Systems and methods for modulating RNA

Abstract

Aspects of the disclosure relate to compositions and methods for modulating RNA. The methods include methods for treating a haploinsufficiency disorder in a subject, the method comprising administering a polypeptide, delivery vehicle, or composition described herein. Also described are delivery vehicles comprising the polypeptides, nucleic acids, and / or expression vectors described herein.

Description

SYSTEMS AND METHODS FOR MODULATING RNACROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional patent Application Serial No. 63 / 657,666, filed June 7, 2024, which is incorporated herein in its entirety.STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under EB035016 awarded by the National Institutes of Health. The government has certain rights in the invention.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on June 6, 2025, is named ARCDP0848WO.xml and is 59,951 bytes in size.BACKGROUND OF THE INVENTION1. Field of the Invention

[0004] The present invention relates generally to the field of chemistry and medicine. More particularly, it concerns the use of a system for modulating RNA.2. Background

[0005] Programmable nucleic acid-binding proteins have revolutionized genome studies and editing technologies (Chandrasegaran and Carroll, 2016; Filipovska et al., 2011; Gootenberg et al., 2018; Hilton et al., 2015; Joung and Sander, 2012; Kearns et al., 2015; Strutt et al., 2018) and are opening up new therapeutic opportunities to treat human diseases (Liao et al., 2017; Monteys et al., 2017). In particular, the CRISPR / Cas9 system, which evolved as a bacterial immune defense mechanism, has transformed the ability to study and manipulate cellular DNA site-specifically (Cong et al., 2013; Jiang et al., 2013; O’Connell et al., 2014; Wiedenheft et al., 2012). A key advantage of CRISPR / Cas systems compared to previous methods (Desjarlais and Berg, 1993; Hockemeyer et al., 2011; Joung and Sander, 2012; Schierling et al., 2012) is that they are easily programmable to target virtually any locus of interest. The CRISPR / Cas system is a ribonucleoprotein complex that uses base pair interactions of a displayed guide RNA (gRNA) to interact with a target nucleic acid sequence. The simple nature of base pair-guided targeting opens up the possibility to program systems to interact with a defined nucleic acid sequence by simply changing the nucleic acid sequence on the guiding strand.

[0006] While targeting DNA directly will have profound clinical ramifications, diseases that involve subtle alterations to many genes will be challenging to target using DNA editing technologies (Fuxman Bass et al., 2015). Additionally, the potential side effects or risks of permanent genetic alteration might not be tolerated. For example, the genes one may want to target to activate an enhanced wound healing response are likely targets that could pose a risk for cancer development, making permanent DNA-based strategy risky. Targeting information flow at the RNA level presents several opportunities for therapeutic intervention, including but not limited to the ability to halt treatment if side effects emerge, the ability to target genes that would be too risky to alter at the DNA level, and the ability to manipulate gene expression without permanent alterations to the host genome. While inhibiting or enhancing transcription at the genome level provides one possibility for controlling gene expression (Du et al., 2017; Fuxman Bass et al., 2015; Qi et al., 2013), recently discovered RNA epitranscriptomic regulatory mechanisms offer a broad range of processes to target, including editing, degradation, transport, and translation of RNA transcripts (Nishikura, 2010; Roundtree et al., 2017; Zhao et al., 2017). Although the mechanisms and consequences of this epitranscriptomic regulatory layer are just beginning to be uncovered, it is apparent that the information flow through RNA is tightly regulated, offering many new opportunities for both basic research discoveries as well as therapeutic development.

[0007] Programmable RNA-targeting tools analogous to the dCas9 DNA-targeting systems hold great promise for studying the mechanisms of epitranscriptomic regulation and for therapeutic applications. The current tools for RNA targeting involve the delivery of large complexes and pose immunogenicity issues. From a basic science perspective, the large size of the delivery vehicle could lead to potential perturbations to the RNA under interrogation, convoluting the study of RNA regulatory mechanisms. From a translational perspective, the large size presents challenges for viral packaging or direct protein delivery. Additionally, while DNA-editing therapies will likely consist of a one-time, irreversible treatment, RNA-targeting therapies will need to be continually administered, making delivery concerns especially important. Moreover, it was recently discovered that 85% of people already have circulating antibodies to CRISPR / Cas proteins (Kim et al., 2018; Wagner et al., 2018), suggesting immunogenicity issues may prove problematic in clinical applications. Therefore, there is a need in the art for improved systems that can target RNA and be delivered efficiently without activating an immune response.SUMMARY OF THE INVENTION

[0008] Provided herein is a polypeptide comprising an effector domain wherein the effector domain comprises the amino acid sequence of one of SEQ ID NOs:l, 2, 21, or 55-58 or comprises an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 1, 2, 21, or 55-58. Also provided is a nucleic acid comprising the amino acid sequence of one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63 or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63. Also provided is one or more expression vector(s) comprising the nucleic acids described herein. The current disclosure further provides for a host cell comprising the nucleic acid or expression vector described herein. Also described herein is a delivery vehicle comprising the polypeptides, nucleic acids, and / or expression vectors described herein. Also described herein is a method of modulating at least one target RNA comprising contacting the target RNA with the polypeptide, the delivery vehicle, or composition described herein. Another method provided is a method for modulating at least one target RNA in a subject, the method comprising administering the polypeptide, the delivery vehicle, or the composition described herein. Further methods include a method for treating a haploinsufficiency disorder in a subj ect, the method comprising administering a polypeptide, delivery vehicle, or composition described herein.

[0009] The polypeptide may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOs:l, 2, 21, or 55-58. In some aspects, the polypeptide comprises the amino acid sequence of at least two, at least 3, or at least 4 of SEQID NOs:l, 2, 21, or 55-58 or amino acid sequences that have at least 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least two, at least 3, or at least 4 of SEQ ID NOs:l, 2, 21, or 55-58. The polypeptide may comprise two regions, wherein each region is a duplicate of one of SEQ ID NOs:l, 2, 21, or 55-58 or wherein each region comprises the amino acid sequence that has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to one of SEQ ID NOs:l, 2, 21, or 55-58.

[0010] The polypeptide may further comprise or exclude one or more of a stabilizer domain, linker, RNA hairpin binding domain, nuclear export signal, and molecular tag. The hairpin binding domain may be amino-proximal to the effector domain. The hairpin binding domain may be carboxy-proximal to the effector domain. The stabilizer domain may be amino- proximal to the RNA hairpin binding domain and / or effector domain. The stabilizer domain may be carboxy-proximal to the RNA hairpin binding domain and / or effector domain. The nuclear export signal may be amino-proximal to the effector domain. The nuclear export signal may be carboxy-proximal to the effector domain. The molecular tag may be carboxy-proximal to the effector domain. The molecular tag may be amino-proximal to the effector domain. The polypeptide may comprise, from N-terminus to the C-terminus, a stabilizer domain, a glycine serine linker, a RNA hairpin binding region, a nuclear export signal, an effector domain, and a molecular tag. Also provided herein is a nucleic encoding the polypeptides described herein.

[0011] Also provided herein is a cell or progeny thereof comprising modulated target RNA, wherein the target RNA has been modulated according to the methods described herein. Also described is a multicellular organism comprising one or more cells according to the cell or progeny thereof. A plant or animal comprising one or more cells according the multicellular organism. Also described is a kit comprising the polypeptide, nucleic acid, expression vector, host cell, delivery vehicle, or the composition described herein.

[0012] The polypeptide may comprise or exclude a RNA hairpin binding domain. The hairpin binding domain may comprise or exclude one that has the amino acid sequence of SEQ ID NOS:38 or an amino acid sequence with at least 80% sequence identity to SEQ ID NOS:38. The hairpin binding domain may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to SEQ ID NO:38.

[0013] The polypeptide may comprise or exclude a stabilizer domain. The stabilizer domain may have the amino acid sequence of at least one of SEQ ID NOS:31-33 or an amino acid sequence having at least 80% sequence identity to one of SEQ ID NOS:31-33. The stabilizer domain may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to one of SEQ ID NOS:31-33.

[0014] The polypeptide may comprise or exclude a nuclear export signal. The nuclear export signal may comprise the amino acid sequence of SEQ ID NO:40 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:40. The nuclear export signal may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to SEQ ID NO:40.

[0015] The polypeptide may comprise or exclude a molecular tag. The molecular tag may comprise the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:41. The molecular tag may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to SEQ ID NO:41.

[0016] The nucleic acid of the disclosure may comprise an amino acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42- 54, or 59-63.

[0017] The nucleic acid may comprise or exclude a TAR hairpin scaffold or a human SLBP hairpin scaffold. The hairpin structure may comprise or exclude the nucleic acid sequence of at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35 or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35. The hairpin structure may comprise a nucleic acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35. The nucleic acid may comprise 1, 2, 3, 4, 5, or 6 hairpin structures, wherein each structure is independently selected from a nucleic acid that has the sequence of one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35 or a sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35.

[0018] The delivery vehicle may comprise or exclude liposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun or one or more nucleic acid vector(s). The current disclosure also provides for a composition comprising the polypeptide, nucleic acid, expression vector, host cell, or delivery vehicle described herein. Further, provided herein is a method of making a polypeptide comprising transferring the nucleic acid or expression vector described herein into a cell and isolating the expressed polypeptide. 31. The delivery vehicle of claim 29 or 30, wherein the delivery vehicle further comprises a RNA targeting molecule comprising a RNA targeting region and at least one hairpin structure, wherein the hairpin structure of the RNA targeting molecule specifically binds to the RNA hairpin binding domain of the polypeptide.

[0019] The RNA targeting region may hybridize to the 5’UTR of a RNA. The RNA targeting region may hybridize to the 3’UTR of a RNA. The target RNA may include or exclude a mammalian RNA. The RNA targeting region may comprise the nucleic acid sequence of one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63 or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63. The RNA targeting region may comprise a nucleic acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42- 54, or 59-63. The RNA targeting molecule may comprise or exclude a TAR hairpin scaffold or a human SLBP hairpin scaffold. The RNA targeting molecule may comprise or exclude at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35. The RNA targeting molecule may comprise or exclude the nucleic acid sequence of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35 or a nucleic acid sequence that has at least 80% sequence identity to one of SEQ ID NOS: 15-19, 22, 25-30,34, or 35. The RNA targeting molecule may comprise a nucleic acid sequence that has or has at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) percent sequence identity to at least one of SEQ ID NOS:15-19, 22, 25-30, 34, or 35.

[0020] The modulating the at least one target RNA may comprise or exclude cleaving, demethylating, methylating, activating translation, repressing translation, promoting degradation, and / or binding to the RNA. The target RNA may or may not be in a prokaryotic or eukaryotic cell. The target RNA may or may not be in a human cell. The target RNA may or may not be in vitro or in vivo. The haploinsufficiency disorder may comprise or exclude epileptic encephalopathy. The haploinsufficiency disorder may comprise or exclude Dravet Syndrome, cancer, lq21.1 deletion syndrome, 5q- syndrome in myelodysplastic syndrome (MDS), 22ql l.2 deletion syndrome, CHARGE syndrome, cleidocranial dysostosis, Ehlers- Danlos syndrome, Frontotemporal dementia caused by mutations in progranulin, GLUT1 deficiency (DeVivo syndrome), haploinsufficiency of A20, haploinsufficiency of PRR12, holoprosencephaly caused by haploinsufficiency in the Sonic Hedgehog gene, Holt-Oram syndrome, Marfan syndrome, Phelan-McDermid syndrome, Polydactyly, FOXP1 Syndrome, ARID1B haploinsufficiency, ARID IB-mediated disorders, or NR4A2 -related syndrome.. The haploinsufficiency disorder may comprise or exclude Dravet Syndrome. The haploinsufficiency disorder may comprise or exclude ARID IB haploinsufficiency or ARID IB- mediated disorders. The target RNA may comprise or exclude a mRNA from the CHD2, SCNla, ARID1B, and / or Navi.1 gene.

[0021] The term “RNA hairpin” refers to a RNA molecule with stem-loop intramolecular base pairing. A hairpin can occur when two regions of the same strand, usually complementary in nucleotide sequence when read in opposite directions, base-pair to form a double helix that ends in an unpaired loop. The disclosure relates to engineered RNA targeting molecules comprising a RNA targeting region and one or more hairpins. Accordingly, the engineered RNA molecules of the disclosure are chimeric molecules that are non-naturally occurring.

[0022] The term “RNA targeting region” refers to a region of the RNA that is capable of hybridizing to a target RNA. The target RNA may be a disease associated RNA or one that is a modulation target according to the current systems and methods.

[0023] The “effector domain” refers to a peptide or polypeptide that has activity directed to RNA. Examples of activity include methylation activity, RNA-binding activity, nuclease activity, and translational activation or repression activity.

[0024] In some aspects, the RNA hairpin binding domain and the effector domain are operably linked. The term “operably linked” refers to two proteins that are linked through either covalent or non-covalent interactions. For example, the two proteins may be covalently linked through a peptide bond. In some aspects, the proteins are non-covalently linked. One or more proteins of the disclosure may be operably linked to another protein through linkage to a pair of accessory proteins that have a strong affinity for each other. Such accessory proteins are known in the art. For example, the SunTag is one such system that includes an antibody with a strong affinity for a peptide. One protein, polypeptide, or domain of the disclosure may be linked to a SunTag peptide and another protein, polypeptide, or domain of the disclosure may be linked to an antibody to allow operable linkage of the two proteins, polypeptides, or domains through the interaction of the SunTag peptide and antibody. Further examples include biotin and avidin / streptavidin and spytag and spy catcher.

[0025] In some aspects, the system is inducible by providing the effector domain and the hairpin binding domain as two unlinked polypeptides that become linked upon the presence of a stimulant. The induction may be, for example, by light induction or by chemical induction. Such inducibility allows for activation of the RNA regulation at a desired moment in time. In some aspects, the effector domain is covalently linked to a first dimerization domain and the RNA hairpin binding domain is covalently linked to a second dimerization domain and wherein the first and second dimerization domain are capable of dimerizing to form a non-covalent or covalent linkage. In some aspects, the dimerization is inducible. In some aspects, the dimerization is induced through binding of the dimerization domains to a ligand. The term inducible refers to dimerization that is formed in response to a stimulus, such as a ligand, a chemical, a temperature change, or light, for example.

[0026] Light inducibility is for instance achieved by designing a fusion complex wherein the first and second dimerization domains comprise CRY2PHR and CIBN. This system is particularly useful for light induction of protein interactions in living cells and is further described in Konermann S, et al. Nature. 2013;500:472-476, which is herein incorporated by reference.

[0027] Suitable dimerization domains and corresponding ligands are known in the art. For example, Liang, F.S., Ho, W.Q., and Crabtree, G.R. (2011). Engineering the ABA plant stress pathway for regulation of induced proximity. Sci. Signal. 4, rs2, which is incorporated by reference, describes suitable dimerization / ligand systems that are useful in aspects of the disclosure. In some aspects, one of the first or second dimerization domain comprises PYR / PYRl-like (PYL1), the other of the first or second domain comprises ABA insensitive 1 (ABH), and the ligand comprises abscisic acid (ABA) or derivatives or fragments thereof. The dimerization domain may be a fragment or portion of the whole protein and may be a substituted or modified. In some aspects, the first and / or second dimerization domain comprises FKBP12 and the ligand comprises FK1012 or derivatives or fragments thereof. In some aspects, one of the first or second dimerization domain comprises FK506 binding protein (FKBP), the other of the first or second domain comprises FKBP-Rap binding domain of mammalian target of Rap mTOR (Frb), and the ligand comprises rapamycin (Rap) or derivatives or fragments thereof.

[0028] In some aspects, the domains of the disclosure are human or are human-derived. In some aspects, the polypeptide is non-immunogenic. A human protein, polypeptide, domain, or nucleic acid refers to a protein, polypeptide, domain, or nucleic acid that is from the human genome, although it may be produced recombinantly in non-human systems. The term “human-derived” refers to a protein, polypeptide, domain, or nucleic acid that is a variant or fragment of a protein, polypeptide, domain, or nucleic acid from the human genome, although it may be produced recombinantly in non-human systems. In some aspects, the fusion protein, conjugate, system, or parts thereof, such as parts i, ii, and / or iii are non-immunogenic and / or non-toxic when expressed in or administered to humans.

[0029] In some aspects, the nucleic acids or polypeptides of the disclosure are synthetic, are non-natural, and / or do not occur naturally in nature. The polypeptides and methods of the disclosure may exclude a Cas9 enzyme or a polypeptide that comprises or comprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500 amino acids that have or have at least 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% sequence identity to a Cas9 protein. Nucleic acids and methods of the disclosure exclude nucleic acids that bind to a Cas9 protein.

[0030] In some aspects, the RNA targeting molecule comprises exactly one hairpin. In some aspects, the nucleic acid comprises at least one hairpin. In some aspects, the nucleic acid comprises exactly two hairpins. In some aspects, the nucleic acid comprises at least two hairpins. In some aspects, the nucleic acid comprises exactly three hairpins. In some aspects, the nucleic acid comprises at least three hairpins. In some aspects, the nucleic acid comprises exactly four hairpins. In some aspects, the nucleic acid comprises at least four hairpins. In some aspects, the nucleic acid comprises exactly five hairpins. In some aspects, the nucleic acid comprises at least five hairpins. In some aspects, the nucleic acid comprises 1-4 hairpins. In some aspects, the nucleic acid comprises 1-3 hairpins. In some aspects, the nucleic acid comprises 1-2 hairpins. In some aspects, the nucleic acid comprises 2-4 hairpins. In some aspects, the nucleic acid comprises 2-3 hairpins. In some aspects, the nucleic acid comprises at least, at most, or exactly 1, 2, 3, 4, 5, or 6 hairpins (or any range derivable therein). In some aspects, the nucleic acid comprises at least one hairpin that does not bind to the RNA hairpin binding protein and at least one hairpin that binds to the RNA hairpin binding protein. In some aspects, the nucleic acid binds to more than one RNA binding protein. In some aspects, the nucleic acid comprises two, three, or four hairpin structures and binds to at least two RNA binding proteins. In some aspects, the effector system comprises at least two regulatory domains, wherein each regulatory domain binds to a different RNA binding molecule.

