Ribozyme-activated rna constructs and uses thereof

Abstract

The present disclosure provides ribozyme-mediated fusion constructs and systems, and methods thereof, for use in a variety of applications, including inducible gene expression systems, gene therapy, and combinatorial screening.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119 to Provisional Application No. 63 / 136,201, filed January 11, 2021, the disclosure of which is incorporated herein by reference.

[0002] This invention was made with government support under grant numbers R01GM123313, R01CA222826, and R01HG009285 awarded by the National Institutes of Health. The Government has certain rights in this invention. [Technical field]

[0003] The present disclosure provides ribozyme-mediated fusion constructs and systems, and methods thereof, for use in a variety of applications, including inducible gene expression systems, gene therapy, and combinatorial screening.

[0004] Ribozymes (ribonucleic acid enzymes) are RNA molecules that have the ability to catalyze certain biochemical reactions, such as cRNA splicing in gene expression, similar to the action of protein enzymes. Ribozymes are involved in various RNA processing reactions, such as RNA splicing, viral replication, and transfer RNA biogenesis. Examples of ribozymes include hammerhead ribozymes, VS ribozymes, leadzymes, and hairpin ribozymes. Ribozymes have been proposed and developed for the treatment of diseases by gene therapy. Overview

[0005] This disclosure provides innovative engineering of endogenous RNA processing machinery to create linked RNA fusion constructs that can be utilized for combinatorial genetic interaction screens and RNA-based readout for inducible gene expression. As shown in the examples herein, separately transcribed sequences that are complementary to each other are fused to a self-cleaving ribozyme. Once transcribed, the transcripts are cleaved by the autocatalytic activity of the ribozyme to create unique ends. Due to the complementary regions, the two transcripts hybridize and the cut ends are juxtaposed, which are recognized by endogenous RNA ligase to create a linked fusion construct. Further demonstrated herein is the applicability of this approach to link two transcripts delivered to cells on different library elements. When linked to an intron sequence, the ribozyme-mediated RNA fusion constructs can be utilized for controllable expression of full-length gene products. RNA fusions (including circularization) have further utility in transcriptome and genome engineering modalities such as RNAi, ASOs, ADAR-recruiting guide RNAs, and guide RNAs in CRISPR-Cas.

[0006] The present disclosure provides ribozyme-activated RNA constructs comprising one or more ribozymes and one or more RNA coding sequences for at least one polypeptide of interest, wherein transcription of the one or more RNA coding sequences for the at least one polypeptide of interest is activated by or dependent on the activity of the one or more ribozymes. In one embodiment, the ribozyme-activated RNA construct further comprises a first artificial RNA element comprising an optional primer region, an optional barcode region, an RNA coding sequence for a polypeptide of interest, and a complementary sequence to a sequence of a second artificial RNA element, and a first self-cleaving ribozyme, and a second artificial RNA element comprising an optional primer region, an optional barcode region, an RNA coding sequence for a polypeptide of interest, and a complementary sequence to a sequence of the first artificial RNA element, and a second self-cleaving ribozyme, wherein cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes provides a hybridization construct comprising a region of dsRNA from the commentary sequence hybridized together, respectively, wherein the hybridization construct can be further ligated by an RNA ligase to form an RNA fusion construct, and wherein expression from the RNA fusion construct produces the at least one polypeptide of interest. In a further embodiment, the first artificial RNA element comprises a barcode sequence or a specific molecular identity (UMI) sequence, and / or the second artificial RNA element comprises a barcode sequence or a UMI sequence. In yet a further embodiment, the barcode sequence or UMI sequence of the first artificial RNA element has a sequence that is different from the barcode sequence or UMI sequence from the second artificial RNA element. In another embodiment, the first artificial RNA element comprises a primer sequence, and / or the second artificial RNA element comprises a primer sequence. In a further embodiment, the primer sequence of the first artificial RNA element is different from the primer sequence from the second artificial RNA element. In another embodiment, the first and second complementary sequences are 30-60 bp in length. In a further embodiment, the first and second complementary sequences are 40-50 bp in length.In another embodiment, the first and second ribozymes are twister ribozymes. In a further embodiment, the first ribozyme is a P3 twister ribozyme. In yet a further embodiment, the second ribozyme is a P1 twister ribozyme. In another embodiment, the RNA ligase is RtcB. In another or further embodiment of any of the above embodiments, the vector or plasmid comprises the first artificial element; wherein the first artificial element is located downstream of a first RNA promoter and a first perturbation element, and / or the vector or plasmid comprises a second artificial element; wherein the second artificial element is located downstream of a second RNA promoter and a second perturbation element. In a further embodiment, the first RNA promoter and / or the second RNA promoter is a polymerase III promoter. In yet a further embodiment, the polymerase III promoter is a hU6 promoter. In another embodiment, the first perturbation element and / or the second perturbation element is a sgRNA utilized in a CRISPR knockout screen.In another embodiment, the ribozyme-activated RNA construct comprises a first artificial RNA element, an RNA coding sequence for a polypeptide of interest, an intron sequence, a sequence of a second artificial RNA element and a complementary sequence to the 3' aptamer, and a first self-cleaving ribozyme, the 3' aptamer interacting with the first self-cleaving ribozyme to stabilize the first self-cleaving ribozyme, the second artificial RNA template is tethered to a second self-cleaving ribozyme, and the 3' aptamer is and a second artificial RNA element that interacts with the second self-cleaving ribozyme to stabilize the second self-cleaving ribozyme, wherein cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes provides a hybridization construct comprising a region of dsRNA from a commentary sequence hybridized together, respectively, wherein the hybridization construct can be further ligated by an RNA ligase, and the intron sequence is removed by the spliceosome to form an RNA fusion construct, wherein expression from the RNA fusion construct produces the at least one polypeptide of interest. In a further embodiment, the intron sequence is derived from dihydrofolate reductase. In another embodiment, the RNA coding sequence for a polypeptide of interest is adjacent to each of the intron sequences. In another embodiment, the RNA coding sequence for a polypeptide of interest encodes a polypeptide / protein selected from insulin, coagulation factor IX, cystic fibrosis transmembrane conductance regulator protein, and dystrophin protein.In another embodiment, the ribozyme-activated RNA construct comprises one or more promoter sequences, one or more RNA coding sequences for at least one polypeptide of interest, one or more ribozymes that are aptazyme-based riboswitches, a 3'UTR sequence that includes the aptazyme-based riboswitch, and a poly(A) sequence, wherein the aptazyme-based riboswitch destabilizes the ribozyme-activated RNA construct when not bound to a target ligand, resulting in a decrease in expression of the at least one polypeptide of interest, and wherein the aptazyme-based riboswitch stabilizes the ribozyme-activated RNA construct when bound to a target ligand, resulting in an increase in expression of the at least one polypeptide of interest. In another embodiment, the aptazyme-based riboswitch is a hammerhead aptazyme. In yet another embodiment, the target ligand is selected from tetracycline, theophylline, and guanine. In another embodiment, the at least one polypeptide of interest is selected from the group consisting of a prodrug activating enzyme, a biological response modifier, a receptor ligand, an immunoglobulin-derived binding polypeptide, a non-immunoglobulin binding polypeptide, an antigenic polypeptide, a genome editing enzyme, and any combination thereof, wherein the multiple polypeptides are separated by a 2A or 2A-like peptide. In a further embodiment, the biological response modifier is an immune enhancing cytokine. In yet a further embodiment, the immune enhancing cytokine is selected from the group consisting of interleukin 1-38, interferon, tumor necrosis factor (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF). In another embodiment, the 2A or 2A-like peptide further comprises a GSG linker moiety. In yet another embodiment, the genome editing enzyme is selected from the group consisting of a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an artificial meganuclease, and an RNA-guided DNA endonuclease (Cas) polypeptide. In yet another embodiment, the one or more promoter sequences are polymerase II (pol-II) promoter sequences.In another embodiment, the one or more promoter sequences comprise sequences for EF1α, hU6, SV40, CMV, RSV, NEUROD2, and / or TBX20. In another embodiment, the poly(A) sequence is a bGH poly(A) sequence.

[0007] The present disclosure also provides a pharmaceutical composition comprising the above ribozyme-activated RNA construct, wherein the ribozyme-activated RNA construct is linearized and comprises a 5' ribozyme, a 5' linking sequence, an internal ribosome entry site (IRES) sequence, an RNA coding sequence for at least one polypeptide of interest, a 3' linking sequence, a 3' ribozyme sequence, and a pharma- ceutically acceptable carrier. In a further embodiment, the linear ribozyme-activated RNA construct lacks a polymerase binding region. In yet another embodiment, the 5' and 3' ribozymes are selected from the group consisting of twister ribozyme, hammerhead ribozyme, hatchet ribozyme, hepatitis delta virus ribozyme, ligase ribozyme, pistol ribozyme, twister sister ribozyme, Vg1 ribozyme, VS ribozyme, and derivatives of any of the above. In another embodiment, the 5' and 3' linking sequences are substrates for a native ligase in situ. In a further embodiment, the native ligase is RtcB. In another embodiment, the IRES comprises any one of the sequences of SEQ ID NOs: 1-1328 and sequences where T is U. In another embodiment, the at least one polypeptide of interest comprises two or more polypeptides of interest separated by a self-cleaving peptide. In yet a further embodiment, the self-cleaving peptide comprises a 2A or 2A-like peptide. In another embodiment, the at least one polypeptide of interest is selected from the group consisting of a prodrug activating enzyme, a biological response modifier, a receptor ligand, an immunoglobulin-derived binding polypeptide, a non-immunoglobulin binding polypeptide, an antigenic polypeptide, a genome editing enzyme, and any combination thereof, wherein the plurality of polypeptides are separated by a 2A or 2A-like peptide. In yet another embodiment, the biological response modifier is an immune enhancing cytokine. In a further embodiment, the immune enhancing cytokine is selected from the group consisting of interleukins 1-38, interferons, tumor necrosis factors (TNF), and granulocyte macrophage colony stimulating factor (GM-CSF).In another embodiment, the 2A or 2A-like peptide further comprises a GSG linker moiety.In yet another embodiment, the genome editing enzyme is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases (TALENs), artificial meganucleases, and RNA-guided DNA endonuclease (Cas) polypeptides. In yet another embodiment, the 5' and 3' ribozyme sequences are independently selected from 5'-GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT-3' (SEQ ID NO: 1354) or 5'-AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC-3' (SEQ ID NO: 1355), and a sequence at least 85-100% identical to any of the above sequences where T is U. In another embodiment, the 5' and 3' linked sequences are independently selected from 5'-AACCATGCCGACTGATGGCAG-3' (SEQ ID NO: 1356) or 5'-CTGCCATCAGTCGGCGTGGACTGTAG-3' (SEQ ID NO: 1357), and a sequence that is at least 85-100% identical to any of the foregoing sequences when T is U.In another embodiment, the IRES sequence is at least 85-100% identical to the sequence 5'--3' (SEQ ID NO: 1358) and T is U.

