporter molecules, poly(A)mines, poly(A)mides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Typical conjugate groups include cholesterols, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance the pharmacodynamic properties, in the context of this disclosure, include groups that improve uptake, enhance resistance to degradation, and / or strengthen sequence-specific hybridization with the target nucleic acid. Groups that enhance the pharmacokinetic properties, in the context of this invention, include groups that improve uptake, distribution, metabolism or excretion of the compounds of the present invention. Representative conjugate groups are disclosed in International Patent Application No.PCT / US92 / 09196, filed Oct. 23, 1992, and U.S. Pat. No. 6,287,860. Conjugate moieties include, but are not limited to, lipid moieties such as a cholesterol moiety, cholic acid, a thioether, e.g., hexy 1-5 -tritylthiol, a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium I,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a poly(A)mine or a polyethylene glycol chain, or adamantane acetic acid, a palmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxy cholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941.

[0108] ‘ ‘Nucleotide edit” or “intended nucleotide edit” refers to a specified deletion of one or more nucleotides at one specific position, insertion of one or more nucleotides at one specific position, substitution of a single nucleotide, or other alterations at one specific position to be incorporated into the sequence of the target gene. Intended nucleotide edits may refer to the edit on the editing template as compared to the sequence on the target strandCRISPR-44538.601of the target gene or may refer to the edit encoded by the editing template on the newly synthesized single stranded DNA that replaces the editing target sequence, as compared to the editing target sequence.

[0109] “Polynucleotide” and “oligonucleotide” can refer to a polymer or oligomer of nucleotide or nucleoside monomers consisting of naturally-occurring bases, sugars and intersugar (backbone) linkages. “Polynucleotide” and “oligonucleotide” can also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases.“Polynucleotide” and “oligonucleotide” can also include polymers or oligomers comprising both deoxy and ribonucleotide combinations or variants thereof in combination with backbone modifications, such as those described herein.

[0110] Longer polynucleotides that are less amenable to chemical synthesis and are typically produced by enzymatic synthesis can also be modified by various means. Such modifications can include, for example, the introduction of certain nucleotide analogs, the incorporation of particular sequences or other moieties at the 5' or 3' ends of molecules, and other modifications. By way of illustration, the mRNA encoding Cas9 is approximately 4 kb in length and can be synthesized by in vitro transcription. Modifications to the mRNA can be applied to, e.g., increase its translation or stability (such as by increasing its resistance to degradation with a cell), or to reduce the tendency of the RNA to elicit an innate immune response that is often observed in cells following introduction of exogenous RNAs, particularly longer RNAs such as that encoding Cas9.

[0111] Numerous such modifications have been described in the art, such as poly(A) tails, 5' cap analogs (e.g., Anti Reverse Cap Analog (ARCA) or m7G(5')ppp(5')G (mCAP)), modified 5' or 3' untranslated regions (UTRs), use of modified bases (such as Pseudo-UTP, 2-Thio-UTP, 5 -Methylcytidine-5' -Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), or treatment with phosphatase to remove 5' terminal phosphates. These and other modifications are known in the art, and new modifications of RNAs are regularly being developed.

[0112] There are numerous commercial suppliers of modified RNAs, including for example, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon and many others. As described by TriLink, for example, 5-Methyl-CTP can be used to impart desirable characteristics, such as increased nuclease stability, increased translation or reduced interaction of innate immune receptors with in vitro transcribed RNA. 5 -Methylcytidine-5 '-CRISPR-44538.601Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as well as Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innate immune stimulation in culture and in vivo while enhancing translation, as illustrated in publications by Kormann et al. and Warren et al. referred to below.

[0113] Chemically modified mRNA delivered in vivo can be used to achieve improved therapeutic effects; see, e.g., Kormann et al., Nature Biotechnology 29,154-157 (2011). Such modifications can be used, for example, to increase the stability of the RNA molecule and / or reduce its immunogenicity. Using chemical modifications such as Pseudo-U, N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substituting just one quarter of the uridine and cytidine residues with 2-Thio-U and 5-Methyl-C respectively resulted in a significant decrease in toll-like receptor (TLR) mediated recognition of the mRNA in mice. By reducing the activation of the innate immune system, these modifications can be used to effectively increase the stability and longevity of the mRNA in vivo; see, e.g., Kormann et al., supra.

[0114] Repeated administration of synthetic messenger RNAs incorporating modifications designed to bypass innate anti-viral responses can reprogram differentiated human cells to pluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30 (2010). Such modified mRNAs that act as primary reprogramming proteins can be an efficient means of reprogramming multiple human cell types. Such cells are referred to as induced pluripotency stem cells (iPSCs), and it was found that enzymatically synthesized RNA incorporating 5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could be used to effectively evade the cell's antiviral response; see, e.g., Warren et al., supra.

[0115] Other modifications of polynucleotides described in the art include, for example, the use of poly(A) tails, the addition of 5' cap analogs (such as m7G(5')ppp(5')G (mCAP)), modifications of 5' or 3' untranslated regions (UTRs), or treatment with phosphatase to remove 5' terminal phosphates — and new approaches are regularly being developed.

[0116] Compositions and techniques applicable to the generation of modified RNAs for use herein have been developed in connection with the modification of RNA interference (RNAi), including small-interfering RNAs (siRNAs). siRNAs present challenges in vivo because their effects on gene silencing via mRNA interference are generally transient, which can require repeat administration. In addition, siRNAs are double- stranded RNAs (dsRNA) and mammalian cells have immune responses that have evolved to detect and neutralize dsRNA, which is often a by-product of viral infection. Thus, there are mammalian enzymes such as PKR (dsRNA-responsive kinase), and potentially retinoic acid-inducible gene I (RIG-I), that can mediate cellular responses to dsRNA, as well as Toll-like receptors (such asCRISPR-44538.601TLR3, TLR7 and TLR8) that can trigger the induction of cytokines in response to such molecules; see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4): 440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012); Burnett et al., Biotechnol J.6(9): 1130-46 (2011); Judge and MacLachlan, Hum Gene Ther 19(2): 111-24 (2008); and references cited therein.10117] Modifications have been developed and applied to enhance RNA stability, reduce innate immune responses, and / or achieve other benefits that can be useful in connection with the introduction of polynucleotides into human cells, as described herein; see, e.g., the reviews by Whitehead K A et al., Annual Review of Chemical and Biomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, Mini Rev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin Mol Ther., 12(2): 158-67 (2010); Deleavey et al., Curr Protoc Nucleic Acid Chem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19 (2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsen et al., Front Genet 3:154 (2012).

[0118] Modified RNAs can be obtained from numerous commercial suppliers, many of which have specialized in modifications designed to improve the effectiveness of siRNAs. A variety of approaches are offered based on various findings reported in the literature. For example, Dharmacon notes that replacement of a non-bridging oxygen with sulfur (phosphorothioate, PS) has been extensively used to improve nuclease resistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery 11:125-140 (2012). Modifications of the 2'-position of the ribose have been reported to improve nuclease resistance of the internucleotide phosphate bond while increasing duplex stability (Tm), which has also been shown to provide protection from immune activation. A combination of moderate PS backbone modifications with small, well-tolerated 2'-substitutions (2'-O-Methyl, 2'-Fluoro, 2'-Hydro) have been associated with highly stable siRNAs for applications in vivo, as reported by Soutschek et al. Nature 432:173-178 (2004); and 2'-O-Methyl modifications have been reported to be effective in improving stability as reported by Volkov, Oligonucleotides 19:191-202 (2009). With respect to decreasing the induction of innate immune responses, modifying specific sequences with 2'-O-Methyl, 2'-Fluoro, 2'-Hydro have been reported to reduce TER7 / TER8 interaction while generally preserving silencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505 (2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additional modifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine, 5-methyluracil, and N6-methyladenosine have also been shown to minimize the immune effectsCRISPR-44538.601mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko, K. et al., Immunity 23:165-175 (2005).

[0119] As is also known in the art, and commercially available, a number of conjugates can be applied to polynucleotides, such as RNAs, for use herein that can enhance their delivery and / or uptake by cells, including for example, cholesterol, tocopherol and folic acid, lipids, peptides, polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther. Deliv. 4:791-809 (2013), and references cited therein.