[0031] In some aspects, the nucleic acid comprises one or more modified nucleotides. In some aspects, the modified nucleotides comprise a modification such as a phosphorothioate, locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, 5’E-VP, or is modified to a morpholino. In some aspects, the modification includes one described herein.

[0032] In some aspects, the effector domain comprises a nuclease, methylase, demethylase, translational activator, translational repressor, single-stranded RNA cleavage activity, doublestranded RNA cleavage activity, or RNA binding activity. In some aspects, the effector domain comprises an activity described herein.

[0033] In some aspects, the effector domain increases translation of a target RNA (e.g., by recruiting the translational machinery). In some aspects, the effector domain increases degradation of a target RNA. In some aspects, the effector domain modifies the localization of a target RNA. In some aspects, the effector domain modifies the processing of the target RNA(e.g., by active nuclease activity on the target or by triggering endogenous epitranscriptomic regulatory pathways).

[0034] In some aspects, the vectors of the disclosure may comprise or further comprise a regulatory element operably linked to the nucleotide of the disclosure. Regulatory elements, in addition to a NLS and NES, as previously described, also include promoters, polyadenylation signals, enhancers, etc. Other regulatory elements are known in the art and described herein and may be used in the aspects of the disclosure. In some aspects, the one or more nucleic acid vectors are optimized for expression in an eukaryotic cell. In some aspects, the expression of the domains, RNA, or polypeptides in the cell or from a vector is constitutive. In some aspects, the expression of the domains, RNA, or polypeptides in the cell or from a vector is conditional. In some aspects, the expression vector further comprises a nucleic acid encoding a RNA targeting molecule. In some aspects, one or more of the vectors are viral vectors. In some aspects, the one or more vectors comprise one or more retroviral, lentiviral, adenoviral, adeno- associated or herpes simplex viral vectors. In some aspects, one or more of the vectors are non-viral vectors. In some aspects, the system or composition is non-viral, which denotes that it does not contain any viral components.

[0035] In some aspects, there is a system or kit comprising one or more of the following components: a polypeptide comprising an effector domain, a polypeptide comprising a RNA binding domain, a polypeptide comprising a stabilizer, a nucleic acid encoding for a effector domain, a nucleic acid encoding for a RNA binding domain, a nucleic acid encoding a stabilizer, a nucleic acid encoding a nucleic acid comprising a RNA targeting region and at least one hairpin structure; a conjugate of the disclosure; a vector of the disclosure, a fusion protein of the disclosure, a recombinant host cell, an expression construct, an engineered viral vector, or an engineered attenuated virus. In certain aspects, a polypeptide of the disclosure is under the control of a heterologous promoter. It is specifically contemplated that any protein or polypeptide function that are used in aspects, may be used a nucleic acid encoding that protein or polypeptide function. Also, any and all polypeptides, proteins, nucleic acid molecules may be contained within a cell or other living organism, such as a virus (for instance, a phage).

[0036] A kit may include one or more components that are separate or together in a suitable container means, such as a sterile, non-reactive container. In some aspects, cells or viruses are provided that contain one or more nucleic acid constructs that encode the polypeptides of the disclosure. The term “promoter” is used according to its ordinary meaning to those in the fieldof molecular biology; it generally refers to a site on a nucleic acid in which a polymerase can bind to initiate transcription. In specific aspects, the promoter is recognized by a T7 RNA polymerase.

[0037] The compositions, vectors, systems, methods, and proteins of the disclosure are useful for a variety of clinical and research-related applications. The aspects of the disclosure may be useful for the treatment of a disease or condition, such as cancer or autoimmunity. In some aspects, the methods and compositions are for the acute treatment of a disease or condition. In some aspects, the methods and compositions are useful for the temporary modulation of RNA. In some aspects exclude permanent modification of gene activity. In some aspects, the methods and compositions are safer due to the acute modulation of RNA and / or due to the ability to control the expression of the system in vivo.

[0038] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

[0039] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0040] As used herein, the terms “or” and “and / or” are utilized to describe multiple components in combination or exclusive of one another. For example, “x, y, and / or z” can refer to “x” alone, “y” alone, “z” alone, “x, y, and z,” “(x and y) or z,” “x or (y and z),” or “x or y or z.” It is specifically contemplated that x, y, or z may be specifically excluded from an embodiment or aspect.

[0041] The words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”), “characterized by” (and any form of including, such as “characterized as”), or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0042] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. The phrase “consisting of’ excludes any element, step, or ingredient not specified. The phrase “consisting essentially of’ limits the scope of described subject matter to the specified materials or stepsand those that do not materially affect its basic and novel characteristics. It is contemplated that embodiments and aspects described in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.”

[0043] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0044] Use of the one or more sequences or compositions may be employed based on any of the methods described herein. Other embodiments and aspects are discussed throughout this application. Any embodiment or aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa.

[0045] It is specifically contemplated that any limitation discussed with respect to one embodiment or aspect of the invention may apply to any other embodiment or aspect of the invention. Furthermore, any composition of the invention may be used in any method of the invention, and any method of the invention may be used to produce or to utilize any composition of the invention. Aspects of an embodiment set forth in the Examples are also embodiments that may be implemented in the context of embodiments discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary of Invention, Detailed Description of the Embodiments, Claims, and description of Figure Legends.

[0046] Other obj ects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments and aspects of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.BRIEF DESCRIPTION OF THE DRAWINGS

[0047] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0048] FIG. 1A-1C. CIRTS-eIF4G (711 - 1599) (4GT1) can increase luciferase expression with mSCNla 3’ UTR targeting gRNA in the SCNla dual luciferase reporting system inHEK293T cells (a) and increase SCNla endogenous protein with gRNA targeting both the 3’ UTR (b) and 5’ UTR (c) in N2a cells. Each figure is normalized to a CIRTS-4GT1 with a nontargeting gRNA. Data are mean ± SEM; n = 3 or more biological replicates.

[0049] FIG. 2A-2C. Further truncations of eIF4G for designed to be tested as CIRTS effectors (a). These new truncations were test for increasing SCNla expression in N2a cells at 3’ UTR gRNA 1 (b) and 5’ UTR gRNA 6 (c). Each figure is normalized to a gRNA-matched CIRTS-GFP negative control. Data are mean ± SEM; n = 2 or more biological replicates.

[0050] FIG. 3. CIRTS-activators CIRTS-4GT3 and CIRTS-NY1 were packaged into lentivirus with corresponding optimal gRNA (gl and g5 respectively) and were used to transduce primary rat cortical neurons and increase SCNla expression. Each figure is normalized to an effector matched CIRTS lentivirus with a non-targeting gRNA. Data are mean ± SEM; n = 3 biological replicates.

[0051] FIG. 4A-4B. CIRTS -NY 1 was also tested around another target related to haploinsufficiency, CHD2, both on a mCHD2 3’ UTR luciferase reporter in HEK293T cells with 3’ UTR-targeting gRNAs (a) and targeting endogenous CHD2 in N2a cells (b).

[0052] FIG. 5A-5B. CIRTS-4GT3 was also tested around the mCHD2 3’ UTR luciferase reporter in HEK293T cells with 3’ UTR-targeting gRNA (a) and targeting endogenous CHD2 in N2a cells (b).

[0053] FIG. 6. CIRTS-based translational activators (CIRTSa) increase target mRNA translation. Representation of CIRTSa-mediated protein expression increase only in haploinsufficient cells that natively express the target. CIRTSa programmably binds to a target mRNA via the complementary gRNA sequence, then likely recruits eukaryotic initiation factors (elFs) which in turn recruit the ribosome to promote translation.

[0054] FIG. 7A-7G. Screening CIRTS-activators on a SCNla reporter and increasing endogenous Navl.l expression, a, Vector design for the dual luciferase reporter, including expression cassettes for the Flue with target 3’ UTR and the untargeted Nluc transcript used for transfection normalization, b, Design for the combined CIRTS protein and gRNA expression vector used in this study, c, Map of the SCNla 3’ UTR, showing the distance in ribonucleotides (nts) from the start of the 3’ UTR to the first base to which each gRNA binds, d, Evaluation of SCNla 3 ’UTR-targeting CIRTS gRNA expressed alongside CIRTS-NY1 increasing Flue expression by dual luciferase assay by two plasmid transfection in HEK293T cells. Each data point is normalized first to the internal Nluc transfection control and thenagainst Flue expression in the CIRTS-NY1 + non-targeting (NT) gRNA control, n = 3 biological replicates for each group, e, Representative western blot for Navi.1 levels 48 hours post-transfection with indicated CIRTS-NYl / gRNA vectors in N2a cells, a-tubulin was used as the loading control and SCNla-g5 was normalized to NT gRNA for quantitation, n = 3 biological replicates for each group, f, Screen for translational effectors fused with CIRTS targeting the SCNla 3’ UTR dual luciferase reporter with SCNla-g5. Each CIRTS was individually normalized to an effector-matched NT gRNA control, n = 3 or 4 biological replicates for each group, g, Representative western blot for CIRTS-4GT1 expressed alongside SCNla-g5 targeting endogenous SCNla transcript in N2a cells 48 hours post-transfection, a- tubulin was used as the loading control and SCNla-g5 was normalized to effector-matched CIRTS expressed with NT gRNA for quantitation. All bar graph values are shown as mean ± SEM with data points. Statistical analysis were performed using one-way ANOVA with post- hoc Dunnett’s multiple comparisons test d vs. NT. * <0.05. Unpaired two-tailed Student’s t test was performed in e, f, and g vs. effector-matched NT. * <0.05, ** <0.01, *** <0.001, **** <0.0001. No asterisk = not significant.

[0055] FIG. 8A-8F. Optimizing CIRTS-eIF4GI through gRNA and protein engineering, a, A SCNla 3’ UTR gRNA panel was tested alongside CIRTS-4GT1 by dual luciferase assay in HEK293T cells. Relative Flue expression was measured 48 hours post-transfection normalized to a transfected CIRTS-4GT1 / NT gRNA control, n = 3 biological replicates, b, Representative endogenous Navl. l western blot 48 hours post-transfection with vector expressing CIRTS- 4GT1 and indicated gRNA. a-tubulin was used as the loading control and each gRNA was normalized to CIRTS-4GT1 expressed with NT gRNA for quantitation, n = 3 biological replicates, c, SCNla 5’ UTR map showing the distance in nucleotides (nts) from the start codon and the first nucleotide on the 5’ UTR each gRNA binds to. d, Representative endogenous Navl. l western blot 48 hours post-transfection with vector expressing CIRTS-4GT1 and indicated 5’ UTR gRNA. a-tubulin was used as the loading control and each gRNA was normalized to CIRTS-4GT1 expressed with NT gRNA for quantitation, n = 4 biological replicates, e, Map demonstrating eIF4GFs domains and their binding partners over the full- length amino acid sequence. Tested truncations are shown below the full-length protein with the domains they possess highlighted, f, Representative endogenous Navl. l western blot 48 hours post-transfection with vector expressing the indicated CIRTS-effector and SCNla-gl a- tubulin was used as the loading control and each CIRTS-4G truncation was normalized to CIRTS-GFP, where GFP was fused to CIRTS as the effector domain, expressed with SCNla-gl. n = 3 or 4 biological replicates. All bar graph values are shown as mean ± SEM with data points. Statistical analyses were performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test vs. NT (a), (b), and (c) or vs. GFP (e) * <0.05, ** <0.01, *** <0.001, ****P<0.0001. No asterisk = not significant.

[0056] FIG. 9A-9I. CIRTS-4GT3 is an improved, general CIRTS-activator for haploinsufficiency-related targets, a, Vectors expressing either CIRTS-NYl / SCNla-g5, CIRTS-4GT3 / SCNla-gl or empty CIRTS / gRNA cassettes were transfected into N2a cells and endogenous Navl. l levels were measured 48 hours later. Representative western image is shown, a-tubulin was used as the loading control and both groups were normalized to the empty vector for quantitation, n = 4 biological replicates b, SCNla mRNA levels were measured by RT-qPCR 48 hours after transfection with vectors expressing either CIRTS-4GT3 or CIRTS- GFP and either on-target SCNla-gl or NT gRNA. GAPDH was used as the reference gene and each group was normalized to the CIRTS-4GT3 / NT condition, n = 3 biological replicates, c, 3’ UTR map for a mouse CHD2 dual luciferase reporter with gRNA positions annotated showing the distance in nucleotides (nts) from the start of the 3’ UTR and the first nucleotide on the 3’ UTR each gRNA binds to. d, CHD2 dual luciferase assay results 48 hours after HEK293T cells were transfected with vectors expressing CIRTS-NY1 and a CHD2 gRNA panel member or e, CIRTS-4GT3 and a CHD2 gRNA panel member. Each replicate was divided by their respective Nluc value and normalized to their effector-matched, NT gRNA control, n = 3 biological replicates, f, Representative endogenous CHD2 western blots completed in N2a cells 48 hours post-transfection with vectors expressing CIRTS-NY1 and indicated gRNA or g, CIRTS-4GT3 and indicated gRNA. a-tubulin was used as the loading control and each gRNA was normalized to effector-matched CIRTS expressed with NT gRNA for quantitation, n = 4 biological replicates, h, Vector map for the CIRTS-4GT3 / gRNA lentivirus expression cassettes and experimental design for primary rat cortical neuron transduction and protein extraction, i, Representative western blot analysis of Navl. l levels in primary cortical rat neurons 2 weeks post-transduction with lentivirus expressing CIRTS- 4GT3 / NT or CIRTS-4GT3 / SCNla-gl at an MOI = 10. a-tubulin was used as the loading control and signal was normalized to CIRTS-4GT3 expressed with NT gRNA for quantitation, n = 3 biological replicates. All bar graph values are shown as mean ± SEM with data points. Statistical analyses were performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test vs. empty vector (a), or vs. NT (d), (e), (f), and (g) or with post hoc Sidak’s multiple comparisons test vs. CIRTS-4GT3 / NT gRNA (b). Statistical analyses were performedusing unpaired two-tailed Student’s t test vs. NT (i). * <0.05, **P<0.01. No asterisk = not significant.

[0057] FIG. 10A-10F. CIRTS-4GT3 in AAV9 delivered by i.c.v. injection to a Dravet syndrome mouse model increases SCNla expression and improves phenotype, a, CIRTS- 4GT3 / SCNla-gl vector map for neuron-specific CIRTS-4GT3 expression via AAV9 delivery and experimental schematic for mouse cohorts used to measure indicated molecular and phenotypic endpoints, b, Representative western blot assay of Navi.1 levels in P20 female Fl mice cortex and hippocampus tissue samples. Genotype for each sample is indicated above western blot as well as treatment with either i.c.v. injection on Pl of AAV9-4GT3-gl or vehicle. Western blot analysis for P20 male Fl mice can be found at Fig. 16a. a-tubulin was used as the loading control, c, Quantitation for western blot analysis shown in Fig. 10b and Fig. 16a. Normalization was carried out in reference to Navl.l levels in wt Fl mice treated with vehicle, n = 2 for wt mice or = 6 biological replicates for SCNla+ / ' mice, d, SCNla mRNA levels measured in both wt and SCNla+ / ' P20 male Fl mice cortex and hippocampus tissue samples after treatment with either AAV9-4GT3-gl or vehicle via i.c.v. injection on Pl. GAPDH was used as the reference gene, n = 5 biological replicates e, Survival rate in female SCNla+ / ' Fl mice treated with either AAV9-4GT3-gl or vehicle via Pl i.c.v. injection after symptoms onset at ~P18 to P50. f, HTS assay performed on male P50 Fl SCNla+ / ' mice treated with either AAV9-4GT3-gl or vehicle via Pl i.c.v. injection. One male mouse treated with vehicle did not survive to P50 and was not included in statistical analysis, n = 9 biological replicates. All bar and scatter plot graph values are shown as mean ± SEM with data points. Statistical analyses were performed using two-way ANOVA with Sidak’s multiple comparisons test between vehicle- and AAV9-4GT3-gl -treated SCNla+ / ' mice (c) and between vehicle- and AAV9-4GT3-gl -treated SCNla+ / +and SCNla+ / ' mice (d). Statistical analyses were performed using Log-rank test (e) between vehicle- and AAV9-4GT3-gl -treated female SCNla+ / ' mice and unpaired two-tailed Student’s Ztest (f) between vehicle- and AAV9-4GT3- gl -treated male SCNla+ / ' mice. * <0.05, ** <0.01, *** <0.001.

[0058] FIG. 11. Overview on technologies to increase gene product. Schematic representing the central dogma for genetic information flow from DNA to mRNA to protein is shown above. Technologies that can theoretically lead to increased correct gene product in haploinsufflcency are listed with advantages and caveats under the molecule or transitional step in the central dogma they most directly influence.