[0008] The present disclosure also provides a vaccine composition comprising a ribozyme-activated RNA construct of the present disclosure, wherein the ribozyme-activated RNA construct is linearized and comprises a 5' ribozyme, a 5' linking sequence, an internal ribosome entry site (IRES) sequence, an RNA coding sequence for at least one antigenic polypeptide, a 3' linking sequence, a 3' ribozyme sequence, and a pharma- ceutically acceptable carrier. In a further embodiment, the linearized ribozyme-activated RNA construct lacks a polymerase binding region. In another embodiment, the 5' and 3' ribozymes are selected from the group consisting of twister ribozyme, hammerhead ribozyme, hatchet ribozyme, hepatitis delta virus ribozyme, ligase ribozyme, pistol ribozyme, twister sister ribozyme, Vg1 ribozyme, VS ribozyme, and derivatives of any of the foregoing. In another embodiment, the 5' and 3' linking sequences are substrates for natural ligases in situ. In a further embodiment, the native ligase is RtcB. In another embodiment, the IRES comprises any one of the sequences of SEQ ID NOs: 1-1328 and the sequences thereof where T is U. In another embodiment, the at least one antigenic polypeptide comprises two or more antigenic polypeptides separated by a self-cleaving peptide. In a further embodiment, the self-cleaving peptide comprises a 2A or 2A-like peptide. In a further embodiment, the 2A or 2A-like peptide further comprises a GSG linker moiety. In yet another embodiment, the 5' and 3' ribozyme sequences are independently selected from 5'-GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT-3' (SEQ ID NO: 1354) or 5'-AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC-3' (SEQ ID NO: 1355), and a sequence at least 85-100% identical to any of the above sequences where T is U.In another embodiment, the 5' and 3' linked sequences are independently selected from 5'-AACCATGCCGACTGATGGCAG-3' (SEQ ID NO: 1356) or 5'-CTGCCATCAGTCGGCGTGGACTGTAG-3' (SEQ ID NO: 1357), and a sequence at least 85-100% identical to either of the preceding sequences when T is U. In another embodiment, the IRES sequence is at least 85-100% identical to 5'--3' (SEQ ID NO: 1358), and the sequence when T is U. In another embodiment, the antigenic polypeptide comprises a SARS-CoV-2 spike protein. In another embodiment, the at least one polypeptide of interest or antigenic polypeptide is comprised within a self-amplifying RNA construct.In a further embodiment, the self-amplifying RNA construct comprises an alphavirus or a paramyxovirus.

[0009] The present disclosure also provides a plasmid or capsid comprising the ribozyme-activated RNA construct of the present disclosure. In one embodiment, the plasmid or capsid is an AAV-based plasmid or capsid. In another embodiment, the plasmid expresses Cas9 protein and gRNA.

[0010] The invention also provides combinatorial screens comprising the ribozyme-activated RNA constructs of any of the above embodiments.

[0011] In certain embodiments, the present disclosure provides a ribozyme-mediated RNA fusion construct or system comprising a first artificial RNA element (wherein the first artificial RNA template is tethered to a first self-cleaving ribozyme) comprising a primer region, a barcode region, and a complementary sequence to the sequence of a second artificial RNA element, and a second artificial RNA element (wherein the second artificial RNA template is tethered to a second self-cleaving ribozyme) comprising a primer region, a barcode region, and a complementary sequence to the sequence of the first artificial RNA element, wherein cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes, respectively, provides a hybridization construct comprising a region of dsRNA from a commentary sequence hybridized together, and wherein the hybridization construct can be further ligated by an RNA ligase to form an RNA fusion construct. In a further embodiment, the first artificial RNA element comprises a barcode region having a different sequence from the barcode region from the second artificial RNA element. In yet a further embodiment, the first artificial RNA element comprises a primer region having a different sequence than the primer region from the second artificial RNA element. In another embodiment, the first and second complementary sequences are 30-60 bp in length. In yet another embodiment, the first and second complementary sequences are 40-50 bp in length. In a further embodiment, the first and second ribozymes are twister ribozymes. In yet a further embodiment, the first ribozyme is a P3 twister ribozyme. In another embodiment, the second ribozyme is a P1 twister ribozyme. In a further embodiment, the RNA ligase is RtcB.

[0012] In another embodiment, the present disclosure also provides a vector or plasmid comprising a first artificial element as described herein, wherein the first artificial element is located downstream of a first RNA promoter and a first perturbation element. In yet another embodiment, the present disclosure also provides a vector or plasmid comprising a second artificial element as described herein, wherein the second artificial element is located downstream of a second RNA promoter and a second perturbation element. In a further embodiment, the first RNA promoter and / or the second RNA promoter is a polymerase III promoter. In yet a further embodiment, the polymerase III promoter is an hU6 promoter. In a particular embodiment, the first perturbation element and / or the second perturbation element is an sgRNA utilized in CRISPR knockout screening.

[0013] In certain embodiments, the present disclosure further provides combinatorial screens comprising the vectors or plasmids described herein.

[0014] In a particular embodiment, the disclosure provides a method for the synthesis of a first artificial RNA element comprising an intron sequence, a complementary sequence to a sequence of a second artificial RNA element, and a 3' aptamer, where the first artificial RNA template is tethered to a first self-cleaving ribozyme, and where the 3' aptamer interacts with and stabilizes the first self-cleaving ribozyme, and a second artificial RNA element comprising an intron sequence, a complementary sequence to a sequence of the first artificial RNA element, and a 3' aptamer, where the second artificial RNA template is tethered to a second self-cleaving ribozyme, and where the 3' aptamer interacts with and stabilizes the first self-cleaving ribozyme. wherein the 3' aptamer interacts with and stabilizes a second self-cleaving ribozyme), wherein cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes, respectively, provides a hybridization construct comprising a region of dsRNA from a commentary sequence hybridized together, and wherein the hybridization construct can be further ligated by an RNA ligase to form an RNA fusion construct. In a further embodiment, the intron sequence is derived from dihydrofolate reductase. In yet another embodiment, a portion of a therapeutic gene or protein is fused to each of the intron sequences. In another embodiment, the therapeutic gene or protein is selected from the human insulin gene, coagulation factor IX, cystic fibrosis transmembrane conductance regulator protein, and dystrophin protein. [Brief description of the drawings]

[0015] Figure 1A-C provide results from preliminary studies. (A) Schematic showing fusion of heterologous barcodes at the RNA level. (B) Gel electrophoresis images following RT-PCR of plasmid transfection with fragL and fragR alone or in combination. (C) Sanger sequencing traces of purified PCR products shown in panel B compared to the expected fusion (SEQ ID NO: 1347).

[0016] Figures 2A-E provide: (A) Schematic showing plasmid design for combinatorial screens in which ribozymes, complementary linkers, barcodes and primers for ribozyme-mediated RNA fusion constructs are cloned downstream of perturbations such as sgRNAs utilized in CRISPR knockout screens. (B) Gel electrophoresis of RT-PCR following plasmid transfection of HEK293T cells with sgRNA_fragL and sgRNA_fragR constructs, alone or in combination. (C) Gel electrophoresis of RT-PCR following plasmid transfection of HEK293T cells with antisense_fragL and antisense_fragR constructs (SEQ ID NO: 1348), alone or in combination. (D-E) Sanger sequencing traces of purified PCXR products from antisense-directed RNA fusions (SEQ ID NO: 1349).

[0017] Figure 3 provides a schematic illustrating the ribozyme-mediated RNA fusion approach for inducible gene expression. The therapeutic payload is split between two constructs. The fragL construct contains the N-terminus of the protein fused to an intron sequence, a complementary region, a self-cleaving ribozyme, a complementary region, an intron sequence, and the C-terminus of the therapeutic protein. Addition of an aptamer-binding ligand disrupts the tertiary interaction between the aptamer and the ribozyme, allowing autocatalytic cleavage of the transcript. The complementary regions hybridize to each other, and the generated ends are ligated by the endogenous RNA ligase RtcB. When the intron sequences are juxtaposed to each other, the cell's splicing machinery can recognize the splice sites, creating a full-length functional protein.

[0018] Figure 4 shows a schematic of RNA fusion from the PoIII promoter. Two constructs are cloned into the px600 AAV backbone, which contains two halves of green fluorescent protein (GFP). Each GFP is linked to a DHFR intron and contains an RNA fusion linker (45 bp complementary region) and either a P3 (GFP-L) or P1 (GFP-R) twister ribozyme. Upon transcription, the ribozyme undergoes self-cleavage to generate a 5' hydroxyl end and a 2',3'-cyclic phosphate end. The complementary linker regions then hybridize and are ligated by RtcB, bringing the intron sequences into close proximity to each other. The endogenous spliceosome machinery then primes the intron to generate a fluorescent GFP molecule.

[0019] Figure 5A-C show: (A) Fluorescence images taken 48 hours after transfection of HEK293FT cells with a plasmid containing one half of GFP (GFP-left or GFP-right) and the RNA fusion machinery bound to an intron sequence recognized by the spliceosome. (B) RNA was isolated from HEK293FT cells shown in panel A and RT-PCR was performed for both halves of the GFP transcript using primers. (C) The PCR products were purified and Sanger sequenced (SEQ ID NO: 1350) to confirm proper ligation of the full-length protein.

[0020] Figure 6A-E show: (A) Fluorescence images taken 48 hours after transfection of HEK293FT cells with plasmids containing one half of GFP (GFP-left or GFP-right) and the RNA fusion machinery linked to different intron sequences. (B) Flow cytometry was performed on transfected cells after 48 hours to quantify the percentage of GFP+ cells. (C) RT-qPCR relative expression of GFP using the DHFR intron after 48 hours. (D) RT-qPCR relative expression of GFP using the pCI construct after 48 hours. (E) Activation percentages were calculated from qPCR comparing relative GFP expression levels in the presence of complementary sequences.