[0120] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605- 2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

[0121] The disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions containing those molecules. As used herein, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5’ and 3’ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.

[0122] ‘ ‘Percent sequence identity” or “percent identity” refers to the percentage of identical nucleotides or amino acids in a linear polynucleotide or polypeptide sequence of a reference (“query”) nucleic acid (or its complementary strand) or polypeptide as compared to a test (“subject”) nucleic acid (or its complementary strand) or polypeptide when the two sequences are optimally aligned. Percent sequence identity may be determined, when the compared sequences are aligned for maximum correspondence, as measured using a sequence comparison algorithm described below and as known in the art, or by visual inspection.CRISPR-44538.601

[0123] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and optionally by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA). An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100.10124] ‘ ‘Reverse Transcriptase editing” or “RT editing” refers to a genome editing system by which the genome of living organisms may be modified. RT editing manipulates the genetic information of a targeted DNA site to essentially “rewrite” the coded sequences.

[0125] “Template armed guide RNA,” or “tagRNA,” refers to a genome-targeting nucleic acid that comprises one or more intended nucleotide edits for incorporation into the target nucleic acid (e.g., DNA). The tagRNA associates with and directs a site-directed RT editor polypeptide to incorporate the one or more intended nucleotide edits into the target nucleic acid (e.g., gene) via RT editing. For example, a tagRNA comprises a Cas9 guide RNA molecule that encodes the crRNA-tracrRNA fused to a primer binding site (PBS) and a reverse transcriptase template nucleic acid sequence. The PBS hybridizes to a desired genomic sequence released by the binding and cleavage of the Cas9 nickase. The 3’ end of the genomic sequence is extended by the reverse transcriptase based on the reverse transcriptase template sequence.

[0126] “Reverse Transcriptase editor” or “RT editor” generally refers to a site-directed RT editor polypeptide comprising an enzymatically impaired endonuclease domain that can nick DNA and is fused to a reverse transcriptase domain and complexed with a tagRNA. RT editing systems are described in PCT Application No. PCT / IB2025 / 052079, entitled, “RTCRISPR-44538.601EDITING COMPOSITIONS AND METHODS,” filed February 26, 2025, and Cas9 variants described in the U.S. Provisional Patent Application entitled, “PAM DIVERSIFICATION OF CAS9 VARIANTS,” filed February 26, 2025, the contents of which are incorporated herein by reference in their entirety. An exemplary RT editor comprises an enzymatically impaired Cas9 endonuclease that can nick DNA and is fused to an engineered reverse transcriptase enzyme and attached to a tagRNA. The tagRNA is capable of programming the nCas9 to recognize a target site with the encoded crRNA-tracrRNA (as does a conventional single guide RNA). The resulting nicked genomic DNA can be extended by the reverse transcriptase based on the tagRNA template sequence to contain a new sequence. Once one strand is recoded, cellular DNA repair pathways can cause conversion of the local DNA sequence to match the new sequence. Such manipulation includes, but is not limited to, insertions, deletions, and base-to-base conversions without the need for double strand breaks (DSBs) or donor DNA templates. For example, RT editing may be performed by a Cas9 CRISPR platform programmed with a tagRNA, such as an enzymatically impaired Cas9 nickase with an appropriate reverse transcriptase.

[0127] A “primer binding site (PBS)” is a single-stranded nucleic acid sequence within a tagRNA that comprises a region of complementarity to the 3’ end of the nicked DNA strand or PAM strand (i.e., the non-target strand or the edit strand). This allows annealing of the free 3’ end of the genomic DNA for extension by the reverse transcriptase based on the template sequence encoded in the tagRNA.

[0128] ‘ ‘Protein,” “polypeptide,” and “peptide” are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. “Polypeptide,” “protein,” and “peptide” refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the E-optical isomer or the D-optical isomer. More specifically, the terms “polypeptide,” “protein,” and “peptide” refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and / or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant.CRISPR-44538.601

[0129] A “protospacer” or “protospacer sequence” refers to a specific sequence in the PAM strand of the target nucleic acid that is complementary to the search target sequence.

[0130] A “protospacer adjacent motif (PAM),” “PAM sequence,” or “P AM-like motif’ refers to a short DNA sequence immediately following the protospacer sequence on the PAM strand of a target nucleic acid (e.g., gene). For example, a PAM sequence can be used by a Cas9 / sgRNA to form an R-loop to interrogate a specific DNA sequence through Watson-Crick pairing of its gRNA with the genome. The PAM specificity may be a function of the DNA-binding specificity of the site-directed polypeptide (e.g., Cas9 protein, via a “protospacer adjacent motif recognition domain” or “PAM-interacting (PI) domain” at the C-terminus of Cas9).

[0131] ‘ ‘Reverse transcriptase template” refers to a ribonucleic acid sequence that is utilized as a substrate for a reverse transcriptase protein that is part of the fusion protein complex as contemplated herein. Such templates provide the necessary information to edit a DNA sequence to support conversions including, but not limited to, base conversions, sequence insertions or sequence deletions.

[0132] As used herein “sequence identity” refers to the extent to which two optimally aligned nucleic acid sequences or amino acid sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. “Identity” 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., fed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0133] A nucleic acid sequence is “substantially identical” to another nucleic acid sequence if it has at least 70% or more sequence identity to the other nucleic acid sequence. For purposes of percent sequence identity between an RNA sequence (e.g., spacer) and a DNA sequence (e.g., target gene protospacer), uracil bases in the RNA are to be considered identical to thymine bases in DNA sequences. The disclosure contemplates nucleic acid sequences that have at least 70%, at least 75% , at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a nucleic acid described herein (e.g., genome targeting nucleic acid). An amino acid sequence is “substantially identical” to another amino acid sequence if it has at least 70% or moreCRISPR-44538.601sequence identity to the other amino acid sequence. The disclosure contemplates amino acid sequences that have at least 70%, at least 75% , at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence described herein (e.g., site-directed polypeptide).

[0134] “sgRNA” refers to single guide RNA used in conjunction with CRISPR associated systems (Cas). sgRNAs are a fusion of crRNA and tracrRNA and contain nucleotides of sequence complementary to the desired target site (Jinek, et al. 2012 (PMID 22745249)). Watson-Crick pairing of the sgRNA with the target site permits R-loop formation, which in conjunction with a functional PAM permits DNA cleavage or in the case of nuclease-deficient Cas9 allows binds to the DNA at that locus.

[0135] ‘ ‘Site-directed polypeptide” refers to a polypeptide used in gene editing, base editing, and RT editing to cleave DNA. The site-directed polypeptide can be administered to a cell or subject as either one or more polypeptides, or one or more mRNAs encoding the polypeptide. A site-directed polypeptide that is used in an exemplary CRISPR / Cas gene editing system can comprise a nuclease domain and a PAM-interacting domain (PI domain). A site-directed polypeptide used as a “base editor (BE)” is also referred to herein as a “site-directed base editor polypeptide” that is capable of modifying a base (e.g., A, T, C, G, or U) within a target nucleic acid sequence (e.g., DNA or RNA). Base editing allows for the conversion of one DNA base pair into another in a target nucleic acid. In contrast to a conventional site-directed polypeptide used for gene editing that comprises at least one nuclease domain, a site-directed base editor polypeptide further comprises at least one deaminase domain. Base editing can change C (cytosine) to T (thymine) or A (adenine) to G (guanine) in the endogenous DNA. In base editing, a genome-targeting nucleic acid (e.g., gRNA) can be designed to target a specific genomic location of interest in the cells of a target tissue, organ, or subject. A site-directed polypeptide used as a “RT editor” is referred to herein as a “site-directed RT editor polypeptide.” RT editing enables complex and precise DNA modifications in the genomes of cells, including insertions, deletions, and all 12 possible base-to-base conversions (A, C, G, T) without double-strand breaks in target DNA, or without donor DNA templates. “RT editing” refers to programmable editing of a target nucleic acid sequence using a site-directed RT editor polypeptide complexed with a genometargeting nucleic acid called a template armed guide RNA (tagRNA), to incorporate an intended nucleotide edit (also referred to herein as a nucleotide change) into the target nucleic acid through target-primed nucleic acid synthesis. In contrast to a conventional site directed polypeptide used for gene editing that comprises at least one nuclease domain, a site-directedCRISPR-44538.601RT editor polypeptide further comprises at least one reverse transcriptase domain. A tagRNA can comprise a guide RNA scaffold, a spacer that specifies the target nucleic acid, a primer binding site (PBS) that is complementary to a target nucleic acid, and a reverse transcriptase template (RTT) that encodes a desired edit. A site-directed RT editing polypeptide-tagRNA complex pairs with a strand of a target nucleic acid (e.g., genomic DNA) and nicks the opposite strand to generate an exposed 3' end that binds the PBS of the tagRNA to form a primer-template complex. The RT engages the primer-template complex and commences RTT reverse transcription. The newly synthesized 3' DNA flap containing the edit is incorporated into the genome, replacing the original DNA sequence and permanently installing the desired edit. Though “site-directed base editor polypeptide” and “site-directed RT editor polypeptide” refer specifically to site-directed polypeptides that have been configured respectively to perform base and RT editing, it should be appreciated that “site-directed polypeptide” generally encompasses site-directed polypeptides that can be used for gene editing, base editing and RT editing.