[0059] FIG. 12A-12F. Additional NaVl. l western blots for dual luciferase screening hits. a, Full western blot used in Fig. 7e quantitation, b, Full western blot used in Fig. 7g quantitation, c-f, western blots for NaVl. l levels 48 hours post-transfection with indicated CIRTS-effector / gRNA vectors in N2a cells, a-tubulin was used as the loading control and each CIRTS-effector / SCNla-g5 was normalized to the matched CIRTS-effector expressed with NT gRNA for quantitation, n = 3 biological replicates for each group. All bar graph values are shown as mean ± SEM with data points. Statistical analyses were performed using unpaired two-tailed Student’s t test vs. matched CIRTS-effector / NT (c-f). No asterisk = not significant.

[0060] FIG. 13A-13G. Extended western blots targeting SCNla 5’ and 3’ UTRs with CIRTS-NY1, eIF4e, and -eIF4GI truncations, a, Full western blot used in Fig. 8b quantitation. b, Full western blot used in Fig. 8d quantitation, c-d, Endogenous NaVl.l western blots 48 hours post-transfection with vectors expressing CIRTS-NY1 (c) or CIRTS-eIF4e (d) and indicated 5’ UTR-targeting gRNA. a-tubulin was used as the loading control and each gRNA was normalized to the matched CIRTS-effector expressed with NT gRNA for quantitation, n = 2 biological replicates, e, Full western blot used in Fig. 8f quantitation, f, NaVl.l western blot 48 hours post-transfection with vectors expressing the indicated CIRTS-effector and SCNla- g6. a-tubulin was used as the loading control and each CIRTS-4G truncation was normalized to CIRTS-GFP / SCNla-g6, n = 2 or 4 biological replicates, g, Additional western blot used in Fig. 13f quantitation. All bar graph values are shown as mean ± SEM with data points. Statistical analyses were performed using one-way ANOVA with Dunnett’s multiple comparisons test vs. NT (c) and (d) or vs. GFP (f) * <0.05, ** <0.01. No asterisk = not significant.

[0061] FIG. 14A-14D. Extended CIRTS-NY1 and -4GT3 CHD2-targeting western blots and RT-qPCR. a, Full western blot used in Fig. 9a quantitation, b, Full western blot used in Fig. 9f quantitation, c, Full western blot used in Fig. 9g quantitation, d, CHD2 mRNA levels were measured by RT-qPCR 48 hours after transfection with vectors expressing CIRTS-4GT3 and either on-target CHD2-g2 or NT gRNA. GAPDH was used as the reference gene and data was normalized to the CIRTS-4GT3 / NT condition, n = 3 biological replicates. All bar graph values are shown as mean ± SEM with data points. Statistical analysis was performed using unpaired two-tailed Student’s t test vs. CIRTS-4GT3 / NT gRNA (d). No asterisk = not significant.

[0062] FIG. 15A-15F. Primary rat cortical neuron lentivirus transduction validation with CIRTS-GFP / NT and CIRTS-NYl / rSCNla-g5 Navl.l western blots, a, Vector map for theCIRTS-GFP / NT gRNA lentivirus expression cassettes and experimental design for primary rat neuron transduction and GFP imaging, b, Representation images of primary rat cortical neurons in the brightfield and GFP channel 6 days post-transduction with different MOIs of CIRTS-GFP / NT lentivirus. Images were captured and processed using the same settings between treated groups, c, Full western blot used in Fig. 9i quantitation, d, Vector map for the CIRTS-NYl / gRNA lentivirus expression cassettes and experimental design for primary rat neuron transduction and protein extraction, e, Western blot analysis of Navi .1 levels in primary cortical rat neurons 2 weeks post-transduction with lentivirus expressing CIRTS-NY1 / NT or CIRTS-NYl / rSCNla-g5 at a MOI = 10. a-tubulin was used as the loading control and signal was normalized to CIRTS-NY1 expressed with NT gRNA for quantitation, n = 3 biological replicates, f, Alignment of partial sequences from mouse and rat SCNla mRNA 3’ UTR with the mouse-targeting SCNla-g5 gRNA and rat-targeting SCNla-rg5 gRNA respectively. Bases modified in the SCNla-rg5 sequence compared to the SCNla-g5 sequence to ensure full targetcomplementarity are highlighted in red boxes. All bar graph values are shown as mean ± SEM with data points. Statistical analysis was performed using unpaired two-tailed Student’s t test vs. NT (i). * <0.05. No asterisk = not significant. The sequences in FIG. 15f correspond to SEQ ID NO:61, 46, 62, and 63, respectively.

[0063] FIG. 16A-16D. Extended In vivo Fl DS mice western blot, survival, and HTS data, a, Additional western blot used in Fig. 10b quantitation performed with cortex and hippocampus tissue from male P21 Fl mice treated with either AAV9-4GT3-gl or vehicle via Pl i.c.v. injection, b, Survival rate in male SCNla+ / - and SCNla+ / +Fl mice treated with either AAV9-4GT3-gl or vehicle via Pl i.c.v. injection to P50. c, Survival rate in female SCNla+ / + Fl mice treated with either AAV9-4GT3-gl or vehicle via Pl i.c.v. injection to P50. d, HTS assay performed on male P50 Fl SCNla+ / +mice treated with either AAV9-4GT3-gl or vehicle via Pl icv injection, n = 5 biological replicates. All scatter plot graph values are shown as mean ± SEM with data points. Statistical analyses were performed using Log-rank test between all groups (b) and (c). No asterisk = not significant.

[0064] FIG. 17A-17C. Linear detection range for western blot primary antibodies and in vivo Navl. l western blot optimization, a, Western blot for Navl.l over a range of total N2a lysate loaded. Signal for each band is plotted below with the total lysate loaded range with the highest linearity shaded, b, Western blot for CHD2 over a range of total N2a lysate loaded. Signal for each band is plotted below with the total lysate loaded range with the highest linearity shaded, c, Representative western blot for SCNla+ / + and SCNla+ / - Fl mouse cortex andhippocampus tissue samples blotted for Navi .1 and a-tubulin with different pre-loading sample heating steps. Statistical analyses were performed using simple linear regression to obtain R? for the dashed line fit to the indicated data points.

[0065] FIG. 18A-18B A, 3’ UTR map for the mouse ARID IB 3’ UTR with gRNA positions annotated showing the distance in nucleotides (nts) from the start of the 3’ UTR and the first nucleotide on the 3 ’ UTR each gRNA binds to. B, Representative endogenous ARID IB western blot completed with N2a cell lysate collected 48 hours post-transfection with vectors expressing CIRTS-4GT3 and indicated gRNA. a-tubulin was used as the loading control and each gRNA was normalized to effector-matched CIRTS expressed with NT gRNA for quantitation, n = 6 or 3 biological replicates. All bar graph values are shown as mean ± SEM with data points. Statistical analyses were performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test vs. CIRTS-4GT3 / NT gRNA (B). *P<0.05, **P<0.01, ***p<0 001, ****P<0.0001. No asterisk = not significant.DETAILED DESCRIPTION

[0066] Epitranscriptomic regulation controls information flow through the central dogma and provides unique opportunities for manipulating cell state at the RNA level. However, both fundamental mechanistic studies and potential translational applications are impeded by a lack of effective methods to target specific RNAs with effector proteins. Here, the inventors present the design and validation of a CRISPR / Cas-inspired RNA targeting system (CIRTS), a new strategy for constructing programmable systems. The inventors show that CIRTS is a simple and generalizable approach to deliver a range of effector proteins, including nucleases, degradation machinery, and translational activators, to target transcripts. CIRTS complexes are not only smaller than naturally-occurring CRISPR / Cas programmable RNA binding systems, but can be built entirely from human parts. The small size and human-derived nature of CIRTS provides a less perturbative method for fundamental RNA regulatory studies as well as a potential strategy to avoid immune issues when applied to epitranscriptome-modulating therapies.I. RNA Effector Domains

[0067] The Table below exemplifies functions of the RNA effector domain.Table 1: Functions of RNA Effector DomainII. RNA Hairpin Binding Domains and Hairpin Structures

[0068] Various RNA hairpin binding domains and hairpin structures that they bind are known in the art and can be used in the systems, compositions, fusion proteins, kits, vectors, and methods of the disclosure. For example, aspects include a RNA hairpin binding domain and hairpin structure according to the following table (Table 2), which lists proteins comprising RNA hairpin binding domains and the hairpin structure to which they specifically bind”Table 2: RNA hairpin binding domain

[0069] It is contemplated that multiple RNA hairpin binding domains and / or the Effector domain may be used in a multiplexed fashion by using RNA hairpin binding domains that bind to different hairpin structures to target multiple different RNAs in the same cell. Different targeted RNAs may be modulated in the same or in different ways. For example, one RNA may be modulation with translational activation, while a second RNA may be modulated with translational repression in the same cell. Therefore, the systems of the disclosure can be usedin a multiplexed fashion for the modulation of at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more RNAs in one cell, tissue, or organisms.III. Stabilizer domain

[0070] Exemplary stabilizer domains include those in the table below as well as variants of those represented by the amino acid sequence below.Table 3: Exemplary Stabilizer Domains

[0071] Stabilizer proteins are proteins that can bind to the gRNA sequence non-specifically, in some embodiments via charge-based interactions, and can be used to stabilize and protect the guiding RNA prior to target engagement.IV. Nucleic Acids

[0072] In certain aspects, there are recombinant nucleic acids encoding the proteins, polypeptides, regulatory domains, or RNA targeting molecules described herein.

[0073] As used in this application, the term “polynucleotide” refers to a nucleic acid molecule that either is recombinant or has been isolated free of total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or fewer in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.

[0074] In this respect, the term “gene,” “polynucleotide,” or “nucleic acid” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar protein (see above).

[0075] In particular aspects, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptides (e.g., a polymerase, RNA polymerase, one or more truncated polymerase domains or interaction components that are polypeptides) that drive gene transcription dependent on polymerase activity from the polymerase domains when the interaction components interact. The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule. .

[0076] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide-encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.

[0077] In certain aspects, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%,97%, 98%, or 99% or higher sequence identity, including all values and ranges there between, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90%, preferably 95% and above, identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.A. Vectors

[0078] Polypeptides may be encoded by a nucleic acid molecule. The nucleic acid molecule can be in the form of a nucleic acid vector. The term “vector” is used to refer to a carrier nucleic acid molecule into which a heterologous nucleic acid sequence can be inserted for introduction into a cell where it can be replicated and expressed. A nucleic acid sequence can be “heterologous,” which means that it is in a context foreign to the cell in which the vector is being introduced or to the nucleic acid in which is incorporated, which includes a sequence homologous to a sequence in the cell or nucleic acid but in a position within the host cell or nucleic acid where it is ordinarily not found. Vectors include DNAs, RNAs, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (for example Sambrook et al., 2001; Ausubel et al., 1996, both incorporated herein by reference). Vectors may be used in a host cell to produce a polymerase, RNA polymerase, one or more truncated polymerase domains or interaction components that are fused, attached or linked to the one or more truncated RNA polymerase domains.

[0079] The term “expression vector” refers to a vector containing a nucleic acid sequence coding for at least part of a gene product capable of being transcribed. In some cases, RNA molecules are then translated into a protein, polypeptide, or peptide. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host organism. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described herein.B. Cells

[0080] The disclosure provides methods for modifying a target RNA of interest, in particular in prokaryotic cells, eukaryotic cells, tissues, organs, or organisms, more in particular in mammalian cells, tissues, organs, or organisms. The target RNA may be comprised in a nucleic acid molecule within a cell. In some aspects, the target RNA is in a eukaryotic cell, such as a mammalian cell or a plant cell. The mammalian cell many be a human, non-human primate, bovine, porcine, rodent or mouse cell. The cell may be a non-mammalian eukaryotic cell such as poultry, fish or shrimp. The plant cell may be of a crop plant such as cassava, com, sorghum, wheat, or rice. The plant cell may also be of an algae, tree or vegetable. The modulation of the RNA induced in the cell by the methods, systems, and compositions of the disclosure may be such that the cell and progeny of the cell are altered for improved production of biologic products such as an antibody, starch, alcohol or other desired cellular output. The modulation of the RNA induced in the cell may be such that the cell and progeny of the cell include an alteration that changes the biologic product produced.

[0081] The mammalian cell may be a human or non-human mammal, e.g., primate, bovine, ovine, porcine, canine, rodent, Leporidae such as monkey, cow, sheep, pig, dog, rabbit, rat or mouse cell. The cell may be a non-mammalian eukaryotic cell such as poultry bird (e.g., chicken), vertebrate fish (e.g., salmon) or shellfish (e.g., oyster, clam, lobster, shrimp) cell. The cell may also be a plant cell. The plant cell may be of a monocot or dicot or of a crop or grain plant such as cassava, com, sorghum, soybean, wheat, oat or rice. The plant cell may also be of an algae, tree or production plant, fruit or vegetable (e.g., trees such as citrus trees, e.g., orange, grapefruit or lemon trees; peach or nectarine trees; apple or pear trees; nut trees such as almond or walnut or pistachio trees; nightshade plants; plants of the genus Brassica; plants of the genus Lactuca; plants of the genus Spinacia; plants of the genus Capsicum; cotton, tobacco, asparagus, carrot, cabbage, broccoli, cauliflower, tomato, eggplant, pepper, lettuce, spinach, strawberry, blueberry, raspberry, blackberry, grape, coffee, cocoa, etc.).

[0082] As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. In the context of expressing a heterologous nucleic acid sequence, “host cell” refers to a prokaryotic or eukaryotic cell, and it includes any transformable organism that is capable of replicating a vector or expressing a heterologous gene encoded by a vector. A host cell can, and has been, used as a recipient for vectors or viruses. A host cell may be“transfected” or “transformed,” which refers to a process by which exogenous nucleic acid, such as a recombinant protein-encoding sequence, is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny.

[0083] Some vectors may employ control sequences that allow it to be replicated and / or expressed in both prokaryotic and eukaryotic cells. One of skill in the art would further understand the conditions under which to incubate all of the above described host cells to maintain them and to permit replication of a vector. Also understood and known are techniques and conditions that would allow large-scale production of vectors, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.C. Expression Systems

[0084] Numerous expression systems exist that comprise at least a part or all of the compositions discussed above. Prokaryote- and / or eukaryote-based systems can be employed for use with an aspect to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. For example, the vectors, fusion proteins, RNA hairpin binding proteins, RNA targeting molecules, Effector domain, and accessory proteins of the disclosure may utilize an expression system, such as an inducible or constitutive expression system. Many such systems are commercially and widely available.

[0085] The insect cell / baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patents 5,871,986, 4,879,236, both herein incorporated by reference, and which can be bought, for example, under the name MAXBAC® 2.0 from INVITROGEN® and BACPACK™ BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH®.

[0086] In addition to the disclosed expression systems, other examples of expression systems include STRATAGENE®’s COMPLETE CONTROL Inducible Mammalian Expression System, which involves a synthetic ecdysone-inducible receptor, or its pET Expression System, an E. coli expression system. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX™ (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGEN® also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would knowhow to express a vector, such as an expression construct, to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide.D. Conjugation of Nucleic Acids and Polypeptides

[0087] Aspects of the disclosure relate to the conjugation of nucleic acids to polypeptides. Methods of conjugation of nucleic acids to polypeptides are known in the art and include those described below. Aspects of the disclosure relate to methods of making nucleic acid- polypeptide molecules and the molecules themselves wherein the nucleic acid has been conjugated to the polypeptide by way of a method described herein. One such example includes click chemistry. The "click reaction", also known as "click chemistry" is a name often used to describe a stepwise variant of the Huisgen 1,3-dipolar cycloaddition of azides and alkynes to yield 1,2, 3 -triazole. This reaction is carried out under ambient conditions, or under mild microwave irradiation, typically in the presence of a Cu(I) catalyst, and with exclusive regioselectivity for the 1,4-di substituted triazole product when mediated by catalytic amounts of Cu(I) salts [V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596; H. C. Kolb, M. Finn, K. B. Sharpless, Angew Chem., Int. Ed. 2001, 40, 2004],

[0088] In other conjugation methods, a mutant form of the human DNA repair protein 06- alkylguanine-DNA alkyltransferase reacts rapidly and specifically with O6-benzylguanin (BG) and also with derivatives that carry a large moiety linked to the benzyl group. With guanine as the leaving group, the benzyl moiety becomes covalently attached to a cysteine in the active site of the enzyme. The enzyme has also been mutagenized to become specific for 06- benzylcytosine (BC) in a similar manner. These enzyme domains (about 20 kDa) are commercially available as SNAP and CLIP tags, respectively.

[0089] A further conjugation method utilizes the Halo tag. The Halo tag makes use of a chemical reaction orthogonal to eukaryotes, i.e. the dehalogenation of haloalkane ligands, thus, leading to highly specific covalent labelling of the tag, and therefore protein, in both live and fixed cells.E. Nucleic Acid Modifications

[0090] The oligonucleotides of the disclosure, such as the RNA targeting molecules and other nucleic acids described herein may have modifications, for example that increase the stability of the nucleic acid. In some aspects, the nucleic acid is an oligonucleotide analog. The term “oligonucleotide analog” refers to compounds which function like oligonucleotides butwhich have non-naturally occurring portions (e.g., that include one or more non-natural residues or linkages). Oligonucleotide analogs can have altered sugar moieties, altered base moieties or altered inter-sugar linkages. The term “oligomers” is intended to encompass oligonucleotides, oligonucleotide analogs or oligonucleosides. Thus, in speaking of “oligomers” reference is made to a series of nucleosides or nucleoside analogs that are joined via either natural phosphodiester bonds or other linkages, including the four atom linkers. Although the linkage generally is from the 3’ carbon of one nucleoside to the 5’ carbon of a second nucleoside, the term “oligomer” can also include other linkages such as 2’ -5’ linkages.