[0021] Figure 7 shows the results of cells isolated 48 hours after transfection and analyzed by flow cytometry. The percentage of GFP+ cells among the total cells present is shown.

[0022] Figures 8A-B show: (A) Schematic of the plasmid design of ribozyme-mediated RNA barcode fusion constructs driven by polymerase II and polymerase III-like H1 promoters; (B) Relative expression measured by quantitative RT-PCR (qRT-PCR) (normalized to GAPDH) of RNA fusion constructs in cells transfected with a negative control (lentiCRISPRv2 plasmid backbone) or both fragL and fragR constructs driven by the U6 or H1 promoters.

[0023] 9A-B provide: (A) Schematic diagram showing the design of ribozyme-mediated circularized RNA barcodes. The 3' end of the fragL construct is modified with a "designer exon" and an intron, while the 5' end of the fragR construct is mediated by an intron followed by a "designer exon." Upon transcription, the ribozyme self-cleaves and hybridizes with the complementary sequence, juxtaposing the designer exon and intron to each other. This is recognized by the spliceosome, which undergoes a back-spiking reaction, splicing out the intron and joining the exon to form the fully circularized construct. (B) Agarose gel images from RT-PCR assays utilizing the primer pairs shown in panel (A) for cells transfected with circ-fragL and circ-fragR constructs.

[0024] FIG. 10 shows the sequences of the designer exons with exon splicing enhancer (ESE) elements indicated and the intron sequences used with the fragL and fragR constructs with key features highlighted (SEQ ID NOs: 1351-1353).

[0025] Figure 11A-B shows (A) a schematic diagram showing the plasmid design of the dual promoter system, where the GFP-pCI-L construct is driven by the U6 promoter and the GFP-pCI-R construct is driven by the CMV promoter, and (B) the relative expression of GFP RNA transcripts measured by qRT-PCT. The fold activation between the full-length and unlinked constructs that do not contain ribozymes and complementary sequences is shown above the two graphs.

[0026] Figure 12A-C shows: (A) a schematic showing the mechanism of inducible gene expression using a tetracycline-responsive hammerhead aptazyme embedded in the 3'UTR of a gene of interest (GOI), (B) insulin ELISA absorbance values ​​from cell culture supernatants harvested 48 hours after addition of DMEM (-) or tetracycline (+) 4-6 hours after gene transfection, and (C) insulin ELISA absorbance values ​​from cell culture supernatants harvested 48 hours after gene transfection with higher concentrations of tetracycline added 4-6 hours after gene transfection.

[0027] Figures 13A-B show (A) a schematic diagram of the various ribozyme locations where the effect on gene expression induction was assessed, and (B) insulin ELISA levels in cell culture supernatants for the plasmid designs shown in panel (A) 48 hours after gene transfer in HEK293T cells.

[0028] Figure 14A-E shows: (A) Schematic of the plasmid design of the ribozyme-mediated inducible SaCas9 construct, (B) SaCas9 mRNA levels measured by quantitative PCR 48 hours after transfection in HEK293T cells, (C) Timeline of the experiments performed to evaluate tetracycline-inducible Cas9-mediated editing of the Pcsk9 gene. Arrows indicate the time points when blood was collected (B), when tetracycline was administered for 3 consecutive days (T), or when livers were harvested (H). (D) PCSK9 serum levels measured at various time points after AAV8 administration, and (E) editing rates measured in the livers of mice injected with the AAV8-SaCas9-Ribo construct at 5 and 7 weeks.

[0029] 15 shows a schematic of an in vitro transcribed RNA delivery system in which a linear RNA construct is circularized in situ. In this embodiment, linear RNA is generated in vitro, and the linear RNA is delivered into a cell where it is circularized in situ. In some embodiments, it is only circularized in situ.

[0030] Figures 16A-D provide (A) a general template for engineering circular RNA (SEQ ID NO: 1329) (where the payload is provided as GFP but can be any polypeptide of interest), (B) a schematic of the circular RNA construct and the resulting GFP expression comparing linear and circular constructs on days 1, 2, and 3, (C) fluorescent expression and photomicrographs of expression, and (D) relative GFP RNA expression over time.

[0031] Figures 17A-C provide (A) a schematic diagram of an exemplary CRISPR / ZF / TALE / gene payload containing circular format, (B) a graph showing the editing efficiency of linear vs. circular constructs containing zinc finger (ZF) proteins, and (C) a graph showing the editing efficiency of constructs containing Cas9 proteins.

[0032] FIG. 18 shows the design of a construct for in situ generation of circularized RNA, including a self-amplifying RNA construct (SEQ ID NO: 1330).

[0033] FIG. 19 provides sequences of IRESs (Table 2) useful in the methods and compositions of the disclosure.

[0034] Figure 20 provides sequences of circular constructs (SEQ ID NOs:1331-1346) useful in the methods and compositions of the disclosure.

[0035] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to a "prodrug" includes a plurality of such prodrugs, a reference to a "chemotherapeutic agent" includes a reference to one or more chemotherapeutic agents and equivalents thereof known to those skilled in the art, and so forth.

[0036] Additionally, the use of "or" means "and / or" unless otherwise stated. Similarly, "include," "including," "including" and "including" are interchangeable and are not intended to be limiting.

[0037] It is understood that where the description of various embodiments uses the term "comprising," those skilled in the art will recognize that in some specific instances the embodiments may alternatively be described using the phrase "consisting essentially of" or "consisting of."

[0038] Unless otherwise limited, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although many methods and reagents are similar or equivalent to those described herein, exemplary methods and materials are disclosed herein.

[0039] All publications mentioned herein are incorporated by reference in their entirety for the purpose of describing and disclosing methodologies that may be used in connection with the teachings herein. Furthermore, with respect to any term presented in one or more publications that is similar or identical to a term explicitly defined in this disclosure, the definition of the term explicitly provided in this disclosure will control in all respects.

[0040] It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments, and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0041] Except in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about." The term "about," as used to describe the present invention, means ±1% in relation to percentages.

[0042] The term "alphavirus" as used herein has its conventional meaning in the art and includes various species such as Venezuelan equine encephalitis (VEE) virus, Eastern equine encephalitis (EEE) virus, Everglades virus (EVE), Mucambo virus (MUC), Pixuna virus (PIX), and Western equine encephalitis virus, all of which are members of the VEE / EEE group of alphaviruses. Other alphaviruses include, for example, Semliki Forest virus (SFV), Sindbis, Ross River virus, Chikungunya virus, SA AR86, Barmah Forest virus, Middleburg virus, O'nyong-nyong virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Wataroa virus, Bambangki virus, Kijilgaha virus, Highlands J virus, Fort Morgan virus, Ndum virus, and Boggy Creek virus. Alphaviruses that are particularly useful in the constructs and methods described herein are the VEE / EEE group alphaviruses.

[0043] The terms "alphavirus RNA replicon," "alphavirus replicon RNA," "alphavirus RNA vector replicon," "vector replicon RNA," and "self-replicating RNA construct" are used interchangeably to refer to an RNA molecule expressing a nonstructural protein gene, such that it is capable of directing its own replication (amplification), and which includes at least 5' and 3' alphavirus replication recognition sequences, a coding sequence for an alphavirus nonstructural protein, and a polyadenylation tract. It may further include one or more elements (e.g., an IRES sequence, a core or mini promoter, a 2A peptide sequence, etc.) to direct expression, i.e., transcription and translation, of a coding sequence of interest. An alphavirus replicon of the present disclosure, in one embodiment, can include 5' and 3' alphavirus replication recognition sequences, a coding sequence for an alphavirus nonstructural protein, and a polyadenylation tract.

[0044] As used herein, the term "adeno-associated virus" or "AAV" refers to a member of the class of viruses associated with the name and belonging to the genus adenovirus of the Parvoviridae family. Multiple serotypes of the virus may be suitable for gene delivery. In some cases, the serotypes can infect cells from various tissue types. Examples of AAV serotypes are AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11. Non-limiting examples of serotypes useful for the purposes disclosed herein include any of the eleven serotypes, such as AAV2 and AAV8.

[0045] As used herein, the terms "circularized" and / or "circular" used in the context of a nucleic acid molecule (e.g., an artificial guide RNA) can generally refer to a nucleic acid molecule that can be represented as a polynucleotide sequence in a circular two-dimensional format with nucleotides one after the other. Wherein the represented polynucleotide is a circular or closed loop. In some embodiments, a circular nucleic acid molecule does not contain a 5' reducing hydroxyl, a 3' reducing hydroxyl, or both that can be exposed to solvent.

[0046] The term "complementary" as used herein refers to Watson-Crick base pairing between nucleotides, and in particular to nucleotides hydrogen bonded to each other at a thymine or uracil residue bound to an adenine residue by two hydrogen bonds and at a cytosine and guanine residue bound to each other by three hydrogen bonds. In general, nucleic acids include nucleotide sequences that are described as having a "percent complementarity" or "percent homology" to a particular second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a particular second nucleotide sequence. This indicates that 8 out of 10 nucleotides, 9 out of 10 nucleotides, or 10 out of 10 nucleotides of a sequence are complementary to the particular second nucleotide sequence.

[0047] The term "encode," when applied to a polynucleotide, can refer to a polynucleotide that, in its natural state or when engineered, is said to encode a polypeptide and can refer to a polynucleotide that can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, from which the coding sequence can be deduced.

[0048] The terms "equivalent" or "biological equivalent" are used interchangeably when referring to a particular molecular, biological or cellular material that has minimal homology while maintaining a desired structure or functionality.

[0049] As used herein, the term "expression" can refer to the process by which a polynucleotide is transcribed into mRNA and / or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression can include splicing of the mRNA in a eukaryotic cell.

[0050] "Homology", "identity" or "similarity" can refer to sequence similarity between two peptides or two nucleic acid molecules. Homology can be measured by comparing positions in each sequence that can be aligned for comparison. For example, if a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present disclosure.