[0136] A “spacer extension sequence” refers to sequence that can modify activity, provide stability and / or provide a location for modifications of a genome-targeting nucleic acid.

[0137] “Split-intein site-directed polypeptide” refers to site-directed polypeptide that has been split into amino-terminal (-N) and carboxy-terminal (-C) segments, which are then fused into a full length polypeptide by a trans- splicing intein. This configuration imparts flexibility to a site-directed polypeptide thereby facilitating a packaging into an adeno-associated virus (AAV).

[0138] “Upstream” and “downstream” define relevant positions of at least two regions or sequences in a nucleic acid molecule orientated in a 5’-to-3’ direction. For example, a first sequence is upstream of a second sequence in a nucleic acid molecule (e.g., DNA) where the first sequence is positioned 5’ to the second sequence and the second sequence is downstream of the first sequence.

[0139] ‘ ‘Vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector, wherein additional nucleic acid segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, andCRISPR-44538.601thereby are replicated along with the host genome. Vectors can be capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors,” or more simply “expression vectors,” which serve equivalent functions. “Operably linked” means that the nucleotide sequence of interest is linked to regulatory sequence(s) in a manner that allows for expression of the nucleotide sequence. “Regulatory sequence” is intended to include, for example, promoters, enhancers and other expression control elements (e.g., poly(A)denylation signals). Such regulatory sequences are well known in the art and are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cells, and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). 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 target cell, the level of expression desired, and the like. LNP Definitions

[0140] As used in the specification and appended claims, unless specified to the contrary, the following lipid nanoparticle terms have the meaning indicated:10141] ‘ ‘Effective amount” or “therapeutically effective amount” refers to that amount of a compound disclosed herein which, when administered to a mammal, preferably a human, is sufficient to effect treatment in the mammal, preferably a human. The amount of a compound which constitutes a “therapeutically effective amount” will vary depending on the compound, the condition and its severity, the manner of administration, and the age of the mammal to be treated, but can be determined routinely by one of ordinary skill in the art having regard to his own knowledge and to this disclosure.

[0142] “Ionizable lipid” refers to lipid species that are positively charged at acidic pH, but are neutral at physiological pH. Examples of ionizable lipids include, but are not limited to, ALC-0315, SM-102, DLin-MC3-DMA (MC3), DODAP, and LP-01.

[0143] ‘ ‘Cationic lipid” refers to lipid species with a permanent positive charge. Examples of cationic lipids include, but are not limited to, DOTMA (l,2-dioleoyl-3-trimethylammonium propane), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DC-Chol (3a-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol hydrochloride), and DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate).CRISPR-44538.601

[0144] ‘ ‘Neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, but are not limited to, phosphotidylcholines such as l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as 1 ,2-Dioleoyl-sn-glycero-3 -phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived.

[0145] “Lipid nanoparticle” refers to particles having at least one dimension on the order of nanometers (e.g., 1-1,000 nm) which include one or more of the compounds of structure (I) or other specified cationic lipids. In some embodiments, lipid nanoparticles are included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid (e.g., mRNA) to a target site of interest (e.g., cell, tissue, organ, tumor, and the like). In some embodiments, the lipid nanoparticles of the invention comprise a nucleic acid. Such lipid nanoparticles typically comprise a compound of Formula (I), (II), (III), or (IV) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids. In some embodiments, the active agent or therapeutic agent, such as a nucleic acid, may be encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells e.g. an adverse immune response.

[0146] In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, nucleic acids, when present in the lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease. Lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 2004 / 0142025,CRISPR-44538.6012007 / 0042031 and PCT Pub. Nos. WO 2013 / 016058 and WO 2013 / 086373, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0147] “Polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG) and the like.

[0148] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.1. Chemically Modified RNA Sequences

[0149] The present disclosure provides a library of chemically modified RNA sequences that can be ligated or otherwise attached to the 3’ end to an RNA sequence of interest (e.g., an mRNA sequence encoding a gene of interest, a vaccine or an RNA pharmaceutical). The chemically modified RNA sequences described herein may be used to tune the stability and translation of the RNA sequence to which it is ligated.

[0150] In some embodiments, the synthetic RNA molecules in the library comprise the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’, wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 (or any range included therein), preferably from 1 to 4; and Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides, and wherein at least one of Ri, R2, R3, R4, Rs, and Re is an adenosine ribonucleotide comprising a modified sugar moiety.

[0151] In some preferred embodiments, one of Ri, R2, R3, R4, Rs, and Re is an adenosine ribonucleotide comprising a modified sugar moiety. In some preferred embodiments, two of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety. In some preferred embodiments, three of Ri, R2,R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety. In some preferred embodiments, four of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety. In some preferred embodiments, five of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety. In some preferred embodiments, all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.CRISPR-44538.601

[0152] The sequences of the present disclosure are not limited to the use of any particular adenosine ribonucleotide comprising a modified sugar moiety. In some preferred embodiments, the modified sugar moiety included in the adenosine ribonucleotide is selected from the group consisting of a locked nucleic acid (LNA) subunit, a 2'0,4'C-ethylene-bridged nucleic acid (ENA) subunit, a tricyclo-DNA (tc-DNA) subunit, a 2' O-methyl subunit, a 2' O-methoxyethyl subunit, a 2'-fluoro subunit, and a 2'-O-[2-(N-methylcarbamoyl)ethyl] subunit, and combinations thereof when the sequences include two or more adenosine ribonucleotides comprising a modified sugar moiety. In some particularly preferred embodiments, the modified sugar moiety included in the adenosine ribonucleotide is selected from the group consisting of a 2' O-methyl subunit and a 2' O-methoxyethyl subunit, and combinations thereof when the sequences include two or more adenosine ribonucleotides comprising a modified sugar moiety. For example, one, two, three, four, five or all of Ri, R2, R3, R4, Rs, and Re may be an adenosine ribonucleotide comprising a 2' O-methyl subunit, one, two, three, four, five or all of Ri, R2, R3, R4, Rs, and Re may be an adenosine ribonucleotide comprising a 2' O-methoxyethyl subunit, or one or more of Ri, R2, R3, R4, Rs, and Re may be an adenosine ribonucleotide comprising a 2' O-methyl subunit and one or more of Ri, R2, R3, R4, Rs, and Re may be an adenosine ribonucleotide comprising a 2' O-methoxyethyl subunit so that Ri, R2, R3, R4, Rs, and Re comprise a combination of adenosine ribonucleotides individually comprising 2' O-methoxyethyl and a 2' O-methyl subunits.