[0091] Oligonucleotide analogs also can include other modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and / or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true nucleic acid species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.

[0092] The present disclosure concerns modified oligonucleotides, i.e., oligonucleotide analogs or oligonucleosides, and methods for effecting the modifications. These modified oligonucleotides and oligonucleotide analogs may exhibit increased chemical and / or enzymatic stability relative to their naturally occurring counterparts. Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds. When present as the protonated acid form, the lack of a negatively charged backbone may facilitate cellular penetration.

[0093] The modified internucleoside linkages are intended to replace naturally-occurring phosphodiester-5’ -methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound. Preferred linkages have structure CH2— RA— NR1 CH2, CH2— NR1— RA— CH2, RA— NR1— CH2— CH2, CH2— CH2— NR1— RA, CH2— CH2— RA— NR1, or NR1— RA— CH2— CH2 where RA is O orNR2.

[0094] Modifications to the nucleic acid molecules of the disclosure may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA synthesizer using methodology commonly known to those skilled in DNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5’ to 3’ sense, an “upstream” synthon is modified at its terminal 3’ site, while a “downstream” synthon is modified at its terminal 5’ site.

[0095] Oligonucleosides linked by hydrazines, hydroxylarnines, and other linking groups can be protected by a dimethoxytrityl group at the 5 ’-hydroxyl and activated for coupling at the 3 ’-hydroxyl with cyanoethyldiisopropyl-phosphite moieties. These compounds can be inserted into any desired sequence by standard, solid phase, automated DNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing a uniform backbone linkage can be synthesized by use of CPG- solid support and standard nucleic acid synthesizing machines such as Applied Biosystems Inc. 380B and 394 and Milligen / Biosearch 7500 and 8800s. The initial nucleotide (number 1 at the 3 ’-terminus) is attached to a solid support such as controlled pore glass. In sequence specific order, each new nucleotide is attached either by manual manipulation or by the automated synthesizer system.

[0096] Free amino groups can be alkylated with, for example, acetone and sodium cyanoboro hydride in acetic acid. The alkylation step can be used to introduce other, useful, functional molecules on the macromolecule. Such useful functional molecules include but are not limited to reporter molecules, RNA cleaving groups, groups for improving the pharmacokinetic properties of an oligonucleotide, and groups for improving the pharmacodynamic properties of an oligonucleotide. Such molecules can be attached to or conjugated to the macromolecule via attachment to the nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to pendent groups extending from a hydroxyl group of the sugar moiety of one or more of the nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is herein incorporated by reference, and in other of the above-referenced patent applications.

[0097] Solid supports may include any of those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass

[0053] , TentaGel Support — an aminopolyethyleneglycol derivatized support

[0054] or Poros — a copolymer of polystyrene / divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides canbe effected via standard procedures

[0055] , As used herein, the term solid support further includes any linkers (e.g., long chain alkyl amines and succinyl residues) used to bind a growing oligonucleoside to a stationary phase such as CPG.1. Locked Nucleotides

[0098] In some aspects, the nucleic acid of the disclosure, such as the nucleic acid comprises a locked nucleic acid. A locked nucleic acid (LNA or Ln), also referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon. The bridge “locks” the ribose in the 3’-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired and hybridize with DNA or RNA according to Watson-Crick base-pairing rules. Such oligomers are synthesized chemically and are commercially available. The locked ribose conformation enhances base stacking and backbone pre-organization. This significantly increases the hybridization properties (melting temperature) of oligonucleotides.2. Ethylene Bridged Nucleotides

[0099] In some aspects, the nucleic acid of the disclosure, such as the nucleic acid comprises one or more ethylene bridged nucleotides. Ethylene-bridged nucleic acids (ENA or En) are modified nucleotides with a 2’-O, 4’C ethylene linkage. Like locked nucleotides, these nucleotides also restrict the sugar puckering to the N-conformation of RNA.3. Peptide Nucleic Acids

[0100] In some aspects, the nucleic acid of the disclosure, such as the nucleic acid comprises one or more peptide nucleic acids. Peptide nucleic acids (PNA or Pn) mimic the behavior of DNA and binds complementary nucleic acid strands. The term, “peptide,” when used herein may also refer to a peptide nucleic acid. PNA is an artificially synthesized polymer similar to DNA or RNA. DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The various purine and pyrimidine bases are linked to the backbone by a methylene bridge (-CH2-) and a carbonyl group (-(C=O)-). PNAs are depicted like peptides, with the N-terminus at the first (left) position and the C-terminus at the last (right) position.

[0101] Since the backbone of PNAs contains no charged phosphate groups, the binding between PNA / DNA strands is stronger than between DNA / DNA strands due to the lack of electrostatic repulsion. PNAs are not easily recognized by either nucleases or proteases, makingthem resistant to degradation by enzymes. PNAs are also stable over a wide pH range. In some aspects, the PNAs described herein have improved cytosolic delivery over other PNAs.4. 5’(E)-vinyl-phosphonate (VP) modification

[0102] In some aspects, the nucleic acid of the disclosure, such as the nucleic acid comprises one or more 5’(E)-vinyl-phosphonate (VP) modifications. 5’-Vinyl-phosphonate modifications (metabolically stable phosphate mimics) have been reported to enhance the metabolic stability and the potency of oligonucleotides.5. Morpholinos

[0103] In some aspects, the nucleic acid of the disclosure, such as the nucleic acid comprises a morpholino. Morpholinos are synthetic molecules that are the product of a redesign of natural nucleic acid structure. Usually 25 bases in length, they bind to complementary sequences of RNA or single-stranded DNA by standard nucleic acid base-pairing. In terms of structure, the difference between Morpholinos and DNA is that, while Morpholinos have standard nucleic acid bases, those bases are bound to methylenemorpholine rings linked through phosphorodiamidate groups instead of phosphates. The figure compares the structures of the two strands depicted there, one of RNA and the other of a Morpholino. Replacement of anionic phosphates with the uncharged phosphorodiamidate groups eliminates ionization in the usual physiological pH range, so Morpholinos in organisms or cells are uncharged molecules. The entire backbone of a Morpholino is made from these modified subunits.V. Delivery Vehicles

[0104] The current disclosure contemplates several delivery systems compatible with nucleic acids that provide for roughly uniform distribution and have controllable rates of release. A variety of different media are described below that are useful in creating nucleic acid delivery systems. It is not intended that any one medium or carrier is limiting to the present invention. Note that any medium or carrier may be combined with another medium or carrier; for example, in one aspect a polymer microparticle carrier attached to a compound may be combined with a gel medium.

[0105] Carriers or mediums contemplated by this disclosure comprise a material selected from the group comprising gelatin, collagen, cellulose esters, dextran sulfate, pentosan polysulfate, chitin, saccharides, albumin, fibrin sealants, synthetic polyvinyl pyrrolidone, polyethylene oxide, polypropylene oxide, block polymers of polyethylene oxide and polypropylene oxide, polyethylene glycol, acrylates, acrylamides, methacrylates including, butnot limited to, 2-hydroxyethyl methacrylate, poly(ortho esters), cyanoacrylates, gelatin- resorcinol-aldehyde type bioadhesives, polyacrylic acid and copolymers and block copolymers thereof.A. Microparticles

[0106] Some aspects of the present disclosure contemplate a delivery system comprising a microparticle. Preferably, microparticles comprise liposomes, nanoparticles, microspheres, nanospheres, microcapsules, and nanocapsules. Preferably, some microparticles contemplated by the present invention comprise poly(lactide-co-glycolide), aliphatic polyesters including, but not limited to, poly-glycolic acid and poly-lactic acid, hyaluronic acid, modified polysaccharides, chitosan, cellulose, dextran, polyurethanes, polyacrylic acids, pseudo- poly(amino acids), polyhydroxybutyrate-related copolymers, polyanhydrides, polymethylmethacrylate, poly(ethylene oxide), lecithin and phospholipids.B. Liposomes

[0107] One aspect of the disclosure contemplates liposomes capable of attaching and releasing nucleic acids conjugates, polypeptides, and fusion proteins as described herein. Liposomes are microscopic spherical lipid bilayers surrounding an aqueous core that are made from amphiphilic molecules such as phospholipids. For example, a liposome may trap a nucleic acid between the hydrophobic tails of the phospholipid micelle. Water soluble agents can be entrapped in the core and lipid-soluble agents can be dissolved in the shell-like bilayer. Liposomes have a special characteristic in that they enable water soluble and water insoluble chemicals to be used together in a medium without the use of surfactants or other emulsifiers. Liposomes can form spontaneously by forcefully mixing phospholipids in aqueous media. Water soluble compounds are dissolved in an aqueous solution capable of hydrating phospholipids. Upon formation of the liposomes, therefore, these compounds are trapped within the aqueous liposomal center. The liposome wall, being a phospholipid membrane, holds fat soluble materials such as oils. Liposomes provide controlled release of incorporated compounds. In addition, liposomes can be coated with water soluble polymers, such as polyethylene glycol to increase the pharmacokinetic half-life. One aspect of the present invention contemplates an ultra high-shear technology to refine liposome production, resulting in stable, unilamellar (single layer) liposomes having specifically designed structural characteristics. These unique properties of liposomes allow the simultaneous storage of normally immiscible compounds and the capability of their controlled release.

[0108] In some aspects, the disclosure contemplates cationic and anionic liposomes, as well as liposomes having neutral lipids. Preferably, cationic liposomes comprise negatively-charged materials by mixing the materials and fatty acid liposomal components and allowing them to charge-associate. Clearly, the choice of a cationic or anionic liposome depends upon the desired pH of the final liposome mixture. Examples of cationic liposomes include lipofectin, lipofectamine, and lipofectace.

[0109] One aspect of the present disclosure contemplates a delivery system comprising liposomes that provides controlled release of at least one molecule described herein. Preferably, liposomes that are capable of controlled release: i) are biodegradable and non-toxic; ii) carry both water and oil soluble compounds; iii) solubilize recalcitrant compounds; iv) prevent compound oxidation; v) promote protein stabilization; vi) control hydration; vii) control compound release by variations in bilayer composition such as, but not limited to, fatty acid chain length, fatty acid lipid composition, relative amounts of saturated and unsaturated fatty acids, and physical configuration; viii) have solvent dependency; iv) have pH-dependency and v) have temperature dependency.

[0110] The compositions of liposomes are broadly categorized into two classifications. Conventional liposomes are generally mixtures of stabilized natural lecithin (PC) that may comprise synthetic identical-chain phospholipids that may or may not contain glycolipids. Special liposomes may comprise: i) bipolar fatty acids; ii) the ability to attach antibodies for tissue-targeted therapies; iii) coated with materials such as, but not limited to lipoprotein and carbohydrate; iv) multiple encapsulation and v) emulsion compatibility.

[0111] Liposomes may be easily made in the laboratory by methods such as, but not limited to, sonication and vibration. Alternatively, compound-delivery liposomes are commercially available. For example, Collaborative Laboratories, Inc. are known to manufacture custom designed liposomes for specific delivery requirements.C. Microspheres, Microparticles and Microcapsules

[0112] Microspheres and microcapsules are useful due to their ability to maintain a generally uniform distribution, provide stable controlled compound release and are economical to produce and dispense. Preferably, an associated delivery gel or the compound-impregnated gel is clear or, alternatively, said gel is colored for easy visualization by medical personnel.

[0113] Microspheres are obtainable commercially (Prolease™, Alkerme's: Cambridge, Mass.). For example, a freeze dried medium comprising at least one therapeutic agent ishomogenized in a suitable solvent and sprayed to manufacture microspheres in the range of 20 to 90 pm Techniques are then followed that maintain sustained release integrity during phases of purification, encapsulation and storage. Scott et al., Improving Protein Therapeutics With Sustained Release Formulations, Nature Biotechnology, Volume 16: 153-157 (1998). Modification of the microsphere composition by the use of biodegradable polymers can provide an ability to control the rate of nucleic acid release. Miller et al., Degradation Rates of Oral Resorbable Implants {Polylactates and Poly glycolates: Rate Modification and Changes in PLA / PGA Copolymer Ratios, J. Biomed. Mater. Res., Vol. 11 :711-719 (1977).

[0114] Alternatively, a sustained or controlled release microsphere preparation is prepared using an in-water drying method, where an organic solvent solution of a biodegradable polymer metal salt is first prepared. Subsequently, a dissolved or dispersed medium of a nucleic acid is added to the biodegradable polymer metal salt solution. The weight ratio of a nucleic acid to the biodegradable polymer metal salt may for example be about 1 : 100000 to about 1 : 1, preferably about 1 :20000 to about 1 :500 and more preferably about 1 : 10000 to about 1 :500. Next, the organic solvent solution containing the biodegradable polymer metal salt and nucleic acid is poured into an aqueous phase to prepare an oil / water emulsion. The solvent in the oil phase is then evaporated off to provide microspheres. Finally, these microspheres are then recovered, washed and lyophilized. Thereafter, the microspheres may be heated under reduced pressure to remove the residual water and organic solvent.

[0115] Other methods useful in producing microspheres that are compatible with a biodegradable polymer metal salt and nucleic acid mixture are: i) phase separation during a gradual addition of a coacervating agent; ii) an in-water drying method or phase separation method, where an antiflocculant is added to prevent particle agglomeration and iii) by a spraydrying method.

[0116] In one aspect, the present invention contemplates a medium comprising a microsphere or microcapsule capable of delivering a controlled release of a nucleic acid for a duration of approximately between 1 day and 6 months. In one aspect, the microsphere or microparticle may be colored to allow the medical practitioner the ability to see the medium clearly as it is dispensed. In another aspect, the microsphere or microcapsule may be clear. In another aspect, the microsphere or microparticle is impregnated with a radio-opaque fluoroscopic dye.

[0117] Controlled release microcapsules may be produced by using known encapsulation techniques such as centrifugal extrusion, pan coating and air suspension. Such microspheres and / or microcapsules can be engineered to achieve desired release rates. For example, Oliosphere™ (Macromed) is a controlled release microsphere system. These particular microsphere's are available in uniform sizes ranging between 5-500 pm and composed of biocompatible and biodegradable polymers. Specific polymer compositions of a microsphere can control the nucleic acid release rate such that custom-designed microspheres are possible, including effective management of the burst effect. ProMaxx™ (Epic Therapeutics, Inc.) is a protein-matrix delivery system. The system is aqueous in nature and is adaptable to standard pharmaceutical delivery models. In particular, ProMaxx™ are bioerodible protein microspheres that deliver both small and macromolecular drugs, and may be customized regarding both microsphere size and desired release characteristics.

[0118] In one aspect, a microsphere or microparticle comprises a pH sensitive encapsulation material that is stable at a pH less than the pH of the internal mesentery. The typical range in the internal mesentery is pH 7.6 to pH 7.2. Consequently, the microcapsules should be maintained at a pH of less than 7. However, if pH variability is expected, the pH sensitive material can be selected based on the different pH criteria needed for the dissolution of the microcapsules. The encapsulated nucleic acid, therefore, will be selected for the pH environment in which dissolution is desired and stored in a pH preselected to maintain stability. Examples of pH sensitive material useful as encapsulants are Eudragit™ L-100 or S-100 (Rohm GMBH), hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, polyvinyl acetate phthalate, cellulose acetate phthalate, and cellulose acetate trimellitate. In one aspect, lipids comprise the inner coating of the microcapsules. In these compositions, these lipids may be, but are not limited to, partial esters of fatty acids and hexitiol anhydrides, and edible fats such as triglycerides. Lew C. W., Controlled-Release pH Sensitive Capsule And Adhesive System And Method. U.S. Pat. No. 5,364,634 (herein incorporated by reference).

[0119] In one aspect, the present invention contemplates a microparticle comprising a gelatin, or other polymeric cation having a similar charge density to gelatin (i.e., poly-L-lysine) and is used as a complex to form a primary microparticle. A primary microparticle is produced as a mixture of the following composition: i) Gelatin (60 bloom, type A from porcine skin), ii) chondroitin 4-sulfate (0.005%-0.1%), iii) glutaraldehyde (25%, grade 1), and iv) l-ethyl-3-(3- dimethylaminopropyl)-carbodiimide hydrochloride (EDC hydrochloride), and ultra-puresucrose (Sigma Chemical Co., St. Louis, Mo.). The source of gelatin is not thought to be critical; it can be from bovine, porcine, human, or other animal source. Typically, the polymeric cation is between 19,000-30,000 daltons. Chondroitin sulfate is then added to the complex with sodium sulfate, or ethanol as a coacervation agent.

[0120] Following the formation of a microparticle, a nucleic acid is directly bound to the surface of the microparticle or is indirectly attached using a "bridge" or "spacer". The amino groups of the gelatin lysine groups are easily derivatized to provide sites for direct coupling of a compound. Alternatively, spacers (i.e., linking molecules and derivatizing moieties on targeting ligands) such as avidin-biotin are also useful to indirectly couple targeting ligands to the microparticles. Stability of the microparticle is controlled by the amount of glutaraldehyde- spacer crosslinking induced by the EDC hydrochloride. A controlled release medium is also empirically determined by the final density of glutaraldehyde-spacer crosslinks.