[0051] Homology can refer to percentage (%) sequence identity with reference sequence. In practice, whether a particular sequence is at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical with any sequence described herein (which may correspond to a particular nucleic acid sequence described herein), such a particular peptide, polypeptide, or nucleic acid sequence can be measured using a conventional computer program, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). When using Bestfit or any other sequence alignment program to measure whether a particular sequence is, for example, 95% identical with a reference sequence, the percentage of identity is calculated over the entire length of the reference sequence, and parameters can be set so that identity gaps of up to 5% of the entire reference sequence are allowed.

[0052] For example, in certain embodiments, the identity between a reference sequence (query sequence, sequence of the present disclosure) and a subject sequence, also referred to as global sequence alignment, can be measured using the FASTDB computer program. In some cases, the parameters used in the FASTDB amino acid alignment for certain embodiments in which identity is narrowly constructed can include: scoring scheme=PAM (percentage of mutations allowed) 0, k-tuple=2, mismatch penalty=1, binding penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500, or the shorter length of the subject sequence. According to such embodiments, if the subject sequence is shorter than the query sequence due to N- or C-terminal deletions rather than internal deletions, the results can be manually corrected to account for the fact that the FASTDB program does not take into account the N- and C-terminal truncations of the subject sequence when calculating the global percentage identity. For subject sequences truncated at the N- and C-terminus, the percentage identity can be corrected by calculating the number of residues of the query sequence that are lateral to the N- and C-terminus of the subject sequence that are not matched / aligned with the corresponding subject residues relative to the query sequence as a percentage of the total bases of the query sequence. The result of the FASTDB sequence alignment can provide a measure of whether the residues are matched / aligned. The percentage can then be subtracted from the percentage identity calculated by the FASTDB program with the specified parameters to obtain a final percentage identity score. The final percentage identity score can be used for the purposes of this embodiment. In some cases, only residues to the N- and C-termini of the subject sequence that are not matched / aligned with the query sequence are considered for the purposes of manually adjusting the percentage identity score. That is, for this manual correction, only query residue positions outside the farthest N-terminal residue of the subject sequence are considered. For example, a 90-residue subject sequence can be aligned with a 100-residue query sequence to measure the percentage identity.Since this deletion occurs at the N-terminus of the subject sequence, the FASTDB alignment does not show a match / alignment of the first 10 residues at the N-terminus. Since the 10 unpaired residues represent 10% of the sequence (number of residues at the N-terminus and C-terminus that are not matched / total number of residues in the query sequence), 10% is subtracted from the percentage identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched, the final percentage identity could be 90%. In another example, a 90 residue subject sequence is compared to a 100 residue query sequence. This time, since the deletion is an internal deletion, there are no residues at the N- or C-terminus of the subject sequence that are not matched / aligned with the query sequence. In this case, the percentage identity calculated by FASTDB is not manually corrected. Again, only residues located outside the N- and C-terminus of the subject sequence that are not matched / aligned with the query sequence are manually corrected to appear in the FASTDB alignment.

[0053] "Hybridization" can refer to a reaction in which one or more polynucleotides react to form a complex stabilized through hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction can constitute a step in a more extensive process, such as the initiation of a PC reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0054] Examples of stringent hybridization conditions include the following: incubation temperature: about 25° C. to about 37° C., hybridization buffer concentration: about 6× SSC to about 10× SSC, formamide concentration: about 0% to about 25%, and wash solution: about 4× SSC to about 8× SSC. Examples of moderate hybridization conditions include the following: incubation temperature: about 40° C. to about 50° C., buffer concentration: about 9× SSC to about 2× SSC, formamide concentration: about 30% to about 50%, and wash solution: about 5× SSC to about 2× SSC. Examples of highly stringent hybridization conditions include: incubation temperature: about 55°C to about 68°C, buffer concentration: about 1x SSC to about 0.1x SSC, formamide concentration: about 55% to about 75%, and wash solution: about 1x SSC, 0.1x SSC, or deionized water. In general, hybridization incubation times are 5 minutes to 24 hours with one, two or more wash steps, and wash incubation times are about 1, 2 or 15 minutes. SSC is a 0.15 M NaCl and 15 mM citrate buffer. It will be understood that equivalents of SSC using other buffer systems can be utilized.

[0055] As used herein, the term "isolated" can refer to a molecular, biological, or cellular material that is substantially free of other materials. In one aspect, the term "isolated" can refer to a nucleic acid, such as DNA or RNA, or a protein or polypeptide (e.g., an antibody or derivative thereof), or a cell or organelle, or a tissue or organ, that is separated from other DNA or RNA, or a protein or polypeptide, or a cell or organelle, or a tissue or organ, that is present in a natural source. The term "isolated" can also refer to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium if produced by recombinant DNA technology, or chemical precursors or other chemicals if chemically synthesized. Also, "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally present as fragments and would not be found in the natural state. In some cases, the term "isolated" is also used to refer to a polypeptide that is isolated from other cellular proteins, meaning to include both purified and recombinant polypeptides. In some cases, the term "isolated" is also used to refer to a cell or tissue that is isolated from other cells or tissues, meaning to include both cultured and artificial cells or tissues.

[0056] "Linking sequence" refers to a sequence complementary to another sequence that allows Watson-Crick base pairing and forms a substrate suitable for ligation by a ligase, such as an RNA ligase. In one embodiment, the 5' and 3' linking sequences are substrates for an RNA ligase, such as, but not limited to, RtcB. When ligated, the 5' and 3' linking sequences circularize the RNA molecule of the present disclosure. Such circularization reduces RNA degradation and improves persistence in vivo.

[0057] "Operably linked" refers to an arrangement of elements such that the components so described are configured to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of affecting the transcription, and possibly the translation, of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression of the coding sequence.

[0058] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs or combinations thereof. Polynucleotides can have any three-dimensional structure and can perform any function. The following are non-limiting examples of polynucleotides: genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, RNAi, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, for example, by conjugation with a labeling component. The term can also refer to both double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the present disclosure that is a polynucleotide can encompass both the double-stranded form and two complementary single-stranded forms that are known or predicted to constitute the double-stranded form. In some embodiments, a polynucleotide can include both RNA and DNA nucleotides.

[0059] The term "polynucleotide sequence" can be an alphabetical representation of a polynucleotide molecule. The alphabetical representation can be entered into a database in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching. In any alphabetical representation, the present disclosure contemplates both RNA and DNA (i.e., "T" is replaced with "U" or vice versa).

[0060] In certain embodiments, a "promoter" may be used to drive transcription of an operably linked nucleic acid. As used herein, a "promoter" refers to a DNA sequence that contains a binding site for RNA polymerase and initiates transcription of a downstream nucleic acid sequence. Promoters for use in this disclosure may be constitutive, inducible, or tissue-specific or temporal promoters. Suitable promoters may be derived from viruses, prokaryotes, and eukaryotes. Suitable promoters may be used to drive expression by any RNA polymerase. Examples of inducible promoters include, but are not limited to, T7 RNA polymerase promoter, T3 RNA polymerase promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG)-regulated promoter, lactose-induced promoter, heat shock promoter, tetracycline-regulated promoter, steroid-regulated promoter, metal-regulated promoter, estrogen receptor-regulated promoter, and the like. Inducible promoters may be regulated by various molecules, such as doxycycline. In one embodiment, the promoter is a prokaryotic promoter selected from the group consisting of T7, T3, SP6, and derivatives thereof.

[0061] As used herein, a "ribozyme" (ribonucleic acid enzyme) is an RNA molecule capable of catalyzing a biochemical reaction. A "self-cleaving ribozyme" is a ribozyme capable of cleaving itself. The ribozyme used in this disclosure can be any small endonucleolytic ribozyme that self-cleaves in a target cell type, including, for example, hammerhead, hairpin, hepatitis delta virus, Valkud satellite, twister, twister sister, pistol, and hatchet. See, for example, Roth et al., Nat Chem Biol. 10(1):56-60 and Weinberg et al., Nat Chem Biol. 2015 August; 11(8):606-10, both of which are incorporated herein by reference. US 2015 / 0056174 provides modified hammerhead ribozymes with enhanced endonucleolytic activity. The ribozyme cleaves the substrate RNA in a sequence-specific manner at the substrate cleavage site. Typically, a ribozyme comprises a catalytic region flanked by two binding regions. The ribozyme binding region hybridizes to the substrate RNA, while the catalytic region cleaves the substrate RNA at the substrate cleavage site to produce a cleaved RNA product. In various embodiments, the 5' or 3' of various constructs can be a twister ribozyme or a twister sister ribozyme. For example, the 5' and 3' ribozymes of various constructs are either P3 or P1 twister ribozymes, but not both P3 or both P1.

[0062] As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid into a host cell, including, for example, calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection (e.g., using commercially available reagents such as LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI® (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT® (OZ Biosciences, France), etc.), or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and other laboratory manuals.Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described in Sambrook, J., Fritsch, EF, and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, (1989); Silhavy, TJ, Bennan, ML, and Enquist, LW, Gene Fusion Experiments; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, (1984); and Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience (1987), each of which is incorporated herein by reference in its entirety. Further useful methods are described in manuals including "Advanced Bacterial Genetics" (Davis, Roth and Botstein, Cold Spring Harbor Laboratory, 1980), "Gene Fusion Experiments" (Silhavy, Berman and Enquist, Cold Spring Harbor Laboratory, 1984), "Molecular Genetics Experiments" (Miller, Cold Spring Harbor Laboratory, 1972), "Laboratory Techniques in Bacterial Genetics" (Maloy, in Jones and Bartlett, 1990), and "A Short Course in Bacterial Genetics" (Miller, Cold Spring Harbor Laboratory 1992), each of which is incorporated herein by reference in its entirety.

[0063] As used herein, the terms "treat," "treating," and "treatment" refer to ameliorating the symptoms associated with a disease or disorder. The terms "treat," "treating," and "treatment" also include preventing or delaying the onset of said disease or disorder and / or reducing the severity or frequency of the symptoms of said disease or disorder.