[0153] In some embodiments, the RNA molecules described herein may comprise further modifications. In some embodiments, the RNA molecules comprise a modified internucleoside linkage between one or more of Ri, R2, R3, R4, Rs, and Re. In some preferred embodiments, the modified internucleoside linkage is a phosphorothioate internucleoside linkage. In some embodiments, there is at least one modified internucleoside linkage in the linked adenosine nucleotides defined by Ri, R2, R3, R4, Rs, and Re. In some embodiments, there are at least two modified internucleoside linkage in the linked adenosine nucleotides defined by Xn-Ri-R2-R3-R4-Rs-R6. In some embodiments, there are at least three modified internucleoside linkage in the linked adenosine nucleotides defined by Xn-Ri-R2-R3-R4-Rs-Re. In some embodiments, there are at least four modified internucleoside linkage in the linked adenosine nucleotides defined by Xn-Ri-R2-R3-R4-Rs-R6. In some embodiments, there are at least five modified internucleoside linkage in the linked adenosine nucleotides defined by Xn-Ri-R2-R3-R4-Rs-R6. In some embodiments, there are six modified internucleoside linkage in the linked adenosine nucleotides defined by Xn-Ri-R2-R3-R4-R5-R6.CRISPR-44538.601

[0154] In some embodiments, the synthetic RNA molecules comprise the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0; Ri, R2, R3, R4, Rs, and R6are adenosine ribonucleotides, and wherein at least four of Ri, R2, R3, R4, Rs, and Re comprise a modified internucleoside linkage, preferably a phosphorothioate internucleoside linkage.

[0155] In some embodiments, Re is the 3’ terminal nucleotide of the RNA molecules described herein. In some embodiments, Re is selected from the group consisting of an adenosine ribonucleotide comprising a 2' O-methyl subunit, an adenosine ribonucleotide comprising a 2' O-methyl subunit and a 3’ inverted T modification, a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification, and a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification and a 3’ inverted T modification.

[0156] In some embodiments, the synthetic RNA molecules comprise the sequence 5’-Xn-RI-R2-R3-R4-RS-R6-3’ wherein: X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0; Ri, R2, R3, R4, and R5are unmodified adenosine ribonucleotides and Re is a modified adenosine comprising a 3’ inverted T modification.

[0157] In some embodiments, the synthetic RNA molecules described herein comprise a 5’ terminal nucleotide comprising a 5’ phosphorylation at the 5’ Xnposition of the molecule. In some preferred embodiments, the 5 ’terminal nucleotide is an adenosine ribonucleotide comprising a 5’ terminal phosphorylation.

[0158] In the RNA molecules described herein, X in the sequence defined by Xn-Ri-R2-R3-R4-R5-R6 may be any ribonucleotide. The ribonucleotides defined by X may be modified ribonucleotides as described above, unmodified ribonucleotides, or combinations thereof. In some embodiments, X is preferably an adenosine ribonucleotide. In some embodiments, X is most preferably an unmodified adenosine ribonucleotide.

[0159] In the RNA molecules described herein X may vary in length. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-200. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-100. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-50. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-20. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-10. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-9. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-8. In someCRISPR-44538.601embodiments, n in the sequence defined by Xn-Ri-Ra-Ra-Rt-Rs-Re is from 1-7. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-6. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-5. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-4. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-3. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 is from 1-2. In some embodiments, n in the sequence defined by Xn-Ri-R2-R3-R4-Rs-R6 islO, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0.

[0160] In some embodiments, the RNA molecules have a sequence corresponding any one of SEQ ID NOs:l-55.2. RNA Sequences of Interest

[0161] The chemically modified RNA sequences of the present disclosure may be ligated or otherwise operably linked to a RNA sequence of interest. Accordingly, the present disclosure provides RNA molecule comprising a chemical modified RNA sequence as described herein operably linked to an RNA sequence of interest. The present invention is not limited to any particular RNA sequence of interest. Suitable RNA sequences of interest include those encoding a protein of interest, a vaccine, of a therapeutic RNA sequence. In some embodiments, the present invention provides methods of producing an RNA sequence comprising ligating a chemically modified RNA sequence as described above to an RNA sequence of interest.

[0162] In some embodiments, the RNA sequence of interest encodes a component of a gene editing system. The disclosure contemplates any genome editing system known to the skilled artisan, including clustered regularly interspaced short palindromic repeats (CRISPR) / Cas nuclease gene editing systems, base editing systems, reverse transcriptase (RT) editing systems, transcription activator-like effector nuclease (TALEN) gene editing systems, and zinc finger nuclease (ZFN) gene editing systems. The gene editing systems, base editing systems, and RT editing systems described herein use a site-directed polypeptide such as a nuclease that is directed by a genome-targeting nucleic acid to modify a target nucleotide sequence of a genome in a precise or pre-determined manner.

[0163] The base editing, gene editing, and RT editing systems described herein comprise site-directed polypeptides, which can be encoded by the RNA molecules described herein, that associate and / or complex with a genome-targeting nucleic acid to edit a genome of a cell in vitro, ex vivo, or in vivo (e.g., in a human subject). In the context of a CRISPR / Cas, CRISPR / Cpfl system, or base editing system, the site-directed polypeptide can bind to aCRISPR-44538.601gRNA that, in turn, specifies the site in the target DNA to which the polypeptide is directed. In the context of an RT editing system, the site-directed polypeptide can bind to a template armed guide RNA (tagRNA) that, in turn, specifies the site in the target DNA to which the polypeptide is directed. In some embodiments, the RNA sequence of interest encodes a site directed polypeptide. In some embodiments, the site directed polypeptide is a nuclease.

[0164] Accordingly, a site-directed polypeptide of a base editing system, gene editing system, and / or RT editing system disclosed herein can comprise one or more nuclease domains (e.g., a domain having endonuclease activity). The site-directed polypeptide can be engineered to have one or more domains having endonuclease activity or a wild-type polypeptide with one or more domains having endonuclease activity (e.g., Cas9 from S. pyogenes). In the gene editing (e.g., CRISPR / Cas or CRISPR / Cpfl systems), base editing, and RT editing systems disclosed herein, the site-directed polypeptide can be an endonuclease, such as a DNA endonuclease. Exemplary Cas proteins (e.g., DNA nucleases) include, without limitation, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csnl and Csxl2), CaslO, Casll, Casl2a / Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2d / CasY, Casl2e / CasX, Cas 12g, Casl2h, Casl2i, Csyl, Csy2, Csy3, Csy4, Csel , Cse2, Cse3, Cse4, Cse5e, Cscl , Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl , Cmr3, Cmr4, Cmr5, Cmr6, Csbl , Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csxl5, Csfl , Csf2, Csf3, Csf4, Cpfl, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, SluCas9, Type II Cas effector proteins, Type V Cas effector proteins, Type VI Cas effector proteins, CARF, DinG, Cpfl, Casl2b / C2cl, Casl2c / C2c3, Casl2b / C2cl, Casl2c / C2c3, SpCas9(K855A), eSpCas9(l.l), SpCas9-HFl, hyper accurate Cas9 variant (HypaCas9), CasB, and homologs, modified or engineered variants, mutants, and / or functional fragments thereof. In some embodiments, the site-directed polypeptide of a base editing system, gene editing system, and / or RT editing system is encoded by an RNA and operably linked to a chemically modified RNA poly(A) tail of the present disclosure.

[0165] The base editing, gene editing and RT editing systems described herein use a genome-targeting nucleic acid that can direct a site-directed polypeptide to a target nucleic acid sequence to edit a target gene in the genome of a cell, tissue, or organism (e.g., human subject). The disclosure is not limited to any particular type of genome-targeting nucleic acid. The skilled artisan will appreciate that the genome-targeting nucleic acid can depend on the type of genomic editing used.CRISPR-44538.601

[0166] The genome-targeting nucleic acid can be an RNA. A guide RNA can comprise at least a spacer sequence that hybridizes to a target nucleic acid sequence of interest, and a CRISPR repeat sequence. In Type II systems, the gRNA also comprises a second RNA called the trans-activating RNA (tracrRNA) sequence. In the Type II guide RNA (gRNA), the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V guide RNA (gRNA), the crRNA forms a duplex. In both systems, the duplex can bind a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex.

[0167] The genome-targeting nucleic acid can provide target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus can direct the activity of the site-directed polypeptide.

[0168] The genome-targeting nucleic acid can be a single-molecule guide RNA (sgRNA). A single-molecule guide RNA (sgRNA) in a Type II system can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension can comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker can link the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension can comprise one or more hairpins. A single-molecule guide RNA (sgRNA) in a Type V system can comprise, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.