[0121] In one aspect, the present invention contemplates microparticles formed by spraydrying a composition comprising fibrinogen or thrombin with a nucleic acid. Preferably, these microparticles are soluble and the selected protein (i.e., fibrinogen or thrombin) creates the walls of the microparticles. Consequently, the nucleic acids are incorporated within, and between, the protein walls of the microparticle. Heath et al., Microparticles And Their Use In Wound Therapy. U.S. Pat. No. 6,113,948 (herein incorporated by reference). Following the application of the microparticles to living tissue, the subsequent reaction between the fibrinogen and thrombin creates a tissue sealant thereby releasing the incorporated compound into the immediate surrounding area.

[0122] One having skill in the art will understand that the shape of the microspheres need not be exactly spherical; only as very small particles capable of being sprayed or spread into or onto a surgical site (i.e., either open or closed). In one aspect, microparticles are comprised of a biocompatible and / or biodegradable material selected from the group consisting of polylactide, polyglycolide and copolymers of lactide / glycolide (PLGA), hyaluronic acid, modified polysaccharides and any other well known material.VI. Proteinaceous and Nucleic Acid Compositions

[0123] The polypeptides or polynucleotides of the disclosure, such as the CIRT fusion proteins, stabilizer polypeptide, linker, RNA hairpin binding domain, NES, effector domain, tag, NLS, RNA targeting molecule, hairpin region of the RNA targeting molecule, helical region, or targeting region of the nucleic acid may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more variant amino acids or nucleic acid substitutions or be at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with at least, or at most 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207,208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids or nucleic acids, or any range derivable therein, of SEQ ID NOs: 1-63.

[0124] The polypeptides or polynucleotides of the disclosure, such as the CIRT fusion proteins, stabilizer polypeptide, linker, RNA hairpin binding domain, NES, effector domain, tag, NLS, RNA targeting molecule, hairpin region of the RNA targeting molecule, helical region, or targeting region of the nucleic acid may include 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more contiguous amino acids, or any range derivable therein, of SEQ ID NOs: 1-63.

[0125] In some aspects, the polypeptide or nucleic acid may comprise amino acids or nucleotide 1 to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76. 77. 78. 79. 80. 81. 82. 83. 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 94. 95. 96. 97. 98. 99. 100.101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138,139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176,177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214,215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271,272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309,310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328,329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347,348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366,367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404,405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423,424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442,443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461,462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480,481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518,519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537,538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575,576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594,595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) of SEQ ID NOs: 1-63.

[0126] In some aspects, th polypeptide may comprise 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104 , 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) contiguous amino acids or nucleic acids of SEQ ID NOs: 1-63.

[0127] In some aspects, the polypeptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104. , 105, 106, 107, 108, 109, 110,111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585,586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 (or any derivable range therein) contiguous amino acids of SEQ ID NOs: l-63 that are at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous with one of SEQ ID NOs: l-63.

[0128] In some aspects, the stabilizer polypeptide may have one or more substitutions that reduce or eliminate binding to endogenous proteins. In some aspects, the stabilizer polypeptide may have one or more substitutions that reduce or eliminate an activity directed to an endogenous protein.

[0129] The polypeptides and nucleic acids of the disclosure, such as the CIRT proteins, stabilizer domain, linker, RNA hairpin binding domain, NES, effector domain, molecular tag, , RNA hairpin region of the RNA targeting molecule, may comprise, comprise at least, or comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146^ 147, 148, 149, 150, 151, 152, 153, 154, 155,156, 157, 158, 159, 160, 161, 162, 163, 164, 165:166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184:185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203^ 204, 205, 206, 207, 208, 209, 210, 211, 212,213, 214, 215, 216, 217, 218, 219, 220, 221, 222 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 24L 242, 243, 244, 245, 246, 247, 248, 249, 250,251, 252, 253, 254, 255, 256, 257, 258, 259, 260^ 261, 262, 263, 264, 265, 266, 267, 268, 269,270, 271, 272, 273, 274, 275, 276, 277, 278, 229 280, 281, 282, 283, 284, 285, 286, 287, 288,289, 290, 291, 292, 293, 294, 295, 296, 297, 298^ 299, 300, 301, 302, 303, 304, 305, 306, 307,308, 309, 310, 311, 312, 313, 314, 315, 316, 317 318, 319, 320, 321, 322, 323, 324, 325, 326,327, 328, 329, 330, 331, 332, 333, 334, 335, 336^ 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355:356, 357, 358, 359, 360, 361, 362, 363, 364,365, 366, 367, 368, 369, 370, 371, 372, 373, 374 375, 376, 377, 378, 379, 380, 381, 382, 383,384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421,422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440,441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459,460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478,479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516,517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535,536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554,555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573,574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592,593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,612, 613, 614, or 615 substitutions.

[0130] The substitution may be at amino acid position or nucleic acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123,124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180,181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218,219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237,238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256,257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313,314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351,352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370,371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389,390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408,409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446,447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465,466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484,485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503,504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522,523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560,561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579,580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, or 615 of one of SEQ ID NO: 1-63.

[0131] The substitution at amino acid position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 12t 122, 123, 124, 125, 126, 127, 128, 129, 130,131, 132, 133, 134, 135, 136, 137, 138, 139, 140 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159:160, 161, 162, 163, 164, 165, 166, 167, 168,169, 170, 171, 172, 173, 174, 175, 176, 177, 178:179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216^ 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235^ 236, 237, 238, 239, 240, 241, 242, 243, 244,245, 246, 247, 248, 249, 250, 251, 252, 253, 254^ 255, 256, 257, 258, 259, 260, 261, 262, 263,264, 265, 266, 267, 268, 269, 270, 271, 272, 273^ 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292^ 293, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311. 312, 313, 314, 315, 316, 317, 318, 319, 320,321, 322, 323, 324, 325, 326, 327, 328, 329, 330^ 331, 332, 333, 334, 335, 336, 337, 338, 339,340, 341, 342, 343, 344, 345, 346, 347, 348, 349^ 350, 351, 352, 353, 354, 355, 356, 357, 358,359, 360, 361, 362, 363, 364, 365, 366, 367, 368^ 369, 370, 371, 372, 373, 374, 375, 376, 377,378, 379, 380, 381, 382, 383, 384, 385, 386, 387 388, 389, 390, 391, 392, 393, 394, 395, 396,397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434,435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453,454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491,492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510,511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529,530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548,549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586,587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605,606, 607, 608, 609, 610, 611, 612, 613, 614, or 650 of SEQ ID NOs: 1-63 may be a substitution with an alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leusine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, or valine.

[0132] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Nonconservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.

[0133] Proteins may be recombinant or synthesized in vitro. Alternatively, a nonrecombinant or recombinant protein may be isolated from bacteria. It is also contemplated thatbacteria containing such a variant may be implemented in compositions and methods. Consequently, a protein need not be isolated.

[0134] The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, and also refers to codons that encode biologically equivalent amino acids.

[0135] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either the 5' or 3' portions of the coding region.

[0136] The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.

[0137] In other aspects, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure with the intent to modify the interactive binding capacity of interaction components. Structures such as, for example, protein interaction domains, nucleic acid interaction domains, and catalytic sites may have amino acids substituted to alter such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with different properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes with appreciable alteration of their biological utility or activity.

[0138] In making such changes, the hydropathic index of amino acids may be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0139] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.

[0140] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0141] In specific aspects, all or part of proteins described herein can also be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available and can be used in accordance with known protocols. See, for example, Stewart and Young, (1984); Tam et al., (1983); Merrifield, (1986); and Barany and Merrifield (1979), each incorporated herein by reference. Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence that encodes a peptide or polypeptide is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.

[0142] One aspect includes the use of gene transfer to cells, including microorganisms, for the production and / or presentation of proteins. The gene for the protein of interest may be transferred into appropriate host cells followed by culture of cells under the appropriate conditions. A nucleic acid encoding virtually any polypeptide may be employed. The generation of recombinant expression vectors, and the elements included therein, are discussedherein. Alternatively, the protein to be produced may be an endogenous protein normally synthesized by the cell used for protein production.VII. SEQUENCESTable 4VIII. EXAMPLES

[0143] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carryout the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.Example 1A. RESULTS

[0144] Shown in FIG 1 A-1C, previously described CIRTS-eIF4G (711 - 1599) here labeled “4GT1”, was tested with gRNA targeting the mouse SCNla 3’ UTR for the ability to increase luciferase expression in the previously described mSCNla dual luciferase assay. The inventors were able to measure significant increased luciferase expression for gRNAs gl and g4. The inventors then retested CIRTS-4GT1 with these gRNA and g5 for the ability to increase endogenous protein expression from the SCNla transcript by western blot of the protein, Navl. l, encoded by the SCNla transcript in N2a cells. The inventors found that gl provided the most significant increase in Navi .1 expression. Likewise, the inventors also tested a gRNA, g6, which is complementary to a region in the SCNla transcript’s 5’ UTR and measured increased Navl. l levels when expressed alongside CIRTS-4GT1.

[0145] For FIG. 2A-2C, four truncations of eIF4G with different protein binding domains were designed and tested as CIRTS effectors targeting the 3’ and 5’ UTRs of SCNla in N2a cells. CIRTS-4GT3 containing the core of eIF4G and binding sites for eIF4a and eIF3 resulted in the most significant increase in Navi .1 expression targeting the 3 ’ UTR. CIRTS-4GT2 with additional eIF4e and RNA binding domains increased Navl. l expression most significantly when targeted to the 5’ UTR.

[0146] Shown in FIG. 3, neurons transduced with CIRTS-activators and SCNla-targeting gRNA were measured to have significantly higher Navi .1 expression than neurons transduced with lentivirus encoding CIRTS-activators and non-targeting gRNA.

[0147] While two gRNA, g2 and g3, increased luciferase expression in the CHD2 3’ UTR reporter both gRNA failed to significantly increase endogenous CHD2 expression in N2a cells by western blot (FIG. 4A-4B).

[0148] CIRTS-4GT3 expressed with CHD2 3’ UTR targeted g2 demonstrated significant increase to luciferase expression from the CHD2 3’ UTR dual luciferase reporter and also to endogenous CHD2 levels measured by western blot in N2a cells (FIG. 5A-5B).B. METHODS1. Transducing neurons

[0149] Primary cultures of rat cortical neurons were prepared as described (citation below) using Neurobasal Media (NBM), 4% (v / v) B27, and 0.125 mM L-glutamine (all from Thermo Fisher Scientific, Waltham, MA). Dissociated cortical neurons from El 8 Sprague Dawley rat pups were plated in six-well plates coated with poly-D-lysine (Sigma, St Louis, MO). For western blots, 0.4 * 106cells were plated in each well and four days after plating the indicated lentivirus-packaged CIRTS vectors with added to each well at an MOI of 10. 2 weeks after transduction cells were lysed and western blots were performed.C. TablesTable 5:Table 6:Table 7: gRNA used in FIG. 1A-1C, 2A-2C, 3, 4A-4B, and 5A-5BTable 8: Additional effector amino acid sequences designed and tested in Fig. 2A-2CExample 2: Engineering a human-based translational activator for targeted protein expression restoration

[0150] Therapeutic modalities to programmably boost protein production are in critical need to address diseases caused by deficient gene expression, especially via haploinsufficiency in neurological tissues. Here the inventors report the identification and engineering of human proteins capable of increasing mRNA translation and fuse them to our CRISPR-Cas Inspired RNA-targeting System (CIRTS) platform to enable programmable, guide RNA (gRNA)-directed translational activation with entirely engineered human proteins. The inventors demonstrate that a compact (601 amino acid) CIRTS translational activator (CIRTS-4GT3), can drive targeted, sustained translation increases up to 100% from endogenous transcripts relevant to epilepsy and neurodevelopmental disorders in Neuro-2a cells and primary neurons. AAV-delivery of CIRTS-4GT3 targeting SCNla mRNA to a Dravet syndrome mouse model, a severe developmental and epileptic encephalopathy caused by SCNla haploinsufficiency, led to increased SCNla translation and improved survivability and seizure threshold. This work validates a new strategy to address SCNla haploinsufficiency and emphasizes the preclinical potential translational activation has to broadly address neurological haploinsufficiency.A. INTRODUCTION

[0151] Genetic haploinsufficiency results from a loss-of-function mutation to one gene copy thereby reducing functional protein expression by approximately half h Haploinsufficiency to any one of approximately 3,000 identified “dosage” sensitive genes drive diverse diseases, including metabolic disorders, heart disease, cancers and neurological disorders2’3. Brain-related genes are the most sensitive to aberrant gene dosage2, and the number of validated causal haploinsufficiency genes identified for epilepsies4and neurodevelopmental disorders5,6is rapidly growing. While a panoply of existing therapeutic technologies can inhibit7, knock down8, knock out9, and degrade10over-active gene products, comparably few are capable of correcting gene under-expression, with even fewer deployable in neurological tissues, presenting a critical unmet therapeutic need for millions of patientsn.

[0152] Current therapeutic approaches for restoring homeostatic protein levels from deficient genes typically involve gene supplementation, gene editing, transcriptional activation, suppressor tRNAs, small molecule protein stabilization, or ASOs for splice-switching, IncRNA degradation or disrupting negative regulatory elements12'15. While each approach is promising in certain contexts, these methods face challenges for targeting haploinsufficiency generally in the brain, such as gene size or oligo-related delivery complications, a significant risk for permanent off-target genomic changes or inducing gene overexpression in off-target cell types eliciting toxicity, or a dependence on target-specific regulatory pathways (Fig. 11). In contrast, targeting translation by upregulating protein production from endogenous mRNAs using a programmable translational activator offers potential advantages, including no size limitations on the targeted gene, protein activation levels that correspond to the mRNA presence and abundance within each cell, aligning with cell-specific needs, and activation levels that are predictable and governed by the regulatory pathways being utilized (Fig. 11). Furthermore, thetranscriptome-level specificity afforded by RNA-targeting is particularly advantageous in the brain where incredible cell-type diversity complicates DNA-targeting therapeutic delivery16-18

[0153] While the clinical potential of programmable translational activation for restoring haploinsufficient gene product expression is enticing, especially in the brain, the ability to target translation to relieve haploinsufficient phenotypes remains unproven. The inventors previously established the CRISPR / Cas-inspired RNA-targeting system (CIRTS) - a compact, programmable gRNA-dependent RNA regulatory technology assembled from human proteins to circumvent the immunogenicity and size limitations of CRISPR systems19-21. Here, the inventors deployed CIRTS to investigate whether activating the translation of a haploinsufficient mRNA was a viable strategy to restore homeostatic gene expression in an animal model of disease (Fig. 6). The inventors targeted SCNla, which is translated into the voltage-gated sodium channel Navl.l, and whose haploinsufficiency results in Dravet syndrome (DS), a severe, early-onset developmental and epileptic encephalopathy (DEE)22-24DS is the most common DEE with an incidence of 1 : 15,500 with SCNla haploinsufficiency implicated in more than 80% of cases and is resistant to management by conventional antiepilepsy drugs with no approved cure25. Beyond the high clinical need for SCNla-related therapeutics, increasing SCNla expression by translational activation could overcome outstanding challenges with known toxicity resulting from SCNla gene therapy in non-target cell types and ongoing work to identify minor cell populations impacted by SCNla haploinsufficiency missed by current delivery methods26-28. Collectively, this makes DS a powerful test case to develop and assess programmable translational activators.

[0154] In this work, the inventors used CIRTS to screen putative human translational effectors fortheir ability to activate SCNla translation, then optimized and minimized a CIRTS translational activator (CIRTSa) construct, CIRTS-4GT3, that increases Navl. l expression approximately 100% in cellulo with a size of only 601 amino acids. The inventors also apply CIRTS-4GT3 to increase expression from a second haploinsufficiency mRNA associated with developmental delay and epilepsy, CHD229,30. Finally, the inventors delivered the optimized SCNla-targeting CIRTS to SCNla haploinsufficient mice via AAV9, which increased neuronal SCNla protein levels and resulted in significantly improved survival and diminished susceptibility to thermal-induced seizures - two hallmarks of the disease pathology. Collectively, this work establishes CIRTS-4GT3 as an optimized programmable translationalactivator and validates translational activation is a viable strategy to restore gene expression in a neurological haploinsufficiency.B. RESULTS1. Identifying CIRTS-activator candidates for SCNla

[0155] Previously, the inventors found that a CIRTS fusion to the N-terminal domain (1- 364) of the m6A reader protein YTHDF1 (CIRTS-NY1) was capable of activating translation from reporter genes and model transcripts19,3h Therefore, the inventors first assessed SCNla activation using CIRTS-NY 1 , focusing on targeting the 3 ’ UTR of the SCN 1 a transcript, as the inventors previously found targeting this region most effectively increased translation with CIRTS-NY119. To facilitate screening and optimization, the inventors constructed a dual luciferase reporter where the mouse 3’ UTR of SCNla is expressed downstream of the Firefly Luciferase (Flue) open reading frame, and an untargeted, destabilized Nano Luciferase (Nluc), is also expressed and is used as a transfection reference (Fig. 2a).