[0064] As used herein, the term "vector" can refer to a nucleic acid construct designed for transfer between different hosts, including but not limited to plasmids, viruses, cosmids, phages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), and the like. In some embodiments, a "viral vector" is defined as a recombinantly produced virus or viral particle that contains a polynucleotide that is delivered to a host cell either in vivo, ex vivo, or in vitro. In some embodiments, the plasmid vector can be prepared from a commercially available vector. In other embodiments, the viral vector can be produced from baculovirus, retrovirus, adenovirus, and AAV. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, and the like. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves. Alphavirus vectors, such as Semliki Forest Virus-based vectors and Sindbis Virus-based vectors, have also been developed for use in gene therapy and immunotherapy. In aspects where gene transfer is mediated by a retroviral vector, vector construct can refer to a polynucleotide comprising said retroviral genome or a portion thereof and a gene of interest.

[0065] This disclosure describes the implementation of an artificial endogenous RNA processing machinery to generate linked RNA fusion constructs that can be used for combinatorial genetic interaction screens and RNA-based readout for inducible gene expression. Separately transcribed sequences are complementary to each other and fused to a self-cleaving ribozyme. Once transcribed, the autocatalytic activity of the ribozyme cleaves the transcripts to create unique ends. Due to the complementary regions, the two transcripts hybridize and the cleaved ends are juxtaposed, which are recognized by an endogenous RNA ligase to generate a linked fusion construct. This disclosure further demonstrates the applicability of this approach to link two transcripts delivered to a cell on different library elements, and when linked to an intron sequence, the RNA fusion construct can be used for controllable expression of a full-length gene product.

[0066] This disclosure describes the use of artificial RNA elements that undergo multiple endogenous processing events to create linked RNA fusion constructs that can be used for a variety of applications, including combinatorial genetic screens or inducible gene expression. These elements, called the left fragment (fragL) and the right fragment (fragR), are engineered to be expressed from both polymerase II and polymerase III promoters. In certain embodiments, the RNA elements can further comprise flanking amplification primers and / or variable barcode regions. As shown in FIG. 1A, the RNA elements contain 45 base pair (bp) complementary regions to each other and are tethered to P3 and P1 self-cleaving twister ribozymes. Upon transcription, the P3 twister ribozyme self-cleaves and generates a 5' hydroxyl group on the fragL construct, while the P1 twister ribozyme generates a 2',3'-cyclic phosphate group at the 3' end of the fragR construct. The complementary regions of the fragments then hybridize the two transcripts, juxtaposing the two ends and allowing recognition by the endogenous ligase RtcB to create a linked fusion construct. By using endogenous processing of these constructs, no additional factors need to be added. Linking two different library elements that are delivered separately to cells but analyzed together downstream and fused is highly useful for a variety of applications. Furthermore, the Twister ribozyme undergoes rapid autocatalytic self-cleavage upon transcription within tens of seconds to minutes, making the half-life of the linked construct longer than the individual transcripts, making this approach robust over multiple time scales.

[0067] The choice of fusion construct is fully tunable and additional classes of ribozymes or transfer RNAs can be used in combination with different ligases and RNA processing enzymes to create higher order fusion junctions.

[0068] We demonstrate the feasibility of this approach through a simple plasmid transfection experiment in which fragL and fragR are cloned into the lentiCRISPRv2 backbone downstream of the U6 promoter. They were delivered to cells individually or in combination, and RNA was isolated 48 hours later. Reverse transcription polymerase chain reaction (RT-PCR) was performed using flanking amplification primers. As shown in Figure 1B, the generated RNA fusion was only observed when both plasmids were co-transfected. The PCR products were then purified and Sanger sequenced to confirm the expected fusion sequence (Figure 1C).

[0069] A major limitation of traditional implementations of genetic interaction screens is the need to physically link multiple perturbations on the same library element to enable genotype-to-phenotype mapping, which complicates library generation and prevents easy combination of different classes of genome and transcriptome engineering toolsets.

[0070] This disclosure describes the engineering of RNA fusion constructs that allow for the coupling of genotype and phenotype at the RNA level, such that multiple libraries (e.g., CRISPR knockout, CRISPR activation / inhibition, translation regions, shRNA, etc.) can be delivered to cells individually but read together. This approach is demonstrated by cloning ribozyme-RNA fusion constructs (called sgRNA fragL and sgRNA fragR) downstream of a U6-driven sgRNA sequence in the lentiCRISPRv2 backbone (Figure 2A). As shown in Figure 2B, the presence of a non-targeting sgRNA transcript at the 5' position of the RNA fusion construct does not alter the RNA processing machinery that generates the linked fusion. Furthermore, the EcoRI restriction site on the sgRNA fragR construct shown in Figure 2C highlights the ability to identify a theoretical barcode placed at this position for perturbation mapping using RNA sequencing.

[0071] The ribozyme-mediated RNA fusion constructs of the present disclosure allow for the linking of genotype and phenotype at the RNA level, such that multiple libraries (e.g., CRISPR knockout, CRISPR activation / inhibition, translated regions, shRNA, etc.) can be delivered to cells individually but read out together. By using this approach with the ribozyme-mediated RNA fusion constructs of the present disclosure, gene interaction screens can be highly multiplexed. This approach allows for the linking of heterologous libraries, such as CRISPR knockout sgRNA libraries and translated region overexpression libraries. Furthermore, unprecedented insight into the regulatory networks that determine cellular function can be realized by using screens that observe the combination of gene activation and inhibition through systems such as CRISPR activation and CRISPR inhibition in cells using the RNA fusion constructs disclosed herein. Screening strategies with RNA fusions are not limited to fitness measurements but can be combined with screens using other readouts such as phenotype, protein expression, or other functional endpoints that can be broadly applied across the biological sciences.

[0072] The RNA fusion constructs of the present disclosure can be used in inducible gene expression systems. Inducible gene expression systems are powerful tools for a wide range of basic and applied research fields, including functional genomics, tissue engineering, biopharmaceutical protein production, and gene therapy. The most common of these systems, such as tetracycline-controlled operons, protein-protein interaction chimera systems, and tamoxifen-controlled recombinant enzyme systems, all require the addition of exogenous proteins. This can cause immunogenic responses in vivo, and delivery and gene transfer of these large size plasmids can be burdensome. The ribozyme-mediated RNA fusion approach using intronic sequences described herein has a wide range of applications. The system of the present disclosure can strongly control gene expression by adding aptazymes to either fragment that respond to molecules such as tetracycline, theophylline, or guanine (see Figure 3).

[0073] The RNA fusion constructs of the present disclosure have great utility in the field of gene therapy to treat a wide range of diseases. In both type 1 and type 2 diabetes, insulin production is limited, so patients typically require exogenous insulin administration when blood glucose levels rise. The inducible ribozyme-mediated RNA fusion system described herein can be adapted to contain two halves of the insulin gene fused to an intron sequence. The two constructs are constitutively present in muscle tissue, but one half is transcribed only when an aptamer-binding ligand, such as a synthetic sugar, is added. This leads to a rapid increase in ribozyme-mediated hybridization and splicing, which produces a full-length functional insulin protein. When the inducer is degraded, one fusion fragment is suppressed and no insulin is produced until more ligand is administered. Thus, the need for painful and cumbersome exogenous insulin administration can be replaced by an endogenous system with precise time control.

[0074] The inducible ribozyme-mediated RNA fusion system described herein can be applied to the generation of inducible gene expression systems for clotting factor IX for hemophilia patients, cystic fibrosis transmembrane permeability regulator protein for cystic fibrosis patients, and dystrophin protein for Duchenne muscular dystrophy patients. In general, any disease caused by poorly expressed or mutated proteins can benefit from the inducible ribozyme-mediated RNA fusion system disclosed herein. This includes, but is not limited to, diseases such as β-thalassemia, severe combined immunodeficiency, spinal muscular atrophy, and age-related macular degeneration.

[0075] The inducible ribozyme-mediated RNA fusion system described herein can be widely applied to gene therapy using the CRISPR / Cas toolset. CRISPR / Cas genome editing is highly adaptable and designed for the study and treatment of genetic diseases, cancer, immune diseases, and infectious diseases. A major limitation in the introduction of these therapies is the inability to control the expression of Cas proteins in vivo. The inducible ribozyme-mediated RNA fusion system described herein can overcome this limitation by fusing two parts of the Cas protein to intronic sequences in the fragL and fragR constructs. Among these, the expression of the Cas protein and its subsequent function can be fully induced under the control of the inducers described herein. This allows precise control of genome editing mediated by the CRISPR / Cas system. It is not limited to gene knockout, but can be broadly adapted to assist in controlled and inducible non-homologous end joining, homology-directed repair, single base exchange, transcriptional control, base editors, PRIME editors, and RNA editing.

[0076] The system can also be utilized (see, e.g., FIG. 12A) to perform experiments to control the expression of Yamanaka factors, including Oct3 / 4, Sox2, Klf4, and c-Myc (OSKM), in vivo. OSK and c-Myc can be cloned into an AAV expression vector, and the aptazyme of choice can be cloned into the 3'UTR. The Oct3 / 4, Sox2, and Klf4 can be cloned into a polycistronic vector and separated by a self-cleaving 2A peptide. c-Myc, a known oncogene, can be cloned into a separate plasmid with its own aptazyme control element. These plasmids can then be packaged into an AAV vector and delivered individually or together based on the reprogramming application at hand.

[0077] Because the ribozyme rapidly cleaves the 3'-poly(A) tail upon transcription, background expression of OSKM is low and iAAV-mediated transduction does not alter the cellular state at baseline expression. Upon delivery of an aptamer-specific ligand, the ribozyme is stabilized and transduced cells will exhibit higher OSKM expression levels based on the dose of ligand delivered. Due to the fast turnover of mRNA transcripts, ribozyme stabilization and resulting gene expression levels are directly dependent on the half-life of the delivered ligand and the chosen dosing regimen, thus allowing for kinetic, pulsatile, and temporal control of OSKM expression. These transcription factors have been thoroughly studied to induce pluripotent states based on their expression levels. The system can be utilized to dynamically reprogram cells in vivo.

[0078] There are also many other "reprogramming factors" that are involved in directing cell phenotype through overexpression. Similar to OSKM, these factors require temporal control to effectively and safely exert their effect on cell state. Thus, the ribozyme-mediated control system can be used to dynamically control the expression of a wide range of genes that have the ability to reprogram cell identity in vivo. These genes to which the system can be applied include, but are not limited to, the genes listed in Table 1.