[0169] The genome-targeting nucleic acid can be an RT editing guide RNA (tagRNA) molecule. A tagRNA can comprise an RNA scaffold, a guide sequence (spacer), a flap binding site (FBS) and an editing template (ET). The tagRNA uses the spacer to direct a site-directed RT-editor polypeptide to a target nucleic acid sequence in a genome (protospacer) and initiates a nuclease- or nickase-mediated strand nick at a nick site, resulting in 3’ end hybridization to the FBS and initiation of reverse transcription by the ET to form a pair of redundant single- stranded DNA flaps. Equilibrium between the edited 3’ flap and unedited 5’ flap mediates cleavage, ligation, DNA repair, and the incorporation of the desired edit encoded in the ET into the genome of a cell.

[0170] The gene editing system may further comprise a donor polynucleotide or sequence. In some embodiments, the donor template is DNA molecule that has homology to a target sequence. The donor template is an exogenous polynucleotide sequence that can be insertedCRISPR-44538.601into a target nucleic acid cleavage site. The donor polynucleotide, a portion of the donor polynucleotide, a copy of the donor polynucleotide, or a portion of a copy of the donor polynucleotide can be inserted into the target nucleic acid cleavage site.

[0171] In some embodiments, the gene editing systems described herein include using site-directed nucleases to cut deoxyribonucleic acid (DNA) at precise target locations in the genome, thereby creating single-strand or double-strand DNA breaks at particular locations within the genome. Such breaks can be and regularly are repaired by natural, endogenous cellular processes, such as homology-directed repair (HDR) and NHEJ, as reviewed in Cox et al., Nature Medicine 21(2), 121-31 (2015). These two main DNA repair processes consist of a family of alternative pathways. NHEJ directly joins the DNA ends resulting from a doublestrand break, sometimes with the loss or addition of nucleotide sequence, which may disrupt or enhance gene expression. HDR utilizes a homologous sequence, or donor sequence, as a template for inserting a defined DNA sequence at the break point. The homologous sequence can be in the endogenous genome, such as a sister chromatid. Alternatively, the donor can be an exogenous nucleic acid, such as a plasmid, a single-strand oligonucleotide, a doublestranded oligonucleotide, a duplex oligonucleotide or a virus, that has regions of high homology with the nuclease-cleaved locus, but which can also contain additional sequence or sequence changes including deletions that can be incorporated into the cleaved target locus. A third repair mechanism can be microhomology-mediated end joining (MMEJ), also referred to as “Alternative NHEJ,” in which the genetic outcome is similar to NHEJ in that small deletions and insertions can occur at the cleavage site. MMEJ can make use of homologous sequences of a few base pairs flanking the DNA break site to drive a more favored DNA end joining repair outcome, and recent reports have further elucidated the molecular mechanism of this process; see, e.g., Cho and Greenberg, Nature 518, 174-76 (2015); Kent et al., Nature Structural and Molecular Biology, Adv. Online doi:10.1038 / nsmb.2961(2015); Mateos-Gomez et al., Nature 518, 254-57 (2015); Ceccaldi et al., Nature 528, 258-62 (2015).

[0172] Accordingly, in some embodiments, the present disclosure provides an RNA sequence of interest that is operably linked to a chemically modified RNA poly(A) sequence as described above. In preferred embodiments, the chemically modified RNA poly(A) sequence is ligated to the RNA sequence of interest. In some embodiments, the RNA sequence of interest may comprise a poly(A) tail and the chemically modified RNA poly(A) sequence is ligated to the 3’ terminal of the preexisting poly(A) tail of the RNA sequence of interest. In some embodiments, the RNA sequence of interest is produced by in vitro transcription of a DNA sequence encoding the RNA sequence of interest. In otherCRISPR-44538.601embodiments, the RNA sequence of interest may be isolated from a cell or organism or may be produced by chemical synthesis.

[0173] In some embodiments, the RNA sequence of interest encodes a protein of interest. In some embodiments, the protein of interest is an enzyme. In some embodiments, the protein of interest is an enzyme that is a component of a gene editing system as described above. In some embodiments, the enzyme is selected from the group consisting of a CRISPR / Cas nuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a nickase, a nickase-reverse transcriptase fusion enzyme, and a dCAS-deaminase fusion enzyme. In some embodiments, the enzyme is a CRISPR / Cas9 nuclease.

[0174] In some embodiments, the present invention provides a gene editing system in which one or more of the components of the gene editing system is an RNA sequence of interest operably linked to a chemically modified poly(A) sequence as described in detail above. In some embodiments, the present invention provides a gene editing system comprising a CRISPR / Cas nuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a nickase, a nickase-reverse transcriptase fusion enzyme, or a dCAS-deaminase fusion enzyme and a guide sequence selected from the group consisting of a gRNA, sgRNA, and template armed guide RNA. In some embodiments, the gene editing system further comprises a donor polynucleotide. In some embodiments, the present invention provides methods of editing a target gene in a genome comprising contacting a cell in vivo or in vitro with the gene editing system under conditions such that the target gene is edited.

[0175] In some embodiments, the protein of interest is a therapeutic protein. Examples of therapeutic proteins include, but are not limited to, antibodies (i.e., antibody heavy and light chains), single chain antibodies, minibodies, hormones and related proteins such as insulin and human growth hormone, erythropoietin, interferons, blood factors such as factor VIII, and cytokines.

[0176] In some embodiments, the RNA sequence of interest encodes an RNA vaccine. See, e.g., Gote et al., A Comprehensive Review of mRNA Vaccines, Int J Mol Sci. 2023 Jan 31;24(3):2700. Examples of mRNA vaccine targets include, but are not limited to, HIV, RSV, Influenza A, Influenza B, seasonal influenza strains, SARS-CoV-2, CMV, cancer associated epitopes and neoepitopes (e.g., vaccine directed against ovarian cancer, colon cancer, melanoma, hepatocellular cancer, genitourinary cancer, squamous cell carcinoma, head and neck cancer, cervical cancer, breast cancer, prostate cancers, glioblastoma, acute myeloid leukemia, and solid tumors), Zika virus, and Epstein-Barr virus. Specific exemplaryCRISPR-44538.601vaccines include, but are not limited to: eOD-GT8 60mer mRNA; Core-g28v2 60mer mRNA vaccine; G505 MD39.3 mRNA, BG505 MD39.3 gpl51 mRNA, and BG505 MD39.3 gpl51 CD4K0 mRNA; mRNA-1345; mRNA-1273.214; mRNA-1020, mRNA-1030, and mRNA-1010; monovalent influenza modRNA vaccine (mIRV), bivalent influenza modRNA vaccine (bIRV AB, bIRV AA, and bIRV BB); quadrivalent influenza modRNA vaccine (qlRV); Seasonal quadrivalent influenza mRNA vaccine CVSQIV; PF-07852352, PF-07836391, PF-07836394, PF-07836395, PF-07836396, and PF-07867246; mRNA NA vaccine; mRNA-1647; mRNA -1215; W_oval vaccine; NCI-4650; BNT113; BNT111; IVAC_W_brel_uID and IVAC_W_brel_uID / IVAC_M_uID; mRNA-1893; Lipo-MERIT; mRNA-4157; mRNA-1653; mRNA-1189; and Moderna and Pfizer SARS-CoV-2 vaccines.

[0177] In some embodiments, the RNA sequence of interest encodes an RNA-based therapeutic molecule. Examples of RNA-based therapeutic molecules include, but are not limited to, antisense oligonucleotides, siRNAs, and miRNAs.3. Delivery of RNA molecules

[0178] The present disclosure further provides pharmaceutical compositions and formulations for delivery of the RNA molecules described above.

[0179] In an embodiment, the pharmaceutical composition comprises any one (or more) of the foregoing RNA molecules and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for oral administration. In other embodiments, the pharmaceutical composition is formulated for injection. In still more embodiments, the pharmaceutical compositions comprise a RNA molecule as disclosed herein and an additional therapeutic agent (e.g., anticancer agent). Non-limiting examples of such additional therapeutic agents are described herein below.