[0156] The inventors cloned dual expression vectors that express both CIRTS-NY1 (Fig. 7b) and a CIRTS gRNA, which the inventors designed to be complementary to sites tiled along the SCNla 3’ UTR (Fig. 7c). The inventors then cotransfected each CIRTS / gRNA expression vector with the SCNla 3’ UTR reporter into HEK293T cells and evaluated target Flue activation normalized to transfection control. From this gRNA screen, the inventors identified SCNla-g5 as the optimal gRNA complimentary to the SCNla 3’ UTR to mediate increased Flue expression by CIRTS-NY1 compared to CIRTS-NY1 expression alongside a nontargeting (NT) gRNA (Fig. 7d). Next, the inventors moved to confirm that the CIRTS- NYl / SCNla-g5 pair can also increase translation from the endogenous SCNla transcript by transfecting the expression vector into mouse Neuro-2a (N2a) cells, which natively express SCNla. Western blot analysis revealed only modest activation of Navi.1 levels compared to transfected CIRTS-NY1 / NT vector (Fig. 7e, Fig. 12a). While these results confirm CIRTS- NYl / SCNla-g5 can target endogenous SCNla and that SCNla translation activation is feasible, they also indicate that the CIRTS-NY1 activator system is not likely to increase Navi .1 protein to therapeutic levels. Because of the limited gRNA flexibility shown by CIRTS- NY1 on SCNla, coupled with the complexity of further engineering NY1 due to the undefined mechanism by which it impacts translation32‘33, the inventors were motivated to expand beyond the CIRTS-NY1 system for CIRTS-mediated translational activation. Therefore, the inventors performed an effector screen, with the goal of identifying more robust and flexible SCNlatranslational activators, including those amenable to optimization through protein and gRNA engineering.

[0157] The inventors selected 11 candidate effector domains from the literature, primarily identified from “tethering” reporter assays, where the protein of interest was fused to an RNA- binding protein that interacts with multiple copies of a target RNA sequence on a reporter gene34The inventors cloned CIRTS-effector domain fusions for each candidate into the dual expression vector with SCN 1 a-g5 and tested each construct using the SCN 1 a 3 ’ UTR luciferase reporter, using a NT gRNA as a control and CIRTS-NY1 as a reference. From this screen, the inventors identified several effectors that mediate significant, gRNA-dependent increases in luciferase expression from the reporter (Fig. 7f). Putative new CIRTS activators include canonical eukaryotic initiation factors such as eIF4a and eIF4e, a C-terminal truncation of eIF4GI previously shown to drive translation in reporter assays, and HSPB1 and Daz4, which although having been previously identified in a high-throughput screen of human RBPs for translational regulators, have not yet been investigated in programmable RBP systems35-37

[0158] The inventors next assessed whether each new CIRTS-activator can increase endogenous Navl. l expression targeting the native mRNA in N2a cells (Fig. 12b-12f). However, only CIRTS-eIF4GI (711-1599) (CIRTS-4GT1) demonstrated consistent and reproducible Navi .1 expression (Fig. 7g, Fig. 12b). While the expression increase imparted by CIRTS-4GT1 was modest and comparable to CIRTS-NY1, the inventors selected CIRTS- 4GT 1 as our lead candidate to attempt to further optimize through gRNA screening and effector engineering, as the eIF4GI protein offers a high potential for further optimization due to greater characterization as a translational effector in the literature compared to YTHDF138-41.2. Optimization of CIRTS-eIF4GI activators targeting SCNla

[0159] To compare the flexibility of landing sites between effectors and to identify improved gRNAs, the inventors tested CIRTS-4GT1 on the SCN1 3’ UTR reporter with the same gRNA panel the inventors initially used for CIRTS-NY1. While only a single guide, SCNla-g5, was capable of activation in the CIRTS-NY1 screen, both gl and g4 showed significant activation when paired with CIRTS-4GT1 (Fig. 8a). Given the challenges observed from validating reporter results with the endogenous transcript, the inventors next tested all three hit gRNAs with CIRTS-4GT1 on endogenous SCNla, which revealed that all three guides increase Navl. l levels (Fig. 8b, Fig. 13a), with gl showing the highest activation, yielding a -50% increase in endogenous Navl.l expression compared to the NT gRNA.

[0160] The inventors next aimed to further map the generality of landing sites for CIRTS-4GT1. eIF4GI is an important member of the eIF4F complex (along with eIF4e and eIF4a), which is integral for scaffolding translation initiation and is thought to canonically act in the 5’ UTR42. The inventors therefore tested whether CIRTS-4GT1 can also promote translation of SCN 1 a from the 5 ’ UTR. Because the 5 ’ UTR of SCN la is much shorter than its 3’ UTR (187 nts compared to 2103 nts), the inventors designed only two gRNAs, g6 and g7, targeting the 5’ UTR, and tested them directly by Western blot on endogenous SCNla in N2a cells. The inventors found that g6 could direct CIRTS-4GT1 to increase Navl. l expression compared to a NT gRNA (Fig. 8c, Fig. 13b). Of note, the inventors found both g6 and g7 failed to activate Navl. l expression with both CIRTS-NY1 and -eIF4e (Fig. 13c-13d), further confirming the increased landing site flexibility offered by CIRTS-4GT1 compared to CIRTS- NY1 and CIRTS-4GT1. The amenability of CIRTS-4GT1 for targeting both 5’- and 3’-UTRs would likely be beneficial when developing potent, specific translational activators for new endogenous targets.

[0161] With improved gRNAs targeting both the 3’ and 5’ UTRs (gl and g6, respectively), the inventors next sought to identify the minimal protein domains required for the 4GT1 effector to increase protein expression. eIF4GI is an important protein for scaffolding and orchestrating initiation factors in the early stages of translation and is composed of many protein-binding domains (Fig. 8d)38. Previously, tethering assays used to investigate eIF4GI truncations both in vitro and in cells demonstrated that the central core of the protein, spanning the middle eIF4a binding domain and eIF3 binding domain, is essential for increasing translation from reporters36,39'41. The inventors hypothesized that the eIF3 interaction is likely critical for activity, as eIF4a itself was a non-functional CIRTS effector. Moreover, the inventors recently demonstrated eIF3 recruitment by RNA-based IRES elements in trans also functions as a viable translational activator43.

[0162] The inventors designed three additional eIF4GI truncations, each retaining the core eIF3 binding domain, and probed how each domain outside of the eIF3 binding domain contributes to increasing Navl.l protein expression as a CIRTS-effector (Fig. 8d). eIF4GI-T2 lacks the C-terminal eIF4A and Mnkl domains, but adds in the eIF4e binding domain and the RNA binding domain (RBD) upstream of this functional core, which, in in vitro tethering assays41, improved translation stimulation in 5’ UTR. eIF4GI-T3 only includes the middle eIF4a and eIF3 binding domains, and eIF4GI-T4 includes only the eIF3 binding domain.

[0163] The inventors tested each CIRTS-delivered effector on endogenous SCNla activation using the 3’ UTR-targeting gl (Fig. 8e, Fig. 13e) and 5’ UTR-targeting g6 (Fig. 13f- 13g), to assess how effectors act on the transcript in different contexts. These experiments revealed that the C-terminal eIF4a and Mnkl binding domains are not major contributors to effector activity in the 3’ UTR, in agreement with previous tethering assays with the 5’ UTR36. Surprisingly, including the eIF4e and RBD on the effector resulted in subpar activation when directed to the 3’ UTR, and removing the middle eIF4a domain dramatically decreased activity, supporting in vitro evidence that eIF4a and eIF3 may bind cooperatively to this eIF4GI region creating a bridge between the bound mRNA and 40S ribosome independently of the RBD41>44. Results in the 5’ UTR also confirmed the C-terminal region after amino acid 1104 does not contribute significantly to activity, but strikingly, including the eIF4e and the RBD was beneficial in this context. Furthermore, deleting the middle eIF4a binding domain also negatively impacted effector function in the 5’ UTR, further confirming that the core eIF4a and eIF3 binding domains together are the main drivers of effector activity.

[0164] From these results, the inventors selected the CIRTS-eIF4G-T3 (CIRTS-4GT3) effector for subsequent studies, as it demonstrated the most robust and general activity across a variety of target locations. Additionally, the inventors anticipate removal of the promiscuous RBD should decrease effector binding to nonspecific RNAs, thus greatly improving specificity, as the CIRTS-4GT3 fusion should therefore depend solely on the gRNA for mRNA- engagement45. The inventors also prioritized further development with CIRTS-4GT3 directed by 3’ UTR SCNla-gl, as targeting the 3’ UTR is likely more versatile as a general gRNA design strategy for future target-development - human 3' UTRs are, on average, longer, less structured, and alternatively spliced to a lesser extent than 5' UTRs46.3. CIRTS-4GT3 is an improved, general translational activator

[0165] To benchmark the performance of CIRTS-4GT3 with CIRTS-NY1, the inventors compared each optimized activator / gRNA pair targeting SCNla in N2a cells by Western blot (Fig. 9a, Fig. 14a) and RT-qPCR analysis (Fig. 9b). The inventors observed that the CIRTS- 4GT3 / SCNla-gl pair drives a 90% increase in expression, outperforming CIRTS- NYl / SCNla-g5, which averaged a 50% increase. The approximate doubling of Navl.l expression observed with CIRTS-4GT3 / SCNla-gl suggests it is a compelling candidate for correcting SCNla under-expression in haploinsufficiency without risking toxicity from protein overexpression. Importantly, CIRTS-4GT3 does not significantly impact target transcript levels during expression activation, confirming that the increase in Navl.l is driven bytranslational activation, in agreement with previous characterization of the CIRTS-NY1 mechanism19,32(Fig. 9b). Furthermore, a slight decrease in mRNA level is observed, consistent with decreased mRNA stability during heavy translation, and which is not recapitulated upon on-target gRNA binding with CIRTS-GFP47

[0166] To assess whether CIRTS-4GT3 is also an improved translational activation system for other targets, the inventors next targeted CHD2, a chromatin remodeling enzyme whose haploinsufficiency results in epilepsy and developmental delay29,30. The inventors used the dual luciferase reporter with the mouse CHD2 3 ’ UTR to test 5 gRNAs tiling along the 3 ’ UTR alongside both CIRTS-NY1 and the new CIRTS-4GT3 to identify functional gRNA / effector pairings (Fig. 9c). The inventors observed CHD2-g2 mediates a significant increase in Flue expression with both CIRTS-NY1 and CIRTS-4GT3, while CHD2-g3 does so only with CIRTS-NY1 (Fig. 9d-9e). The inventors moved to validate that these activator / gRNA pairs can activate endogenous CHD2 translation by transfection in N2a cells and Western blot analysis (Fig. 9f-9g, Fig. 14b-14c). Comparing all activator / gRNA pairs to effector-matched non-targeting (NT) gRNA pairs revealed that CIRTS-4GT3 / CHD2-g2 led to a significant (50%) increase to CHD2 expression, while CIRTS-4GT3 / CHD2-g3 only weakly did so, in agreement with the luciferase assay. However, CIRTS-NY1 did not significantly increase CHD2 expression with either gRNA. Thus, CIRTS-4GT3 is an improved gRNA-dependent translational activator for a second disease-relevant target in N2a cells. Additionally, the inventors confirmed that activation with CIRTS-4GT3 / CHD2-g2 does not significantly change CHD2 mRNA levels in N2a cells (Fig. 14d), again observing a slight decrease in mRNA stability upon targeting with CIRTS-4GT3.4. CIRTS-4GT3 targeting SCNla is deployable by lentivirus in primary rat cortical neurons

[0167] Next, the inventors aimed to evaluate the functionality of CIRTS-4GT3’s in a neuronal context and test whether SCNla translation activation is sustained when CIRTS- 4GT3 and gRNA are constitutively expressed in rat primary cortical neurons after viral delivery. The inventors designed a CIRTS lentiviral vector based on previously reported CRISPR / gRNA expression systems48(Fig. 9h). To confirm CIRTS expression and optimize delivery, the inventors first transduced primary rat cortical neurons at 4 days in vitro (4 DIV) with a CIRTS-GFP / NT-gRNA lentiviral vector. Fluorescence microscopy at 10 DIV showed that a multiplicity of infection (MOI) of 10 results in approximately 100% transduction (Fig. 15a-15b)

[0168] After confirming CIRTS-GFP is stably expressed in primary neurons, the inventors generated lentiviral vectors expressing CIRTS-4GT3 and either SCNla-gl or NT-gRNA. SCNla-gl was fully complementary to the rat SCNla 3’ UTR and the sequence was unmodified compared to past experiments. The inventors transduced neurons on 4 DIV with one of the two lentiviral systems and then measured Navl.l levels by Western blot analysis at 18 DIV (Fig. 9i, Fig. 15c). As expected, neurons treated with lentivirus encoding on-target gRNA expressed approximately 50% more Navl. l than neurons transduced with CIRTS- 4GT3 / NT gRNA, confirming that CIRTS-4GT3 functions in a primary neuron context, with measurable effects at 2 weeks post-transduction. Despite the worse performance of CIRTS- NY1 in preliminary assays, because YTHDF1 has been shown to actively regulate translation in neurons49, the inventors also tested CIRTS-NYl / SCNla-g5 delivered by lentivirus transduction to rat primary cortical neurons. Indeed, these experiments revealed a significant gRNA-dependent increase in Navl. l expression at 2 weeks-post transduction (Fig. 15d-15f), revealing that CIRTS-NY1 is also a viable translational activator in primary neurons, though CIRTS-4GT3 was still prioritized for future experiments due to its greater potential target breadth and higher activation efficiency.5. CIRTS-4GT3 Improves Phenotype in Dravet Mouse Model

[0169] With evidence that virally delivered CIRTS-4GT3 targeting SCNla mRNA has the potential to drive sustained Navl.l levels in primary neurons, the inventors next sought to determine if CIRTS-4GT3 could boost Navl. l expression in vivo and improve phenotype in a SCNla haploinsufficiency model. Heterozygous knockout of SCNla in mice (SCNla+ / ‘) faithfully recapitulates many aspects of Dravet Syndrome (DS) in human patient populations50. These symptoms include marked early life mortality primarily due to sudden death in epilepsy (SUDEP) and a susceptibility to hyperthermic-induced seizures (HTS), which corroborates the 15-20% mortality in DS patients before 20 years of age and fever being a common trigger for DS-related epileptic episodes51. Crucially, DS mouse models are also very sensitive to Navl.l levels; previous studies demonstrate triggered re-expression or viral delivery of SCNla rescues DS phenotypes, with even a 25% boost in expression contributing to measurable phenotypic improvements52. These aspects make SCNla+ / ' mice a valuable testbed for validating CIRTS -activators as a viable strategy for addressing haploinsufficiency on both a molecular and phenotypic level.

[0170] Previous work in DS mouse models characterized GABAergic inhibitory interneurons in the hippocampus and cortex as a crucial cell type impaired by reduced Navl.lexpression and key driver of the epileptic phenotype53. These cells have been the choice target for DS genetic-targeting therapies in order to best balance phenotypic improvements with mitigating off-target effects; however, Navl.l is also expressed in other neuronal cell types with undetermined contribution to the DS phenotype that are likely missed by interneuronspecific delivery and expression strategies26'28. The inventors sought to overcome this limitation and leverage the intrinsic selectivity for cells expressing SCNla transcript afforded by our mRNA-targeting approach and designed a CIRTS-based therapy for broad neuronal delivery. To this end, the inventors designed and generated an AAV9 expressing CIRTS-4GT3 driven by a pan-neuronal hSYN promoter and SCNla-gl under a constitutive hU6 promoter (AAV9-4GT3-gl) (Fig. 10a).

[0171] Genetic background is important for DS mice to develop the epileptic phenotype. Previous work has established SCNla+ / ' mice have no overt phenotype on a 129S6 / SvEvTac background, but if crossed with C57BL / 6J their Fl offspring (129S6 / SvEvTac x C57BL / 6J) will exhibit symptom onset at approximately P21 and experience approximately 50% mortality with death by SUDEP typically occurring in the third and fourth weeks of life.

[0172] In order to evaluate the viability of this strategy to correct SCNla+ / ' phenotype in these F 1 offspring, the inventors first sought to test whether AAV9-4GT3-gl increases Navi .1 expression in the cerebral cortex and hippocampus by P21. To ensure broad neuronal AAV transduction and CIRTS-4GT3 expression by this time point the inventors choose to deliver AAV9-4GT3-gl by neonatal intracerebroventricular (i.c.v.) injection as this method has been demonstrated to be effective for cerebral and hippocampal AAV9-mediated transgene expression within P21 previously54Western blot analysis of cortico-hippocampal samples from Fl P21 mice confirmed specific CIRTS expression in animals treated with AAV9-4GT3- gl compared to animals treated with vehicle, demonstrating sustained CIRTS-4GT3 expression in the brain (Fig. 10b, Fig. 16a). Correspondingly, Navl. l protein levels were modestly increased (-30%) in AAV9-CIRTS-treated mice compared to control (Fig. 10c, Fig. 16a). As expected, SCNla mRNA levels were not significantly affected by AAV9-4GT3-gl treatment in either genotype, further confirming the increase in Navl. l levels in vivo were likely due to increased translation (Fig. 10d). This improvement produces Navl. l levels below that of wildtype; however, given that even modest increases in Navi .1 have been shown to measurably ameliorate DS-like symptoms, the inventors were nevertheless encouraged to pursue phenotypic studies to further characterize the potential of SCNla-targeting CIRTS-4GT3 as anovel therapeutic modality. The inventors therefore tested if the Navl. l expression boost measured resulted in functional protein capable of impacting the epileptic phenotype.