[0079] The system is further adjustable since the AAV serotype used can be changed without the need to change the expression plasmid. Different serotypes can be used to specifically target tissues, such as AAV8 for liver, AAV9 for skeletal muscle, and AAV-PHP.B for the central nervous system. Furthermore, to further improve the specificity of the partial reprogramming system, in addition to the wide range of serotypes already available, artificial recombinant AAVs can also be utilized to specifically target different cell types.

[0080] Table 1 JPEG2024502620000002.jpg208137JPEG2024502620000003.jpg190136

[0081] Utilizing the systems described herein for in vivo control of reprogramming factors allows the systems to be utilized in a wide range of applications.

[0082] In general, transient expression of Yamanaka factors in vivo has been demonstrated to ameliorate aging hallmarks. The disclosed system, including OSKM and 3'-UTR aptazymes, can be packaged into AAVs designed to have broad tropism throughout the body or to target specific organs via engineered AAVs. This can then be administered to a subject to transduce the target organ for a short period of time. Ligands specific to the aptamer sequence can then be administered at the desired dose and treatment regimen to achieve cyclic expression of OSKM. Physiological changes induced by this approach may include reduced DNA damage response with age, reduced expression of genes associated with aging and stress, and changes in epigenetic modifications that occur with age. These molecular changes at the cellular level have important implications for the systematic alleviation of aging. Furthermore, for certain diseases associated with aging, such as Hutchinson-Gilford progeria syndrome, this strategy may be an important therapeutic option to systematically alleviate the physiological hallmarks of aging and simultaneously extend the lifespan of affected individuals.

[0083] At the tissue-specific level, the disclosed system may demonstrate significant therapeutic benefits, as manipulation of the AAV capsid can be utilized for cell-specific targeting of inducible reprogramming strategies. In the central nervous system, transient expression of OSK may be utilized to restore young DNA methylation patterns and transcriptomes in retinal ganglion cells, promoting axonal regeneration after injury and aiding in vision recovery in the elderly and those suffering from visual disorders such as glaucoma. Similarly, targeting the disclosed system to specific brain regions (e.g., hippocampus) may be an important tool for improving memory through specific targeting of dentate gyrus cells. In the cardiovascular system, targeting the disclosed system to cardiomyocytes may cause dedifferentiation of these postmitotic cells. This enabled regenerative capacity may broadly improve cardiac function and significantly improve cardiomyocyte recovery after traumatic events such as myocardial infarction. Administration of the inducible OSKM constructs described herein, followed by treatment of affected individuals with inducible ligands, may dramatically improve recovery from cardiovascular events. Furthermore, myofiber- and liver-specific transient expression mediated by the disclosed system has the potential to promote muscle regeneration in vivo, with broad implications in both aging and disease conditions.

[0084] Apart from the induced, AAV-aptazyme-mediated expression of OSKM, the methods and compositions of the present disclosure can be applied to other reprogramming transcription factors (TFs) as well (Table 1). Depending on the desired outcome, TFs can be delivered individually or in combination with matching aptazyme sequences or individual aptazymes to allow for temporal control of gene expression. These artificial TFs can be applied in health and disease settings, with even broader impacts across the field of regenerative medicine. The iAAV partial reprogramming approach of the present disclosure has broad applications across a variety of organ systems and disease settings.

[0085] RNA is inherently transient, and this transience impacts its activity both as an interacting moiety and as a template. Circularization of RNA improves persistence, but simple, scalable approaches to achieve this are lacking. The present disclosure provides compositions and methods of in situ circularized RNA (icRNA) for sustained protein translation utilizing the autocatalytic RNA circularization described herein. Specifically, in vitro transcribed linear RNAs are provided that have an internal ribosome entry site bound to a messenger RNA of interest, which is then flanked by ribozymes. Upon delivery of these linear RNAs to cells, autocatalytic cleavage of the ribozyme generates in situ circular molecules, leaving ends that are ligated by an endogenous RNA ligase (e.g., the ubiquitous endogenous RNA ligase RtcB) (see, e.g., FIG. 15). This scalable icRNA system has broad applications in basic science and therapeutic applications.

[0086] The icRNA system is exemplified herein in three contexts: first, sustained protein expression via the system using GFP as a test protein (Figures 16A-D); second, improved genome targeting via zinc finger nucleases; and third, genome targeting via CRISPR with a deimmunized Cas9 protein (Figures 17A-C).

[0087] The compositions herein can be used to treat a disease or condition in a subject.For example, the ribozyme-activated RNA constructs of the present disclosure can be administered to treat the diseases described herein.

[0088] The pharmaceutical composition can include a first active ingredient. The first active ingredient can include a ribozyme-activated RNA construct of the present disclosure. The pharmaceutical composition can be formulated in a unit dose form. The pharmaceutical composition can include a pharma- ceutically acceptable excipient, diluent, or carrier. The pharmaceutical composition can include a second, third, or fourth active ingredient.

[0089] The compositions described herein may include an excipient. In some cases, the excipient may include a pharma- ceutically acceptable excipient. The excipient may include a cryopreservative, such as DMSO, glycerol, polyvinylpyrrolidone (PVP), or any combination thereof. The excipient may include a cryopreservative, such as sucrose, trehalose, starch, a salt of any of these, a derivative of any of these, or any combination thereof. The excipient may include a pH agent (to minimize oxidation or degradation of the components of the composition), a stabilizing agent (to prevent denaturation or degradation of the components of the composition), a buffering agent (to improve temperature stability), a solubilizing agent (to improve protein solubility), or any combination thereof. The excipient may include a surfactant, a sugar, an amino acid, an antioxidant, a salt, a non-ionic surfactant, a solubilizing agent, a triglyceride, an alcohol, or any combination thereof. The excipient can include sodium carbonate, acetate, citrate, phosphate, polyethylene glycol (PEG), sorbitol, sucrose, trehalose, polysorbate 80, sodium phosphate, sucrose, disodium phosphate, mannitol, polysorbate 20, histidine, citrate, albumin, sodium hydroxide, glycine, sodium citrate, trehalose, arginine, sodium acetate, acetate, HCl, disodium edetate, lecithin, glycerin, xanthan gum, soy isoflavone, polysorbate 80, ethyl alcohol, water, teprenone, or any combination thereof. In some cases, the carrier or diluent can include an excipient. In some cases, the carrier or diluent can include water, a salt solution (e.g., saline), alcohol, or any combination thereof.

[0090] Non-limiting examples of suitable excipients can include buffers, preservatives, stabilizers, binders, compression agents, lubricants, chelating agents, dispersion enhancers, disintegrants, flavors, sweeteners, and colorants.

[0091] In some cases, the excipient may be a buffer. Non-limiting examples of suitable buffers may include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate. As buffers, sodium bicarbonate, potassium bicarbonate, magnesium hydroxide, magnesium lactate, magnesium gluconate, aluminum hydroxide, sodium citrate, sodium tartrate, sodium acetate, sodium carbonate, sodium polyphosphate, potassium polyphosphate, sodium pyrophosphate, potassium pyrophosphate, disodium hydrogen phosphate, dipotassium hydrogen phosphate, trisodium phosphate, tripotassium phosphate, potassium metaphosphate, magnesium oxide, magnesium hydroxide, magnesium carbonate, magnesium silicate, calcium acetate, calcium glycerophosphate, calcium chloride, calcium hydroxide, and other calcium salts or combinations thereof may be used in pharmaceutical preparations.

[0092] In some cases, the excipient may include a preservative. Non-limiting examples of suitable preservatives include antioxidants such as α-tocopherol and ascorbate, and antimicrobial agents such as parabens, chlorobutanol, and phenol. Antioxidants may further include, but are not limited to, EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, p-aminobenzoic acid, glutathione, propyl gallate, cysteine, methionine, ethanol, and N-acetylcysteine. In some cases, the preservative can include validamycin A, TL-3, sodium orthovanadate, sodium fluoride, Na-tosyl-Phe-chloromethylketone, Na-tosyl-Lys-chloromethylketone, aprotinin, phenylmethylsulfonyl fluoride, diisopropylfluorophosphate, kinase inhibitors, phosphatase inhibitors, caspase inhibitors, granzyme inhibitors, cell adhesion inhibitors, cell division inhibitors, cell cycle inhibitors, lipid signaling inhibitors, protease inhibitors, reducing agents, alkylating agents, antibacterial agents, oxidase inhibitors, or other inhibitors.

[0093] In some cases, the pharmaceutical formulation can include a binder as an excipient. Non-limiting examples of suitable binders include starch, pregelatinized starch, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamide, polyvinyloxoazolidone, polyvinyl alcohol, C12-C18 fatty acid alcohol, polyethylene glycol, polyol, sugar, oligosaccharide, and combinations thereof.

[0094] Binders that can be used in the pharmaceutical formulations can be selected from starches such as potato starch, corn starch, wheat starch, etc.; sugars such as sucrose, glucose, dextrose, lactose, maltodextrin, etc.; natural and synthetic gums; gelatin; cellulose derivatives such as microcrystalline cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, methyl cellulose, ethyl cellulose, etc.; polyvinylpyrrolidone (povidone); polyethylene glycol (PEG); waxes; calcium carbonate; calcium phosphate; alcohols such as sorbitol, xylitol, mannitol, etc.; and water; or combinations thereof.

[0095] In some cases, pharmaceutical formulations can include lubricants as excipients.Non-limiting examples of suitable lubricants can include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oil, sterotex, polyoxyethylene monostearate, talc, polyethylene glycol, sodium benzoate, sodium lauryl sulfate, magnesium lauryl sulfate, and light mineral oil.The lubricants that can be used in pharmaceutical formulations can be selected from metal stearates (such as magnesium stearate, calcium stearate, and aluminum stearate), fatty acid esters (such as sodium stearyl fumarate), fatty acids (such as stearic acid), fatty alcohols, glyceryl behenate, mineral oils, paraffin, hydrogenated vegetable oils, leucine, polyethylene glycol (PEG), metal lauryl sulfates (such as sodium lauryl sulfate and magnesium lauryl sulfate), sodium chloride, sodium benzoate, sodium acetate, and talc, or combinations thereof.

[0096] In some cases, pharmaceutical formulations can include dispersion enhancers as excipients. Non-limiting examples of suitable dispersion enhancers include starch, alginic acid, polyvinylpyrrolidone, guar gum, kaolin, bentonite, refined wood cellulose, sodium starch glycolate, isomorphous silicate, and microcrystalline cellulose as a high HLB emulsifier surfactant.