[0180] In certain embodiments, an RNA molecule as described herein is administered in a local rather than systemic manner, for example, via injection of the RNA molecule directly into an organ, often in a depot preparation or sustained release formulation. In specific embodiments, long acting formulations are administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Furthermore, in other embodiments, the RNA molecule is delivered in a targeted drug delivery system, for example, in a liposome coated with an organ specific antibody. In such embodiments, the liposomes are targeted to and taken up selectively by the organ. In yet other embodiments, the RNA molecule as described herein is provided in the form of a rapid release formulation, in the form of an extended release formulation, or in the form of an intermediate releaseCRISPR-44538.601formulation. In yet other embodiments, the RNA molecule described herein is administered topically.

[0181] In treatment methods according to embodiments of the disclosure, an effective amount of RNA molecule as described herein is administered to a subject suffering from or diagnosed as having such a disease, disorder, or medical condition. Effective amounts or doses may be ascertained by methods such as modeling, dose escalation studies or clinical trials, e.g., the mode or route of administration or drug delivery, the pharmacokinetics of the agent, the severity and course of the disease, disorder, or condition, the subject's previous or ongoing therapy, the subject's health status and response to drugs, and the judgment of the treating physician.

[0182] In some embodiments, RNA molecules of the disclosure are administered in a single dose. In an embodiment, the single dose is administered orally. In another embodiment, the single dose is administered by injection. However, other routes are used as appropriate. In some embodiments, RNA molecules of the disclosure are administered in multiple doses. In some embodiments, dosing is about once, twice, three times, four times, five times, six times, or more than six times per day. In other embodiments, dosing is about once a month, once every two weeks, once a week, or once every other day. In another embodiment RNA molecules of the disclosure and another agent (e.g., an additional anticancer agent) are administered together about once per day to about 6 times per day. In another embodiment the administration of RNA molecules of the disclosure and an agent continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.

[0183] Administration of RNA molecules of the disclosure may continue as long as necessary. In some embodiments, RNA molecules of the disclosure are administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 days. In some embodiments, RNA molecules of the disclosure are administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, RNA molecules of the disclosure are administered chronically on an ongoing basis, e.g., for the treatment of chronic effects.

[0184] In some embodiments, the RNA molecules of the disclosure are administered in individual dosage forms. It is known in the art that due to intersubject variability in RNA molecule pharmacokinetics, individualization of dosing regimen is necessary for optimal therapy.CRISPR-44538.601

[0185] In some embodiments, the RNA molecules described herein are formulated into pharmaceutical compositions. In specific embodiments, pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the disclosed RNA molecules into preparations which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any pharmaceutically acceptable techniques, carriers, and excipients are used as suitable to formulate the pharmaceutical compositions described herein: Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkinsl999).

[0186] Provided herein are pharmaceutical compositions comprising one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, and a pharmaceutically acceptable carrier. In certain embodiments, the RNA molecules described are administered as pharmaceutical compositions in which one or more RNA molecules selected from RNA molecules as described herein are mixed with other active ingredients, as in combination therapy.

[0187] A pharmaceutical composition, as used herein, refers to a mixture of one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. In certain embodiments, the pharmaceutical composition facilitates administration of the RNA molecule to an organism. In some embodiments, therapeutically effective amounts of one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, are administered in a pharmaceutical composition to a mammal having a disease, disorder or medical condition to be treated. In specific embodiments, the mammal is a human. In certain embodiments, therapeutically effective amounts vary depending on the severity of the disease, the age and relative health of the subject, the potency of the RNA molecule used and other factors. The RNA molecules described herein are used singly or in combination with one or more therapeutic agents as components of mixtures.CRISPR-44538.601

[0188] In one embodiment, one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, are formulated in aqueous solutions. In specific embodiments, the aqueous solution is selected from, by way of example only, a physiologically compatible buffer, such as Hank’s solution, Ringer’s solution, or physiological saline buffer. In other embodiments, one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, are formulated for transmucosal administration. In specific embodiments, transmucosal formulations include penetrants that are appropriate to the barrier to be permeated. In still other embodiments wherein the RNA molecules described herein are formulated for other parenteral injections, appropriate formulations include aqueous or nonaqueous solutions. In specific embodiments, such solutions include physiologically compatible buffers and / or excipients.

[0189] In another embodiment, RNA molecules described herein are formulated for oral administration. RNA molecules described herein are formulated by combining the active RNA molecules with, e.g., pharmaceutically acceptable carriers or excipients. In various embodiments, the RNA molecules described herein are formulated in oral dosage forms that include, by way of example only, tablets, powders, pills, dragees, capsules, liquids, gels, syrups, elixirs, slurries, suspensions and the like.

[0190] In one embodiment, the oral dosage forms, such as a pill, capsule or tablet, comprises one or more suitable layers or coatings. In specific embodiments, concentrated sugar solutions are used for coating the dosage form. The sugar solutions, optionally contain additional components, such as by way of example only, gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs and / or pigments are also optionally added to the coatings for identification purposes. Additionally, the dyestuffs and / or pigments are optionally utilized to characterize different combinations of active RNA molecule doses.

[0191] In certain embodiments, therapeutically effective amounts of at least one of the RNA molecules described herein are formulated into other oral dosage forms. Oral dosage forms include push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. In specific embodiments, push fit capsules contain the active ingredients in admixture with one or more filler. Fillers include, by way of example only, lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In other embodiments, soft capsules, containCRISPR-44538.601one or more active RNA molecule that is dissolved or suspended in a suitable liquid. Suitable liquids include, by way of example only, one or more fatty oil, liquid paraffin, or liquid polyethylene glycol. In addition, stabilizers are optionally added.

[0192] In still other embodiments, the RNA molecules described herein are formulated for parental injection, including formulations suitable for bolus injection or continuous infusion. In specific embodiments, formulations for injection are presented in unit dosage form (e.g., in ampoules) or in multi dose containers. Preservatives are, optionally, added to the injection formulations. In still other embodiments, the pharmaceutical compositions are formulated in a form suitable for parenteral injection as sterile suspensions, solutions or emulsions in oily or aqueous vehicles. Parenteral injection formulations optionally contain formulatory agents such as suspending, stabilizing and / or dispersing agents. In specific embodiments, pharmaceutical formulations for parenteral administration include aqueous solutions of the active RNA molecules in water soluble form.

[0193] Methods for the preparation of compositions comprising the RNA molecules described herein include formulating the RNA molecule(s) with one or more inert, pharmaceutically acceptable excipients or carriers to form a solid, semi-solid or liquid composition. Solid compositions include, but are not limited to, powders, tablets, dispersible granules, capsules, cachets, and suppositories. Liquid compositions include solutions in which a RNA molecule is dissolved, emulsions comprising a RNA molecule, or a solution containing liposomes, micelles, or nanoparticles comprising a RNA molecule as disclosed herein. Semi-solid compositions include, but are not limited to, gels, ointments, suspensions and creams. The form of the pharmaceutical compositions described herein include liquid solutions or suspensions, solid forms suitable for solution or suspension in a liquid prior to use, or as emulsions. These compositions also optionally contain minor amounts of nontoxic, auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and so forth.

[0194] In some embodiments, pharmaceutical compositions comprising one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, illustratively takes the form of a liquid where the agents are present in solution, in suspension or both. Typically when the composition is administered as a suspension, the first portion of the agent is present in solution and a second portion of the agent is present in particulate form, in suspension in a liquid matrix. In some embodiments, a liquid composition includes a gel formulation. In other embodiments, the liquid composition is aqueous.CRISPR-44538.601

[0195] In certain embodiments, aqueous suspensions contain one or more polymers as suspending agents. Polymers include water-soluble polymers such as cellulosic polymers, e.g., hydroxypropyl methylcellulose, and water-insoluble polymers such as cross-linked carboxyl-containing polymers. Certain pharmaceutical compositions described herein comprise a mucoadhesive polymer, selected for example from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methylmethacrylate), poly(A)crylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran.

[0196] Pharmaceutical compositions also, optionally, include solubilizing agents to aid in the solubility of one or more RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail. The term "solubilizing agent" generally includes agents that result in the formation of a micellar solution or a true solution of the agent. Certain acceptable nonionic surfactants, for example polysorbate 80, are useful as solubilizing agents, as ophthalmically acceptable glycols, poly glycols, e.g., polyethylene glycol 400, and glycol ethers.