[0173] A second Fl cohort was treated with AAV-CIRTS or vehicle, and were monitored for survival until P50, to assess the impact AAV9-4GT3-gl treatment had on early-life mortality in SCNla+ / -mice. Among SCNla+ / -mice, the inventors observed a dramatic difference in mortality between control males and females, with control female mice displaying the commonly reported 50% mortality for this model: 9 out of 18 female mice, as compared to only 1 out of 9 males dropping out before P50 (Fig. lOe and Fig. 16b). While sexual dimorphisms in disease presentation are notuncommon in epilepsies, there has been conflicting evidence for the extent this DS model presents these differences51,55,56. Due to the phenotypic differences observed in this study, the inventors evaluated male and female mice separately for all subsequent phenotypic evaluation, in agreement with several recent publications using this model55-57Remarkably, for female SCNla+ / -mice, AAV9-4GT3-gl treatment resulted in a significantly reduced mortality rate of 13% (n = 2 / 15), compared to 50% for vehicle-treated female SCNla+ / -mice survival until P50 (n = 9 / 18) (Fig. lOe). The inventors did not observe any deaths in the female or male SCNla+ / +AAV9-CIRTS or vehicle treatment groups, underscoring the efficacy and safety potential for this strategy (Fig. 16b-16c).

[0174] Next, the inventors evaluated if AAV9-4GT3-gl treatment impacted HTS susceptibility in SCNla+ / -mice as the temperature threshold at which seizures are observed is another important measurable phenotype for DS severity22. After only one vehicle-treated male SCNla+ / -dropped out in the Fl survival cohort, the inventors tested the remaining mice by HTS assay in order to compare the temperature threshold for an induced seizure at P50 between treatment groups. The inventors found SCNla+ / -mice treated with AAV9-4GT3-gl possessed a significantly higher temperature threshold (41.5 °C) for induced seizures compared to mice treated with vehicle (40.1 °C) (Fig. 101). Additionally, the inventors recorded no seizures in SCNla+ / +mice from either treatment group (Fig. 16d). Altogether these results confirm that SCNla-targeting CIRTS-4GT3 improves functional, neuronal Navl. l expression in vivo and has exciting potential as a preclinical modality to address DS.C. DISCUSSION

[0175] In this work, the inventors optimized CIRTS-4GT3, a 601 aa protein that can activate translation from two different haploinsufficiency-related mRNAs, is easily deliverable bytherapeutically tractable single viral vectors and, to our knowledge, represents the first translational activator system shown to positively impact a haploinsufficiency phenotype in vivo. For DS specifically, CIRTS-4GT3 joins an exciting panel of emerging preclinical and early clinical therapeutics aimed at alleviating SCNla haploinsufficiency, while possessing a unique attribute blend in the field58. Current strategies to address SCNla haploinsufficiency act primarily on either the DNA-level or RNA-level. Genomic technologies include gene supplementation using dual-AAVs or adenovirus due to SCNla" s large size, or gene activation with CRISPR / dCas9 or Zinc finger transcriptional activator fusions26-59-62. RNA-targeting has also been demonstrated to be effective via ASO-based technologies that either improve productive SCNla mRNA splicing, or knockdown a repressive IncRNA52,63. CIRTS-4GT3 uniquely combines several advantages from both groups, specifically, transcriptome-level specificity of RNA-targeting and the ability to be genetically encoded and delivered broadly by AAV for sustained Navi .1 expression rescue in one dose. The advantages of transcriptomelevel specificity is especially apparent when comparing the increased mortality reported when using the pan-neuron hSYNl promoter to express Navl. l itself in a similar DS mouse model with the improvement to survival reported in this study when expressing CIRTS-4GT3 instead from the same promoter26. However, future work will be needed to investigate neuron population-specific SCNla activation from AAV9-4GT3-gl transduction and whether panneuron or GAB Aergic inhibitory interneuron-specific expression of CIRTS-4GT3 has differing impact on disease phenotype.

[0176] While this work supports the ability of translational activation to address SCNla haploinsufficiency in a heterozygous knockout DS model, many diverse SCNla mutations have been identified in patient populations. The majority of cases, and on average more severe, involve mutations which lead to no or truncated Navl.l expression, which will likely be amenable to translational activation50. Future studies will be needed to examine how translational activation impacts DS derived from SCNla missense mutations as a pool of mutant RNA would also be present and likely not cleared by nonsense mediated decay as in many truncation variants64If unwanted targeting of the mutant mRNA diminished activation of the healthy mRNA then modalities such as shRNA or orthogonal CIRTS-degraders could potentially be included in the AAV vector to achieve mutant- selective knockdown to bias the targetable mRNA population. Furthermore, much like other developed SCNla haploinsufficiency therapies, translational activation would not be amenable to other rareSCNla-related DEEs derived from gain-of-function mutations which demonstrate dominantnegative pathology24

[0177] The phenotypic improvements measured in this study imparts vital preclinical support to the burgeoning translational activation field and advances its development as an impactful strategy to boost protein expression. Over the last decade, programmable translational activation in mammalian cells has made large strides and various programmable translational activation technologies have been developed65,66. Current protein-based translational activation platforms function primarily through fusion of translation-activating effector proteins with RNA-binding peptide repeats such as engineered PUF or PPR domains or with gRNA-dependent CRISPR-based proteins31,67,68. CIRTS has clinical advantages due to its compact size (dCasl3d-4GT3 would be 1,391 aa, and a 30-nt targeting PPR-4GT3 would be -1,444 aa, both over the AAV packaging limit if inserted into the vector design used in this study) and its human origin, potentially mitigating the immunogenicity concerns associated with microbial-derived CRISPR-based technologies, which likely interfere with long-term treatments needed for haploinsufficiency20,21. For effectors, eIF4GI and its domain truncations have emerged as a leading choice for translational-activation as they have been recently demonstrated to also be effective at boosting translation from endogenous mRNA with both dCasl3d- and PPR-based fusions targeting the 5’ UTR687,9. This current work contributes new evidence that eIF4GI truncations are also functional in activating translational when targeted to the 3’ UTR as well as the minimal domains required to drive this effect. The findings in this work highlight eIF4GI as a potent translational activator and motivates future efforts elucidating by what mechanism eIF4GI domain delivery to endogenous mRNA coordinates increased translation, especially in the 3’ UTR, as crosslinking between eIF4GI’s binding partner eIF3 and highly translated mRNA’s 3’ UTRs has been recently reported suggesting similar pathways may be natively active70,71.

[0178] Oligo-based translational activator technologies which combine an RNA translationactivating domain including SINE B (SINEUPs) or IRES domains (taRNAs) directly with a gRNA also offer a potential avenue to increase target translation43-72-74. However, SINEUPs are restricted to gRNA landing sites overlapping with the start codon, which may limit target flexibility depending on accessibility within this region or unavoidable sequence similarity with off-target transcripts. taRNAs, like CIRTS, offer greater gRNA design flexibility as they generally function by landing in either the 5’ and 3’ UTR, but have not demonstrated to function in vivo beyond LNP-mediated delivery to the liver and likely require furtherdevelopment to achieve sustained in vivo activity. Overall, boosting protein expression will likely not be “one size fits all” and a variety of therapeutic modalities with different attributes will be necessary to address haploinsufficiency broadly.

[0179] Beyond DS, CIRTS-4GT3 also has the potential to be applied to improve protein expression in other haploinsufficiencies. The pipeline demonstrated for gRNA design and screening by dual luciferase assay could allow for the rapid development of CIRTS- 4GT3 / gRNA pairs for a broad panel of targets. Interestingly, while the correlation between CIRTS-4GT3 function in the dual luciferase assay and on endogenous transcripts was strong, the inventors found that the results from dual luciferase screening did not always translate to endogenous targeting for CIRTS-NY1 and the other translational activator domains tested. Since many studies to identify translational effectors in the literature depend on similar, often less stringent, high-throughput luciferase assays these results highlight the need for orthogonal, follow-up assays to confirm effectors can modify translation on endogenous transcripts. This observation also highlights the potential - and challenges - of targeting RNA regulation for therapeutic purposes, as nuances in cell-specific contexts can be critical for ultimate efficacy.

[0180] In sum, this work validates a new protein-based translational activation modality with flexible, programmable targeting with proven efficacy in vivo on a high-value therapeutic target. This work confirms that targeting translational activation has therapeutic value, at least in a SCNla heterozygous knockout DS model, indicating the more work on translational activation technology development is warranted. The CIRTS platform is well positioned to offer a strategy for gene expression control in situations where gene replacement may be difficult to implement, or DNA-changes may be undesirable. Future work further improving the platform’s efficacy will likely yield potent preclinical candidates to address the great need for therapeutics to address haploinsufficiency as a general disease mechanism.D. METHODS1. Ethical Statement

[0181] This research complies with all relevant ethical regulations. Animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Chicago (Protocol Numbers 72613 and 72016).2. General Cloning

[0182] Gibson Assembly (GA) was used to clone all plasmids used for transient transfection in this study. Primers and gBlocks were ordered from IDT. PCR fragments used for GibsonAssembly were amplified with Q5 DNA Polymerase (NEB). After GA, plasmids were transformed into chemically competent DHIOP E. coli and after a 1 h outgrowth in 2xYT media, were plated on antibiotic agar plates to select single plasmid sequences. Plasmids were sequenced by the University of Chicago Comprehensive Cancer Center DNA Sequencing and Genotyping Facility.

[0183] All effector sequences (see Table 11) were amplified from gBlocks for cloning into CIRTS plasmids. CHD2 3’ UTR region was cloned from gBlock and SCNla 3’ UTR was amplified from reverse transcription reactions completed with total RNA isolated from Neuro 2a (ATCC) cells. All plasmids used in this study are listed in Table 9 with links to fully annotated vectors and are available upon request. Key plasmids will be made available through Addgene.3. Cloning Viral Vectors

[0184] Cloning for lentiviral and AAV transgene plasmids was conducted via restriction enzyme (all from NEB) cloning to replace parts in published vectors with the indicated promoters, or CIRTS protein or gRNA expression cassettes. For lentivirus vectors, lentiCRISPR v2 (addgene #52961) was digested with PacI and Nhel to clone gRNA cassettes and Xbal and BamHI for CIRTS protein open-reading frames following manufacturer’s protocols. Ligation was carried out with T4 DNA ligase (NEB) at 16 °C for 16 hours. Ligated plasmid was then transformed into chemically competent NEB stable E. coli for plating and antibiotic selection at 30 °C. Similarly, for CIRTS-AAV the pX601-SaCas9-gRNA vector (Addgene #61591) was modified to replace the CMV promoter with hSYN by Xbal and Agel digestion and SaCas9-gRNA was replaced by CIRTS-4GT3-gRNA by Agel and EcoRI digestion.4. Mammalian Cell Culture

[0185] HEK293T (ATCC) and Neuro-2a (ATCC) were maintained using DMEM (L- glutamine, high glucose, sodium pyruvate, phenol red; Corning) supplemented with 10% fetal bovine serum (FBS; Gibco), and 1% penicillin / streptomycin (Gibco). Cells were grown at 37 °C and 5% CO2. For transfections, 1% penicillin / streptomycin was omitted. For all experiments, cells had undergone fewer than 18 passages. All cell lines tested negative for mycoplasma contamination.5. Plasmid Transient Transfection

[0186] For experiments in HEK293T, cells were plated on a white-walled 96-well plate (Corning) 24 hours before transfection in a final volume of 80 L per well and grew to 70% confluency by time of transfection. 12 ng reporter plasmid and 250 ng gRNA-CIRTS plasmids were transfected for each condition. Transfections were completed with 0.5 pL lipofectamine 2000 (Invitrogen) per well and Opti-MEM I Reduced Serum Medium (ThermoFisher Scientific) for dilution of the plasmids and lipofectamine following manufacturer’s instructions. Approximately 24 hours after transfection 40 uL of media was removed from each well and replaced with 40 pL fresh DME : + 10% FBS.

[0187] For experiments in Neuro-2a, cells were plated on a 12-well plate (Coming) 24 hours before transfection in a final volume of 1 mL per well and grew to 70% confluency by time of transfection. 1250 ng gRNA-CIRTS plasmid were transfected for each condition using 1.75 pL Lipofectamine 3000 (Invitrogen) and 2 pL P3000 reagent per well with Opti-MEM I Reduced Serum Medium used for dilution according to manufacturer’s instructions. Media was fully exchanged in each well with 1 mL fresh DMEM + 10% FBS approximately 5 hours after transfection.6. gRNA Sequence Design

[0188] A guide RNA (gRNA) sequence represents the reverse-complementary RNA sequence for the desired binding site on the target mRNA. In order to generate gRNA sequences with a higher chance of success for specific target engagement the inventors recommend comparing the results for several available gRNA prediction programs listed below as well as filtering out suggested gRNA that 1) contain a run of 5 or more Uracil as this represents a transcription termination sequence for RNA polymerase III encoded on the DNA; 2) contain no significant internal secondary structure precluding sequence availability to pair with the target as predicted by programs such as RNAfold; 3) contain no significant off-target binding sites in the transcriptome as predicted by NCBI Blast analysis.

[0189] Three programs were used in combination to generate gRNA sequence candidates. Candidate gRNA sequences were suggested for a given target mRNA by both Cas 13 design (where the generated 23 nt gRNA were extended to 40 nt on the target by prioritizing the parameters above) and Soligo tool offered in the Sfold package where the oligo length was set to 40 nts. Finally, regions in the target mRNA with existing structures requiring low energy to open (higher probability that the target region will be unpaired) were predicted with RNAfold.The two gRNA sequence lists rank ordered by Casl3design and Soligo respectively were compared for overlap with priority given to similar sequences overlapping in each list that were predicted to bind in a low energy target region predicted by RNAfold. For sequences of all gRNA used in this study see Table 10.

[0190] RNAfold75: http: / / rna.tbi.univie.ac.at / cgi-bin / RNAWebSuite / RNAfold.cgi

[0191] Cas 13 design76: https: / / casl3design.nygenome.org /

[0192] Soligo77: https: / / sfold.wadsworth.org / cgi-bin / soligo.pl7. Dual Luciferase Reporter Assay

[0193] 48 hours after transfection, luciferase activity was assayed according to protocols previously developed and described by Baker et. al. with only minor alterations1. 3x firefly assay buffer and 3x nano luciferase assay buffers were prepared fresh before every experiment and protected from light until used. Nano luciferase salts were prepared fresh weekly and Triton lysis buffer was prepared fresh monthly.

[0194] Plates were removed from the incubator and left to cool to room temperature for 15 minutes. After plates had cooled, 40 pL 3x firefly assay buffer was added and mixed vigorously to aid cell lysing. Plates were then incubated for 10 minutes while mixing at 400 rpm on a benchtop orbital shaker. After 10 minutes, the firefly luciferase activity was read using a Biotek Synergy plate reader with an integration time of 0.6 seconds. All luciferase reads were made twice in quick succession and later averaged before taking the ratio between luciferase activities. After the firefly signal was read, 60 pL of 3x nano luciferase assay buffer was added per well and mixed. The plates were then left to mix again for 10 minutes while shaking at 400 rpm. After 10 minutes, the second luciferase signal was measured. Firefly readouts were then normalized to the corresponding nano luciferase readout by dividing the firefly RLU by nano luciferase RLU for each individual well. Fold-changes were then calculated by averaging the luciferase ratio for the negative control and dividing all biological replications by the negative control’s luciferase ratio.8. Western blotting

[0195] 48 hours after transfection for Neuro-2a experiments or 2 weeks after transduction for primary rat neuron experiments, media was removed from each well and the treated cells were washed with ice cold PBS and lysed in ice cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, pH 7.4) supplementedwith protease inhibitors and phosphatase inhibitors (Cell Signaling, 5872S). After 10 min incubation at room temperature with rocking, for experiments measuring Navi .1 levels whole lysates were collected and frozen at -80C, but for experiments measuring CHD2 levels lysates were first centrifuged and the supernatant collected to remove debris. In Navl.l experiments, lysates were later thawed on ice and the DNA pellet was concentrated by light centrifugation and removed. 1 pL Pierce™ Universal Nuclease for Cell Lysis (Thermo Scientific) was then added to lower lysate viscosity and incubated on ice for at least 10 minutes.

[0196] Total protein concentration was measured by BCA assay (Thermo Scientific). The total protein amount loaded was confirmed to be within the linear range of detection for each antibody to detect each target protein for at least a 2x increase in protein amount (Fig. 17a- 17b). For Navi.1 experiments 16 pg (Neuro-2a) or 25 pg (primary rat neuron) total protein was mixed with 4X protein loading buffer (50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 0.05% bromophenol blue, 100 mM DTT) and allowed to incubate at room temperature for 10 minutes. For CHD2 experiments 15 pg total protein was mixed with 4X protein loading buffer and heated at 90 °C for 5 minutes. Prepared lysates were loaded on 8% SDS-PAGE gels and run at 100V during stacking and then 150V until the dye front reached the bottom of the gel.

[0197] The proteins were transferred onto a methanol activated PVDF membrane (pore size 0.45 pm; Immobilon-P from Millipore) using a wet transfer system (Bio-rad). Membranes were blocked with 3% BSA in TBST (200 mM Tris, 1500 mM NaCl, 0.01% Tween 20, pH 7.5) buffer for 1 h at room temperature, incubated with primary antibody (see Table 13 for antibodies and dilutions used in this study) in 3% BSA- TBST at 4 °C overnight. Membranes were then washed with TBST buffer three times for 10 minutes each, followed by corresponding HRP-conjugated secondary antibody in 3% BSA-TBST incubation 1 h at room temperature. The loading control a-tubulin was visualized using 1 :5000 HRP-conjugated anti- a-tubulin antibody (Proteintech, HRP-66031). Membranes were imaged on a LLCOR Odyssey XF imager after incubation with SuperSignal West Pico PLUS chemiluminescent substrate (Thermo Scientific). Full western membrane images can be found in the Source data file.