[0097] In some cases, the pharmaceutical formulation may include a disintegrant as an excipient. In some cases, the disintegrant may be a non-effervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as corn starch, potato starch, pregelatinized starches and modified starches thereof, sweeteners, clays such as bentonite, microcrystalline cellulose, alginates, sodium starch glycolate, agar, guar, locust bean, karaya, pectin, and gums such as tragacanth. In some cases, the disintegrant may be an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include a combination of sodium bicarbonate and citric acid and a combination of sodium bicarbonate and tartaric acid.

[0098] In some cases, the excipient may include flavorings. The flavorings incorporated in the outer layer may be selected from synthetic flavor oils and flavor aromatics; natural oils; extracts from plants, leaves, flowers, and fruits; and combinations thereof. In some cases, the flavorings may be selected from the group consisting of cinnamon oil; oil of wintergreen; peppermint oil; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oils such as lemon oil, orange oil, grape oil, and grapefruit oil; and fruit essences including apple, peach, pear, strawberry, raspberry, cherry, plum, pineapple, and apricot.

[0099] In some cases, the excipient can include a sweetener. Non-limiting examples of suitable sweeteners include glucose (corn syrup), dextrose, invert sugar, fructose, and mixtures thereof (when not used as a carrier); saccharin and its various salts such as sodium salt; dipeptide sweeteners such as aspartame; dihydrochalcone compounds, glycyrrhizin; Stevia rebaudiana (stevioside); chloro derivatives of sucrose such as sucralose; and sugar alcohols such as sorbitol, mannitol, and syritol.

[0100] The compositions may include combinations of active agents (e.g., ribozyme-activated RNA constructs, compounds or compositions of the present disclosure) with inert (e.g., detectable agents or labels) or active (adjuvants, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, etc.) naturally occurring or non-occurring carriers, and may include pharma- ceutically acceptable carriers. Carriers also include pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldolic acids, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination and comprise 1-99.99% by weight or volume, alone or in combination. Exemplary protein excipients include serum albumins, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, and casein. Representative amino acids / antibody components that also function as buffers include alanine, arginine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, and aspartame, etc. Carbohydrate excipients are also contemplated within the scope of the present technology, examples of which include, but are not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), and myo-inositol.

[0101] In some embodiments, the pharmaceutical composition can be formulated in milligrams (mg), milligrams per kilogram (mg / Kg), copy number, or molecule number. In some cases, the composition can include about 0.01 mg to about 2000 mg of the active agent. In some cases, the composition can include about 0.01 mg, 0.1 mg, 1 mg, 10 mg, 100 mg, 500 mg, 1000 mg, 1500 mg, or about 2000 mg of the active agent.

[0102] As used herein, subject, host, individual, and patient may be used interchangeably to refer to either eukaryotic or prokaryotic organisms. In some cases, a subject may refer to an animal, such as a mammal. A mammal may be administered the ribozyme-activated RNA constructs of the present disclosure or the compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, macaques, etc.), livestock (e.g., dogs and cats), farm animals (e.g., horses, cows, goats, sheep, pigs), and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal may be of any age or at any stage of development (e.g., adult, teenager, child, infant, or mammal in utero). The mammal may be male or female. The mammal may be a pregnant female. In some embodiments, the subject is a human. In some embodiments, the subject has or is suspected of having a cancer or neoplastic disease. In other embodiments, the subject has or is suspected of having a disease or disorder associated with abnormal protein expression. In some cases, the human may be about 1 day to about 10 months of age, about 9 months to about 24 months of age, about 1 year to about 8 years of age, about 5 years to about 25 years of age, about 20 years to about 50 years of age, about 1 year to about 130 years of age, or about 30 years to about 100 years of age or older. The human may be about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 years of age or older. The human may be less than about 1, 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 years of age.

[0103] In some embodiments, a method of treating a human in need thereof can include administering to the human a ribozyme-activated RNA construct of the present disclosure. In some embodiments, the compositions herein can be used to treat diseases and conditions. The disease or condition can include a neurodegenerative disease, a muscle disorder, a metabolic disorder, an eye disorder, or any combination thereof. The disease or condition may be cystic fibrosis, albinism, alpha-1-antitrypsin deficiency, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), asthma, beta thalassemia, Cadacill syndrome, Charcot-Marie-Tooth disease, chronic obstructive pulmonary disease (COPD), distal spinal muscular atrophy (DSMA), Duchenne / Becker muscular dystrophy, dystrophic epidermolysis bullosa, epidermolysis bullosa, Fabry disease, factor V Leiden-related disease, familial adenomatous polyposis, galactosemia, Gaucher disease, glucose-6-phosphate dehydrogenase, hemophilia, hereditary hematochromatosis, Hunter syndrome, Huntington's disease, Hurler syndrome, inflammatory bowel disease (IBD), hereditary polyaggregation syndrome, liver congenital amaurosis The diseases or conditions may include disorders such as Lesch-Nyhan syndrome, Lynch syndrome, Marfan syndrome, mucopolysaccharidoses, muscular dystrophies, myotonic dystrophy types I and II, neurofibromatosis, Niemann-Pick disease types A, B, and C, NY-eso1-associated cancer, Parkinson's disease, Peutz-Jeghers syndrome, phenylketonuria, Pompe disease, primary ciliary body disease, prothrombin mutation-associated diseases such as prothrombin G20210A mutation, pulmonary hypertension, retinitis pigmentosa, Sandhoff disease, severe combined immunodeficiency syndrome (SCID), sickle cell anemia, spinal muscular atrophy, Stargardt disease, Tay-Sachs disease, Usher syndrome, X-linked immunodeficiency, and various forms of cancer (e.g., breast and ovarian cancer associated with BRCA1 and 2). In some cases, the disease or condition may include mucoposaccharide disease type I (MPSI). In some cases, the MPS I may include Hurler syndrome, Hurler-Scheie syndrome, Scheie syndrome, or any combination thereof.The disease or condition may include muscular dystrophy, ornithine transcarbamylase deficiency, retinitis pigmentosa, breast cancer, ovarian cancer, Alzheimer's disease, pain, Stargardt macular dystrophy, Charcot-Marie-Tooth disease, Rett syndrome, or a combination thereof. Administration of the composition may be sufficient to (a) decrease expression of the gene compared to expression of the gene prior to administration, (b) edit at least one point mutation in a subject (e.g., a subject in need thereof), (c) edit at least one stop codon in the subject to produce a stop codon read-through, (d) produce an exon skip in the subject, or (e) any combination thereof. The disease or condition may include muscular dystrophy. Muscular dystrophy may include myotonic, Duchenne, Becker, limb-girdle, facioscapulohumeral, congenital, oculopharyngeal, distal, Emery-Dreyfus, or any combination thereof. The disease or condition may include pain, such as chronic pain. The pain may include neuropathic pain, nociceptive pain, or a combination thereof. Nociceptive pain may include visceral pain, somatic pain, or a combination thereof.

[0104] A vector can be utilized to deliver the ribozyme-activated RNA construct of the present disclosure. The vector can include DNA, such as double-stranded DNA or single-stranded DNA. The vector can include RNA. In some cases, the RNA can include one or more base modifications. The vector can include a recombinant vector. In some cases, the vector can be a vector modified from a naturally occurring vector. The vector can include at least a portion of a non-naturally occurring vector. Any vector can be utilized. In some cases, the vector can include a viral vector, a liposome, a nanoparticle, an exosome, an extracellular vesicle, or any combination thereof. In some embodiments, the plasmid vector can be prepared from a commercially available vector. In other embodiments, the viral vector can be produced from a baculovirus, a retrovirus, an adenovirus, an AAV, or a combination thereof. In one embodiment, the viral vector is a lentiviral vector. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and alphaviral vectors, etc. Infectious tobacco mosaic virus (TMV)-based vectors can be used to produce proteins and have been reported to express Griffithsin in tobacco leaves. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. In aspects where gene transfer is mediated by a retroviral vector, the vector construct can refer to a polynucleotide that includes the retroviral genome or a portion thereof and a gene of interest. In some cases, the vector can include both a promoter and a cloning site to which a polynucleotide can be operably linked. Such vectors can transcribe RNA in vitro or in vivo and are commercially available.In some cases, the viral vector can include an adenoviral vector, an adeno-associated viral vector (AAV), a lentiviral vector, a retroviral vector, any portion thereof, or any combination thereof. In some cases, the nanoparticle vector can include a polymer-based nanoparticle, an amino lipid-based nanoparticle, a metal nanoparticle (such as a gold-based nanoparticle), any portion thereof, or any combination thereof. In some cases, the vector can include an AAV vector. The vector can be modified to include a modified VP1 protein (e.g., an AAV vector modified to include a VP1 protein). The AAV can include serotypes such as AAV1 serotype, AAV2 serotype, AAV3 serotype, AAV4 serotype, AAV5 serotype, AAV6 serotype, AAV7 serotype, AAV8 serotype, AAV9 serotype, AAV10 serotype, AAV11 serotype, any derivative thereof, or any combination thereof.

[0105] In some embodiments, the vector can include a nucleic acid encoding a linear precursor of a ribozyme-activated RNA construct of the present disclosure. In some embodiments, the nucleic acid can include a linear precursor of a ribozyme-activated RNA construct of the present disclosure. In some cases, the nucleic acid can be double-stranded. In some cases, the nucleic acid can be DNA or RNA. In some cases, the nucleic acid can include multiple copies of a ribozyme-activated RNA construct of the present disclosure. For example, the nucleic acid can include two, three, four, five, or more copies of a ribozyme-activated RNA construct of the present disclosure. In some cases, the nucleic acid can include a U6 promoter, a CMV promoter, or any combination thereof.

[0106] Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Generally, expression vectors useful in recombinant DNA techniques are in the form of plasmids. As used herein, "plasmid" and "vector" can be used interchangeably. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions. Typically, a vector or plasmid contains sequences that direct the transcription and translation of the relevant genes, a selectable marker, and sequences enabling autonomous replication or chromosomal integration. Suitable vectors contain a 5' region of the gene with transcription initiation controls and a 3' region of the DNA fragment that controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it should also be understood that such control regions may be derived from genes that are not native to the species selected as the production host.