[0197] Furthermore, pharmaceutical compositions optionally include one or more pH adjusting agents or buffering agents, including acids such as acetic, boric, citric, lactic, phosphoric and hydrochloric acids; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate and trishy droxymethylaminomethane; and buffers such as citrate / dextrose, sodium bicarbonate and ammonium chloride. Such acids, bases and buffers are included in an amount required to maintain pH of the composition in an acceptable range.

[0198] Compositions also, optionally, include one or more pharmaceutically acceptable salts in an amount required to bring osmolality of the composition into an acceptable range. Such pharmaceutically acceptable salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable pharmaceutically acceptable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0199] Other pharmaceutical compositions optionally include one or more preservatives to inhibit microbial activity. Suitable preservatives include mercury-containing substances such as merfen and thiomersal; stabilized chlorine dioxide; and quaternary ammonium RNA compounds such as benzalkonium chloride, cetyltrimethylammonium bromide and cetylpyridinium chloride.

[0200] Compositions may include one or more surfactants to enhance physical stability or for other purposes. Suitable nonionic surfactants include polyoxyethylene fatty acidCRISPR-44538.601glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40.

[0201] Compositions may include one or more antioxidants to enhance chemical stability where required. Suitable antioxidants include, by way of example only, ascorbic acid and sodium metabisulfite.

[0202] In certain embodiments, aqueous suspension compositions are packaged in singledose non-reclosable containers. Alternatively, multiple-dose reclosable containers are used, in which case it is typical to include a preservative in the composition.

[0203] In certain embodiments, the formulations described herein comprise one or more antioxidants, metal chelating agents, thiol containing RNA molecules and / or other general stabilizing agents. Examples of such stabilizing agents, include, but are not limited to: (a) about 0.5% to about 2% w / v glycerol, (b) about 0.1% to about 1% w / v methionine, (c) about 0.1% to about 2% w / v monothioglycerol, (d) about 1 mM to about 10 mM EDTA, (e) about 0.01% to about 2% w / v ascorbic acid, (f) 0.003% to about 0.02% w / v polysorbate 80, (g) 0.001% to about 0.05% w / v. polysorbate 20, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrins, (1) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc; or (n) combinations thereof.

[0204] Packaging materials for use in packaging pharmaceutical compositions described herein include those found in, e.g., U.S. Pat. Nos. 5,323,907, 5,052,558 and 5,033,252.Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment. For example, the container(s) includes one or more RNA molecules described herein, optionally in a composition or in combination with another agent as disclosed herein. The container(s) optionally have a sterile access port (for example the container is an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). Such kits optionally comprise a RNA molecule with an identifying description or label or instructions relating to its use in the methods described herein.

[0205] For example, a kit typically includes one or more additional containers, each with one or more of various materials (such as reagents, optionally in concentrated form, and / or devices) desirable from a commercial and user standpoint for use of a RNA molecule described herein. Non-limiting examples of such materials include, but not limited to, buffers, diluents, filters, needles, syringes; carrier, package, container, vial and / or tube labels listing contents and / or instructions for use, and package inserts with instructions for use. A set ofCRISPR-44538.601instructions will also typically be included. A label is optionally on or associated with the container. For example, a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself, a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert. In addition, a label is used to indicate that the contents are to be used for a specific therapeutic application. In addition, the label indicates directions for use of the contents, such as in the methods described herein. In certain embodiments, the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a RNA molecule provided herein. The pack for example contains metal or plastic foil, such as a blister pack. Alternatively, the pack or dispenser device is accompanied by instructions for administration, or the pack or dispenser is accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert. In some embodiments, compositions containing a RNA molecule provided herein formulated in a compatible pharmaceutical carrier are prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.

[0206] In some embodiments, a pharmaceutical composition has a RNA molecule described above and a pharmaceutically acceptable carrier including, for example, any adjuvant, carrier, excipient, glidant, sweetening agent, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonic agent, solvent, or emulsifier which has been approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0207] Disclosed herein are various LNP compositions for delivering biologically active RNA molecules as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly (A) tail, including gene editing system cargoes. Such LNP compositions include an “ionizable amine lipid” or “cationic lipid,” along with, for example, cholesterol, a polymer conjugated lipid, and a helper lipid. “Lipid nanoparticle” or “LNP” refers to, without limiting the meaning, a particle that comprises a plurality of (i.e., more than one) LNP components physically associated with each other by intermolecular forces.

[0208] In some embodiments, the LNP composition comprises a RNA molecule as described herein, e.g., an RNA sequence of interest operably linked to chemically modifiedCRISPR-44538.601Poly(A) tail, an ionizable lipid or cationic lipid, and one or more additional lipid components selected from the group consisting of neutral lipids and / or helper lipids (collectively helper lipids), polymer conjugated lipids, and cholesterol or a derivative thereof.

[0209] In some embodiments, the ionizable lipid is selected from ALC-0315, SM-102, DLin-MC3-DMA (MC3), DODAP, and LP-01.

[0210] In some embodiments, the cationic lipid is selected from DOTMA (l,2-dioleoyl-3-trimethylammonium propane), DOTAP (l,2-dioleoyl-3-trimethylammonium propane), DC-Chol (3a-[N-(N',N'-dimethylaminoethane)carbamoyl] cholesterol hydrochloride), and DOSPA (2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate).

[0211] In some embodiments, the helper lipid is selected from the group consisting of 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1 ,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1 ,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3 -phosphocholine (C16 Lyso PC), 1 ,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1 ,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1 ,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1 ,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-didocosahexaenoyl-sn-glycero-3 -phosphoethanolamine, 1 ,2-dioleoyl-sn-glycero-3 -phospho-rac-(l -glycerol) sodium salt (DOPG), and mixtures thereof. In some embodiments, the helper lipid is DOPE or DSPC.

[0212] Embodiments of the present disclosure provide lipid compositions described according to the respective molar ratios of the component lipids in the composition. All mol-% numbers are given as a fraction of the lipid component of the lipid composition or, more specifically, the LNP compositions. In certain embodiments, the mol-% of the ionizable lipid may be from about 20 mol-% to about 70 mol-%. In certain embodiments, the mol-% of the ionizable lipid may be at least 20 mol-%, at least 30 mol-%, at least 40 mol-%, or at least 50 mol-%.CRISPR-44538.601

[0213] In certain embodiments, the mol-% of the helper lipid may be from about 0 mol-% to about 60 mol-%. In certain embodiments, the mol-% of the helper lipid may be from about 20 mol-% to about 50 mol-%. In certain embodiments, the mol-% of the helper lipid may be about 40 mol-%. In certain embodiments, the mol-% of the helper lipid may be about 30 mol-%.

[0214] In certain embodiments, the mol-% of the neutral lipid may be from about 0 mol-% to about 80 mol-%. In certain embodiments, the mol-% of the neutral lipid may be from about 20 mol-% to about 60 mol-%. In certain embodiments, the mol-% of the neutral lipid may be from about 30 mol-% to about 50 mol-%. In certain embodiments, the mol-% of the neutral lipid may be from 30 mol-% to about 40 mol-% or from about 35% mol-% to about 45 mol-%. In certain embodiments, the mol-% of the neutral lipid is adjusted based on RNA molecule of Formula (I), (IA), (IB), (lA-a), (lA-b), (IB-a), (IB-b), (lA-al), (lA-bl), (IB-al), or (IB -bl), helper lipid and / or PEG lipid concentrations to bring the lipid component to 100 mol-%.

[0215] In certain embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 10 mol-%. In certain embodiments, the mol-% of the PEG lipid may be from about 0.5 mol-% to about 4 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 0.5 mol-% to about 2 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 1.5 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 1.0 mol-%. In certain embodiments, the mol-% of the PEG lipid may be about 0.5 mol-%.

[0216] Accordingly, in some embodiments, the present disclosure provides a pharmaceutical composition comprising a RNA molecule as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail, and a pharmaceutically acceptable carrier.