[0198] When conducting the Navl.l western blot for the cortex and hippocampus tissue samples the inventors saw that Navi .1 presented as two bands, one at the correct 250 kDa size and one above it. Both bands seemed to be approximately half as intense in the SCNla+ / ' samples as the SCNla+ / +samples leading us to believe that both bands represented different Navl. l folding variants or complexes since our sample processing steps were only mildly denaturing as to avoid unwanted aggregation. To highlight this fact, most examples of Navl.lblots in the literature include mildly heating the samples before loading, and when this is performed only one band appears at the incorrect, larger size though the correct correlation between genotype and band intensity is maintained (Fig. 17c). Since our original western blot method faithfully reflects the correct protein size and sample genotype, the inventors decided to move forward quantifying both bands together for future in vivo Navl.l western blot analysis.9. Quantification of signal intensity on western blots

[0199] All image analysis was performed in EmpiriaStudio. The chemiluminescence channel image was processed such that the box area drawn over each band was equal and individually centered on the band(s) intensity being measured. The mean intensity is then divided by the corresponding loading control intensity. The given intensity ratio is then normalized to the ratio of the negative control (set at 1) for final quantification and reporting. Quantification was only performed within target and loading control blot pairs and then combined across separate blot pairs for biological replicates to make western blot quantitation figures.10. RT-qPCR

[0200] For experiments in Neuro-2a cells, plating and transfection were performed as previously described. Total RNA was harvested 48 h after transfection and isolated using the RNeasy Mini Kit (QIAGEN). After isolation, RNA was reverse transcribed to cDNA using the PrimeScript RT Reagent Kit (TaKaRa). All qPCR reactions were run at 20 pL volumes in a 384-well plate (Applied Biosystems) with at least 3 biological replicates and 2 technical replicates for each biological replicate using PowerUp SYBR Master Mix (Thermo Scientific) and amplified on a QuantStudio 6 (Thermo Scientific). The qPCR primers were either identified based on previous publications or verified for specificity using NCBI Primer BLAST. Expression levels were calculated using the housekeeping control gene (GAPDH) cycle threshold (Ct) value and the gene of interest Ct value after averaging both technical replicates. The relative expression level of one gene was determined by 2A(-ACt), where ACt = Ct (gene of interest) - Ct (GAPDH). Relative expression level for targeted gene was obtained upon dividing the targeted gene expression level of cells experiments treated with the on-target CIRTS gRNA by those treated by the non-targeting (NT) CIRTS gRNA or vehicle for in vivo experiments. All qPCR primers can be found in Table 12.11. Viral Production and Purification

[0201] For lentivirus production, HEK293T cells were plated on a 10 cm dish so that they would be approximately 70% confluent 24 hours later. The next day, media was replaced with 6 mL DMEM + 5% FBS and cells were transfected with four plasmids: VSV-G envelope plasmid (Addgene #12259), pRSV-Rev (Addgene #12253), pMDLg / pRRE (Addgene #12251), and lentiCRISPR v2 modified to replace CRISPR protein and gRNA cassettes with CIRTS equivalents described in a 2:2:2:4 pg ratio using 3 pg acidified polyethylenimine (PEI). 3 hrs after transfection, media was replaced with DMEM + 10% FBS. At 24 hrs after transfection, virus-containing media was collected and replaced. At 48 hrs after transfection, virus-containing media was again collected and combined with the media taken previously. The virus-containing media was then centrifuged at 500 g for 5 min and the supernatant was filtered through a 0.45 pm PES filter to remove cellular debris. The virus was then concentrated with PEG-it Virus Precipitation Solution (System Biosciences, LV810A-1) following manufacturer’s instructions and viral titer was estimated via qPCR via Lentivirus Titer Kit (Applied Biological Materials, LV900). Virus was then used immediately or frozen at -80C in single use aliquots.

[0202] For AAV production, CIRTS-4GT3 / SCNla-gl was packaged into AAV9 by PackGene Biotech at a concentration of 1E13 GC / mL. Viral aliquots were kept at -80 °C and used immediately upon thawing.12. Primary neuronal culture and Lentiviral Transduction

[0203] Primary cultures of rat cortical neurons were prepared as described using Neurobasal Media (NBM), 4% (v / v) B27, and 0.125 mM 1-glutamine (Thermo Fisher Scientific, Waltham, MA). Dissociated cortical neurons from El 8 Sprague Dawley rat pups were plated in six-well plates coated with poly-D-lysine (Sigma, St Louis, MO) with 2 mL prepared media. For western blots, 0.4 x io6cells were plated in each well. At day 4 of culture, lentivirus at an MOI of 10 was diluted in 1 mL neuron media for each well to be transduced. 1 mL media was removed from the treated well and replaced with the media with virus. After transduction, 1 mL media was fed to the neurons approximately every 5 days. Transduction was confirmed for lentivirus encoding CIRTS fused with GFP by imaging by an inverted epifluorescence microscope (Leica DMi8) with a 10x objective, a Hamamatsu Orca-Flash 4.0 camera, and a 300 W Xenon light source (Sutter Lambda XL). Wells treated with lentivirus encoding CIRTS-4GT3 or CIRTS-NY1 with on-target or NT gRNA were processed as described in Western Blotting.13. Animals

[0204] Heterozygous SCNla+ / ' mice used as breeders to establish the colony used in this study, were 129S6 / SvEvTac-ScnlatmlKea / Mmjax (RRID:MMRRC_037107-JAX) obtained from the Mutant Mouse Resource and Research Center (MMRRC) at The Jackson Laboratory, an NIH-funded strain repository, and was donated to the MMRRC by Jennifer Kearney, Ph.D., Northwestern University51. The colony was propagated by crossing these SCNla+ / ' breeders with wildtype 129S6 / SvEvTac mice (Taconic). For experiments, SCNla+ / ' mice on the 129S6 / SvEvTac background were crossed with wild type C57BL / 6N (Charles River) mice to generate Fl C57 / 126S9 offspring. Genotyping was performed via PCR as described by the MMRRC and Jackson Laboratory (see Table 12 for primer sequences).

[0205] All mice were maintained on a 12: 12-hr light: dark cycle and had unrestricted access to food and water. Mice were euthanized using CO2 followed by cervical dislocation. For western blot analysis, cortical and hippocampal tissues were collected, combined and homogenized via a KIMBLE dounce tissue grinder (Sigma-Aldrich) in RIPA buffer + protease and phosphatase inhibitors prepared as described in Western Blotting. Samples were then sonicated for 5 sec at 25% amplitude and centrifuged at 14,000 g for 10 min at 4 °C. The supernatant was collected and used immediately for western blot as described previously or frozen at 80 °C. For RT-qPCR analysis, cortical and hippocampal tissues from one hemisphere were immediately placed in 500 pL RNAlater (Thermo Fisher Scientific) until RNA extraction by RNeasy Mini Kit (QIAGEN) following manufacturer’s instructions for RNA extraction from tissues. Downstream RT-qPCR was performed as previously described.14. i.c.v. injections

[0206] At postnatal day 1 (Pl), neonates were anesthetized on ice for ~3 min. 4 pL of viral suspension or vehicle control with added 0.05% Fast Green FCF dye (Sigma-Aldrich) to visualize successful intraventricular injection was injected into the right ventricle using a Model 701 RN 10 pL Syringe and 33 gauge, Small Hub RN Needle (Hamilton, 7635-01 and 7803-15) mounted on a micromanipulator (WPL M3301R). The pup’s head was positioned exposing its right side, and the injection site was located by estimating roughly 1 / 2 the distance along an imaginary line between Lambda and the center of the eye. The needle was lowered to a depth of 2mm. Successful injection was confirmed by visualizing the characteristic wingedshape of the dye representing the ventricles on either hemisphere. After injection, mice were placed on a warming pad and returned to the mother in the cage once recovered.15. Hyperthermia-induced Seizure (HTS) Assay

[0207] Mice were fitted with a rectal probe (RET-3; Physitemp) secured by medical tape and allowed to acclimate to the test chamber for 10 min. The probe was connected to a TCAT 2DF animal temperature controller (Physitemp) to continuously monitor the mouse’s internal body temperature. After 10 min, a heat lamp above the test chamber was turned on and the mouse’s body temperature was continuously monitored to increase by 0.5 °C every 2 min until onset of a tonic-clonic seizure, identified by erratic, uncontrolled movements and / or loss of posture, or a max internal body temperature of 42.5 °C was maintained for 3 min (as has been described previously78,79). Either temperature seizure first occurred, or the experiment was seizure free was recorded and mice were immediately placed in a recovery chamber with unrestricted food and water for at least 10 min.16. Statistics and reproducibility

[0208] All data are presented as mean ± SEM with individual data points. Statistical significance was assessed by unpaired two-tailed Student’s t test, one-way analysis of variance (ANOVA), two-way analysis of variance (ANOVA), or log-rank test with appropriate post hoc tests as described in the figure legends. P < 0.05 is considered statistically significant. Exact P values are provided in the source data file. All experiments were performed three or more times independently under identical or similar conditions, except when indicated in the figure legends.E. TablesTable 9: Representative plasmids used in this studyTable 10: CIRTS gRNA sequencesTable 11: CIRTS effectors amino acid sequencesTable 12: qPCR primer sequencesTable 13: Antibodies and dilutions used for western blotsREFERENCES1. Rice, A. M. & McLysaght, A. Dosage-sensitive genes in evolution and disease. BMC Biol 15, 78 (2017). https: / / doi.org: 10.1186 / sl2915-017-0418-y2. Lek, M. et al. 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[0209] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims. The references cited herein, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

Claims

CLAIMS1. A polypeptide comprising an effector domain wherein the effector domain comprises the amino acid sequence of one of SEQ ID NOs:l, 2, 21, or 55-58 or comprises an amino acid sequence with at least 80% sequence identity to one of SEQ ID NOs: 1, 2, 21, or 55-58.

2. The polypeptide of claim 1, wherein the polypeptide comprises one or more of a stabilizer domain, linker, RNA hairpin binding domain, nuclear export signal, and molecular tag.

3. The polypeptide of any one of claims 1-2, wherein the hairpin binding domain is amino- proximal to the effector domain.

4. The polypeptide of any one of claims 1-3, wherein the stabilizer domain is amino- proximal to the RNA hairpin binding domain and / or effector domain.

5. The polypeptide of any one of claims 1-4, wherein the nuclear export signal is amino- proximal to the effector domain.

6. The polypeptide of any one of claims 1-5, wherein the molecular tag is carboxy - proximal to the effector domain.

7. The polypeptide of any one of claims 1-6, wherein the polypeptide comprises, from N- terminus to the C-terminus, a stabilizer domain, a glycine serine linker, a RNA hairpin binding region, a nuclear export signal, an effector domain, and a molecular tag.

8. The polypeptide of any one of claims 1-7, wherein the polypeptide comprises one or more RNA hairpin binding domain(s).

9. The polypeptide of claim 8, wherein the hairpin binding domain(s) comprises the amino acid sequence of SEQ ID NOS:38 or an amino acid sequence with at least 80% sequence identity to SEQ ID NOS:38.

10. The polypeptide of any one of claims 1-9, wherein the polypeptide comprises a stabilizer domain.

11. The polypeptide of claim 10, wherein the stabilizer domain comprises the amino acid sequence of at least one of SEQ ID NOS:31-33 or or an amino acid sequence having at least 80% sequence identity to SEQ ID NOS:31-33.

12. The polypeptide of any one of claims 1-11, wherein the polypeptide comprises a nuclear export signal.

13. The polypeptide of claim 12, wherein the nuclear export signal comprises the amino acid sequence of SEQ ID NO:40 or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:40.

14. The polypeptide of any one of claims 1-13, wherein the polypeptide comprises a molecular tag.

15. The polypeptide of claim 14, wherein the molecular tag comprises the amino acid sequence of SEQ ID NO:41, or an amino acid sequence that has at least 80% sequence identity to SEQ ID NO:41.

16. The polypeptide of one of claims 1-15, wherein the polypeptide comprises at least one substitution relative to SEQ ID NOs:l, 2, 21, or 55-58 .

17. A nucleic acid encoding the polypeptide of any one of claims 1-16.

18. A nucleic acid comprising the amino acid sequence of one of SEQ ID NOS:3-19, 21- 30, 34-36, 39, 42-54, or 59-63, or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63.

19. The nucleic acid of claim 18, wherein the nucleic acid further comprises at least one hairpin structure.

20. The nucleic acid of claim 18 or 19, wherein the nucleic acid comprises a TAR hairpin scaffold or a human SLBP hairpin scaffold.

21. The nucleic acid of claim 19 or 20, wherein the hairpin structure comprises the nucleic acid sequence of at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35 or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35.

22. One or more expression vector(s) comprising the nucleic acid of any one of claims 17- 21.

23. A host cell comprising the nucleic acid of any one of claims 17-21 or expression vector of claim 22.

24. A delivery vehicle comprising at least one polypeptide of any one of claims 1-16, and / or at least one nucleic acid of any one of claims 17-21, and / or expression vector of claim 22.

25. The delivery vehicle of claim 24, wherein the delivery vehicle comprises liposome(s), particle(s), exosome(s), microvesicle(s), a gene-gun or one or more nucleic acid vector(s).

26. The delivery vehicle of claim 24 or 25, wherein the delivery vehicle further comprises a RNA targeting molecule comprising a RNA targeting region and at least one hairpin structure, wherein the hairpin structure of the RNA targeting molecule specifically binds to the RNA hairpin binding domain of the polypeptide.

27. The delivery vehicle of claim 26, wherein the RNA targeting region hybridizes to the 5’UTR of a RNA.

28. The delivery vehicle of claim 27, wherein the RNA targeting region hybridizes to the 3’UTR of a RNA.

29. The delivery vehicle of claim 27 or 28, wherein the RNA targeting region hybridizes to the 5’UTR or 3’UTR of a mammalian mRNA.

30. The delivery vehicle of any one of claims 24-29, wherein the RNA targeting region comprises the nucleic acid sequence of one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63 or a nucleic acid sequence having at least 80% sequence identity to one of SEQ ID NOS:3-19, 21-30, 34-36, 39, 42-54, or 59-63.

31. The delivery vehicle of any one of claims 24-30, wherein the RNA targeting molecule comprises a TAR hairpin scaffold or a human SLBP hairpin scaffold.

32. The delivery vehicle of any one of claims 24-31, wherein the RNA targeting molecule comprises the nucleic acid sequence of at least one of SEQ ID NOS: 15-19, 22, 25-30, 34, or 35 or a nucleic acid sequence that has at least 80% sequence identity to one of SEQ ID NOS : 15- 19, 22, 25-30, 34, or 35.

33. The delivery vehicle of claim 32, wherein the RNA targeting molecule comprises SEQ ID NO:34 or 35.

34. A composition comprising the polypeptide of any one of claims 1-16, nucleic acid of any one of claims 17-21, expression vector of claim 22, host cell of claim 23, or delivery vehicle of any one of claims 24-33.

35. A method of making a polypeptide comprising transferring the nucleic acid of any one of claims 17-21 or expression vector of claim 22 into a cell and isolating the expressed polypeptide.

36. A method of modulating at least one target RNA comprising contacting the target RNA with the polypeptide of any one of claims 1-16, the delivery vehicle of any one of claims 24- 33, or the composition of any one of claim 34.

37. A method for modulating at least one target RNA in a subject and / or for treating a haploinsufficiency disorder in a subject, the method comprising administering the polypeptide of any one of claims 1-16, the delivery vehicle of any one of claims 24-33, or the composition of claim 34 to the subject.

38. The method of claim 36 or 37, wherein modulating the at least one target RNA comprises cleaving, demethylating, methylating, activating translation, repressing translation, promoting degradation, and / or binding to the RNA.

39. The method of any one of claims 36-38, wherein the target RNA is in a prokaryotic or eukaryotic cell.

40. The method of claim 39, wherein the target RNA is in a human cell.

41. The method of claim 39 or 40, wherein the target RNA is in vitro or in vivo.

42. The method of any one of claims 36-41, wherein the haploinsufficiency disorder comprises epileptic encephalopathy.

43. The method of any one of claims 36-42, wherein the haploinsufficiency disorder comprises Dravet Syndrome, cancer, lq21.1 deletion syndrome, 5q- syndrome in myelodysplastic syndrome (MDS), 22ql l.2 deletion syndrome, CHARGE syndrome, cleidocranial dysostosis, Ehlers-Danlos syndrome, Frontotemporal dementia caused by mutations in progranulin, GLUT1 deficiency (DeVivo syndrome), haploinsufficiency of A20, haploinsufficiency of PRR12, holoprosencephaly caused by haploinsufficiency in the SonicHedgehog gene, Holt-Oram syndrome, Marfan syndrome, Phelan-McDermid syndrome, Polydactyly, FOXP1 Syndrome, ARID1B haploinsufficiency, ARID IB-mediated disorders, or NR4A2 -related syndrome.

44. The method of claim 43, wherein the haploinsufficiency disorder comprises Dravet Syndrome.

45. The method of any one of claims 37-44, wherein the target RNA comprises a mRNA from the CHD2, SCNla, ARID1B, and / or Navi.1 gene.

46. The method of any one of claims 36-45, wherein the subject is a human subject.

47. A cell or progeny thereof comprising modulated target RNA, wherein the target RNA has been modulated according to any one of claims 36-41.

48. A multicellular organism comprising one or more cells according to claim 47.

49. A plant or animal comprising one or more cells according to claim 47.

50. A kit comprising the polypeptide of any one of claims 1-16, the delivery vehicle of any one of claims 24-33, or the composition of claim 34.

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