[0217] In some embodiments, the present disclosure provides a lipid nanoparticle comprising a RNA molecule as described herein, e.g., an RNA sequence of interest operably linked to chemically modified Poly(A) tail. In some embodiments, the present disclosure provides a lipid nanoparticle comprising components of a gene editing system as described above. In some embodiments, the LNP comprises a gene-editing nuclease and a guide sequence. In some embodiments, the lipid nanoparticle comprises an ionizable or cationic lipid and one or more additional lipids selected from the group consisting of helper lipids, cholesterol, and polymer conjugated lipids.ExamplesCRISPR-44538.601

[0218] The following are examples of the present invention and are not to be construed as limiting.Example 1

[0219] Chemically modified RNA poly(A) oligonucleotides were synthesized. The sequences of the oligonucleotides are provided in Table 1.CRISPR-44538.601CRISPR-44538.601CRISPR-44538.601Example 2

[0220] This example provides a demonstration of the ability of chemically modified poly(A) tails to tune mRNA translation. The chemically modified poly(A) oligonucleotides were ligated to a mRNA encoding luciferase (the DNA sequence for the luciferase is provided as SEQ ID NO:57). The ligation of chemically modified oligos to the 3’ end of mRNA was initiated by incubating mRNA acceptor and oligo donor at 1:40 molar ratio in the presence of T4 RNA ligase 1. The reactions were conducted at 25 °C for 30 minutes. The reactions were purified with LiCl precipitation or oligod(T) based chromatography and polished by ultrafiltration. The reaction components are provided in Table 2. To assess the ligation efficiency of chemically modified poly(A) tail, the ligated mRNA was digested with RNase T1 which specifically degrades single-stranded RNA at G residues. The released chemically modified poly(A) oligonucleotides were captured and purified using Dynabeads™ Oligo(dT)25. The cleaned-up sample was resolved with RP-HPLC for ligation efficiency assessment. All modified mRNA samples demonstrated ligation efficiency exceeding 90%. FIG. 1 A-D provides data showing the ligation efficiency of selected oligos.

[0221] The resulting mRNA sequences were then formulated in LNPs and introduced into mice for live imaging of luciferase expression. FIG. 2 provides live images of expression of luciferase at 4, 24, 48, 72, 96, 120 and 168 hours after administration of ENPs containing the mRNA sequences to mice. FIG. 3 provides a graph showing measured luciferase activityCRISPR-44538.601over time while FIG. 4 presents the results as total luciferase activity integrated as area under the curve. FIG. 5 provides a bar graph showing the log fold change in luciferase activity at 4 hours. FIGs. 6A and 6B provide the results of a pharmocokinetics analysis of luciferase activity for selected chemically modified poly(A) sequences. As can be seen, the chemically modified poly(A) tails can be utilized to tune mRNA translation.

[0222] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0223] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CRISPR-44538.601ClaimsWhat is claimed is:

1. A synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’ wherein:X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0, preferably from 1 to 4;Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides;wherein at least one of Ri, R2, R3, R4, Rs, and Re is an adenosine ribonucleotide comprising a modified sugar moiety.

2. The synthetic RNA molecule of claim 1, wherein at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.

3. The synthetic RNA molecule of any one of claims 1 to 2, wherein the modified sugar moiety is selected from the group consisting of a locked nucleic acid (LNA) subunit, a 2'0,4'C-ethylene-bridged nucleic acid (ENA) subunit, a tricyclo-DNA (tc-DNA) subunit, a 2' O-methyl subunit, a 2' O-methoxyethyl subunit, a 2'-fluoro subunit, and a 2'-O-[2-(N-methylcarbamoyl)ethyl] subunit, and combinations thereof when at least two of Ri, R2,R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.

4. The synthetic RNA molecule of claim 3, wherein the modified sugar moiety is selected from the group consisting of a 2' O-methyl subunit and a 2' O-methoxyethyl subunit, and combinations thereof when at least two of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a modified sugar moiety.

5. The synthetic RNA molecule of claim 4, wherein at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a 2' O-methyl subunit.CRISPR-44538.6016. The synthetic RNA molecule of claim 4, wherein at least two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides comprising a 2' O-methoxyethyl subunit.

7. The synthetic RNA molecule of any one of claims 1 to 6, wherein at least one, two, three, four, five or all six of Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides that comprise a modified internucleoside linkage.

8. The synthetic RNA molecule of claim 7, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

9. The synthetic RNA molecule of any one of claims 1 to 8, wherein X is an adenosine ribonucleotide.

10. The synthetic RNA molecule of any one of claims 1 to 9, wherein the RNA molecule has a 5’ terminal nucleotide and the 5’ terminal nucleotide comprises a 5’ phosphorylation.

11. The synthetic RNA molecule of any one of claims 1 to 10, wherein the RNA molecule terminates at Re and Re is selected from the group consisting of an adenosine ribonucleotide comprising a 2' O-methyl subunit, an adenosine ribonucleotide comprising a 2' O-methyl subunit and a 3’ inverted T modification, a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification, and a 3’ adenosine ribonucleotide with a 2’methoxyethyl modification and a 3’ inverted T modification.

12. A synthetic RNA molecule of any one of SEQ ID NOs: 1-48.

13. A synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’ wherein:X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0;Ri, R2, R3, R4, Rs, and Re are adenosine ribonucleotides; andwherein at least four of Ri, R2, R3, R4, Rs, and Re comprise a modified internucleoside linkage, preferably a phosphorothioate internucleoside linkage.CRISPR-44538.60114. A synthetic RNA molecule of SEQ ID NO:49 or 52.

15. A synthetic RNA molecule comprising the sequence 5’-Xn-Ri-R2-R3-R4-R5-R6-3’ wherein:X is a ribonucleotide and n is 1-200, 1-100, 1-50, 1-20, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0; andRi, R2, R3, R4, and R5 are unmodified adenosine ribonucleotides and Re is a modified adenosine comprising a 3' inverted T modification.

16. A synthetic RNA molecule of SEQ ID NO:54 or 55.

17. A RNA molecule comprising a RNA sequence of interest operably linked to a poly(A) tail comprising the synthetic RNA molecule of any one of claims 1 to 16.

18. The RNA molecule of claim 17, wherein the RNA sequence of interest encodes a therapeutic protein.

19. The RNA molecule of claim 17, wherein the RNA sequence of interest encodes an enzyme.

20. The RNA molecule of claim 19, wherein the enzyme is a therapeutic protein.

21. The RNA molecule of claim 19, wherein the enzyme is a component of a gene-editing system.

22. The RNA molecule of claim 21, wherein the enzyme is selected from the group consisting of a CRISPR / Cas nuclease, a zinc-finger nuclease, a transcription activator-like effector nuclease, a nickase, a nickase-reverse transcriptase fusion enzyme, and a dCAS-deaminase fusion enzyme.

23. The RNA molecule of claim 22, wherein the nuclease is a CRISPR / Cas nuclease.

24. A lipid nanoparticle comprising the RNA molecule of any one of claims 17 to 23.CRISPR-44538.60125. A pharmaceutical composition comprising the RNA molecule of any one of claims 17 to 23 and a pharmaceutically acceptable carrier.

26. A gene editing system comprising:an enzyme of claim 22 or 23; anda guide sequence selected from the group consisting of a gRNA, sgRNA, and template armed guide RNA.

27. The gene editing system of claim 26, further comprising a donor polynucleotide.

28. A lipid nanoparticle comprising the gene editing system of claims 26 or 27.

29. The lipid nanoparticle of claim 28, wherein the lipid nanoparticle comprises an ionizable or cationic lipid and one or more additional lipids selected from the group consisting of helper lipids, cholesterol, and polymer conjugated lipids.

30. A method of editing a genome comprising introducing a gene editing system or lipid nanoparticle of any of claims 26 to 29 into a cell under conditions such that the genome of the cell is edited.

31. The method of claim 30, wherein the cell is in vivo.

32. A method of making a RNA molecule comprising ligating a poly(A) tail comprising the synthetic RNA molecule of any one of claims 1 to 16 to an RNA sequence of interest.

33. A method of treating a subject, comprising administering to the subject an RNA molecule of claim 19 or 20, LNP of claim 24, or pharmaceutical composition of claim 25.

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