[0107] Typically, the vector or plasmid contains sequences directing transcription and translation of the gene fragment, a selection marker, and sequences allowing autonomous replication or chromosomal integration. A suitable vector contains a 5' region of the gene with transcription initiation controls, and a 3' region of the DNA fragment that controls transcription termination. Both control regions may be derived from genes homologous to the transformed host cell, although it should also be understood that such control regions may be derived from genes that are not native to the species selected as the production host.

[0108] Initiation control regions or promoters that are useful for driving the expression of related coding regions in desired host cells are numerous and well known to those skilled in the art. Virtually any promoter that can drive these genetic elements is suitable for use in the present disclosure. For example, pol III promoter, U6 promoter, CMV promoter, T7 promoter, and H1 promoter can be used to drive expression. Termination control regions can be derived from various genes that are native to the preferred host.

[0109] The administration of the ribozyme-activated RNA constructs of the present disclosure can be performed continuously or intermittently in one dose throughout the course of treatment. The method of determining the most effective means and dosage can vary depending on the composition used for treatment, the purpose of the treatment, the target cell being treated, and the subject being treated. Single or multiple administrations can be performed with the dosage level and pattern selected by the treating physician. The appropriate dosage form and method of administration of the drug can vary and depend on the disease or condition. The route of administration can vary depending on the composition used for treatment, the purpose of the treatment, the health or disease stage of the subject being treated, and the target cell or tissue. Non-limiting examples of routes of administration include oral administration, nasal administration, injection, and topical application.

[0110] Administration can refer to methods that can be used to enable delivery of a compound or composition (such as a DNA construct, a viral vector, etc.) to a desired site of biological action. These methods can include topical administration (lotion, cream, ointment, etc.) to an external surface such as the skin. These methods can include parenteral administration (including intravenous, subcutaneous, intrathecal, intraperitoneal, intramuscular, intravascular or injection), oral administration, inhalation administration, intraduodenal administration, and rectal administration. In some cases, the subject can administer the composition without supervision. In some cases, the subject can administer the composition under the supervision of a medical professional (e.g., a physician, nurse, physician assistant, attending physician, hospice worker, etc.). In some cases, a medical professional can administer the composition. In some cases, a cosmetic professional can administer the composition.

[0111] Administration or application of the compositions disclosed herein provides at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53 , 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 days of treatment period can be performed continuously or non-continuously. In some cases, the treatment period can be about 1-30 days, about 2-30 days, about 3-30 days, about 4-30 days, about 5-30 days, about 6-30 days, about 7-30 days, about 8-30 days, about 9-30 days, about 10-30 days, about 11-30 days, about 12-30 days, about 13-30 days, about 14-30 days, about 15-30 days, about 16-30 days, about 17-30 days, about 18-30 days, about 19-30 days, about 20-30 days, about 21-30 days, about 22-30 days, about 23-30 days, about 24-30 days, about 25-30 days, about 26-30 days, about 27-30 days, about 28-30 days, or about 29-30 days.

[0112] Administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 times per day. In some cases, administration or application of the compositions disclosed herein can be performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 times per week. In some cases, administration or application of a composition disclosed herein is at least 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, 109, 109, 109, 108, 109, 109, 102, 104, 105, 106, 107, 108 You can do this 2, 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, or 90 times.

[0113] In some cases, the composition can be administered or applied as a single dose or divided doses. In some cases, the composition described herein can be administered at a first time point and a second time point. In some cases, the composition can be administered such that the first administration is administered 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 16 hours, 20 hours, 1 day, 2 days, 4 days, 7 days, 2 weeks, 4 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year or more after the other administration.

[0114] Also described herein are kits and manufacturer's documentation that include the ribozyme-mediated RNA fusion constructs or inducible ribozyme-mediated RNA fusion systems described herein. Such kits can include carriers, packages, or containers that are compartmentalized to receive one or more containers, such as vials or tubes, each of which contains one of the separate elements used in the methods described herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The containers can be formed from a variety of materials, such as glass or plastic.

[0115] For example, the container can contain one or more RNA fusion constructs described herein, optionally in a composition or in combination with another reagent disclosed herein. Optionally, the container has a sterile access port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic injection needle). Optionally, such a kit contains a compound disclosed herein with identifiable instructions, labels, or instructions for its use in the methods described herein.

[0116] Kits typically include one or more other containers each containing various materials (e.g., reagents, optionally in concentrated form, and / or equipment) desirable from a commercial and user standpoint for use of one or more of the compounds described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes, carriers, packaging, containers, vial and / or tube labels describing contents and / or instructions, and inserts providing instructions for use.

[0117] A label can be placed on or associated with a container. A label can be affixed to a container when the letters, numbers, or other characters forming the label are attached, molded, or etched onto the container itself. A label can be associated with a container when it is in an article or carrier that holds the container, e.g., as a package insert. A label can be used to indicate that the contents are to be used for a particular therapeutic application. The label can also indicate how to use the contents, e.g., as described herein. These other therapeutic agents can be used in amounts indicated, for example, in the Physician's Desk Reference (PDR) or as otherwise determined by one of skill in the art.

[0118] The following examples are intended to illustrate but not limit the disclosure, and are typical of those that may be used, although other procedures known to those skilled in the art may alternatively be used. EXAMPLES

[0119] Generation of RNA fusion constructs using plasmids A simple plasmid transfection experiment was performed in which fragL and fragR were cloned into the lentiCRISPRv2 backbone downstream of the U6 promoter. These were delivered to cells individually or in combination, and RNA was isolated 48 hours later. Reverse transcription polymerase chain reaction (RT-PCR) was performed using flanking amplification primers. As shown in Figure 1B, RNA fusion was observed when both plasmids were transfected together but not alone. PCR products were then purified and Sanger sequenced to confirm the expected fusion sequence (see Figure 1C).

[0120] Small RNAs and polymerase III promoters RNA fusion constructs with polymerase III (pol-III) promoters Ribozyme-RNA fusion constructs were cloned downstream of the U6-driven sgRNA sequence (designated sgRNA fragL and sgRNA fragR) in the lentiCRISPRv2 backbone (see FIG. 2A). As shown in FIG. 2B, the presence of a non-targeting sgRNA transcript at the 5' position of the RNA fusion construct did not alter the RNA processing machinery that generated the ligated fusion. Furthermore, the EcoRI restriction site on the sgRNA fragR construct shown in FIG. 2C highlights the ability to identify a theoretical barcode placed at that position for perturbation mapping using RNA sequencing.

[0121] RNA fusion constructs for combinatorial screening The constructs were designed to be suitable for combinatorial screening by orienting the fragL and fragR sequences in the antisense orientation to the transcripts (designated anti_fragL and anti_fragR) controlled by the lentiviral 5' long terminal repeat promoter. The design prevents the degradation of the transcripts during lentiviral production due to the fast cleavage rate of the twister ribozyme implemented in the system. The correct concatenated RNA fusion construct sequences in the antisense orientation were confirmed by RT-PCR and Sanger sequencing (see Figure 2D-E). Furthermore, the anti_fragL and anti_fragR constructs were cloned into a plasmid containing a fluorescent reporter, allowing for the rapid selection of cells in which the RNA fusion was produced. Cells that received both plasmids were easily identified by fluorescence and underwent fluorescence-activated cell sorting to screen for only those cells that received both fragments that could produce the RNA fusion construct.

[0122] Since it is desirable for the RNA fusion system to be modular across a variety of polymerase III promoters, we demonstrated that these constructs can be fused to each other when driven by the H1 promoter, which showed both polymerase II and polymerase III activity (Figure 8A). As shown in Figure 8B, the presence of the RNA fusion construct can be detected when driven by the H1 promoter. This shows that the system is flexible and suitable for a variety of promoters that may be present in genome engineering constructs for various purposes.

[0123] Use of RNA fusion constructs for combinatorial screening The use of EcoRI restriction sites (see Figure 2C) allows variable barcodes to be cloned into each sgRNA or other perturbation, so that the constructs can be identified by the barcode. This ensures that each cell in the screen receives a unique barcode pair that serves as a unique molecular identifier (UMI), with all UMIs collapsed in downstream counts. This library is further subjected to next generation sequencing (NGS) to generate a lookup table matching barcodes to perturbations. This library is transduced at an intermediate multiplicity of infection such that most cells receive two library elements. Cells are then sorted for GFP / tdTomato double positive fluorescence, screening for cells that receive two barcodes. Cells were collected and RNA was isolated at multiple time points of the screen (days 3, 14, 21, and 28) to ensure robust fitness measurements. UMIs from the collected RNA were then selectively reverse transcribed, amplified, and sequenced. The barcodes identified at each time point were then mapped to the first generated lookup table, providing a genotype-to-phenotype mapping across the entire experiment.

[0124] Ribozyme-mediated RNA fusion using large molecular weight RNA and polymerase II promoters. To further explore the broad utility of the ribozyme-mediated RNA fusion approach, we next investigated fusion expression driven by polymerase II (pol-II) promoters. RNA polymerase II is responsible for the transcription of most cellular genes (mRNAs) and pol-II promoters, such as EF1α, SV40, CMV, and RSV, as well as tissue-specific promoters such as NEUROD2 in the CNS and TBX20 in the aorta, which are important for effective gene therapy translation. We hypothesized that a ribozyme-mediated RNA fusion approach could be engineered into the AAV backbone to precisely control the expression of various gene therapy modalities. A general schematic of the approach is shown in Figure 3. Ligand-responsive aptamer sequences are attached to the 3' end of fragL and the 5' end of the fragR construct. These aptamers make tertiary interactions with the ribozyme, stabilizing the catalytic loop so that self-cleavage does not occur. In the absence of cleavage, the two fragments never hybridize and no linked RNA fusion transcript is generated. Addition of a ligand disrupts the structural conformation of the aptamer and is unable to stabilize the ribozyme, which then undergoes self-cleavage to generate unique ends, allowing the complementary regions of the two fragments to hybridize to each other and be ligated by RtcB ligase.

[0125] To allow the inducible system to generate a full-length transcript and subsequent protein without additional features, an intron sequence was fused between the therapeutic payload and the ribozyme-mediated fusion construct (see Figure 3). When the two fragments hybridize and are joined, features of the two introns such as 5' and 3' splice sites, branch points, and polypyrimidine tracts are recognized by the cellular splicing machinery. Trans-splicing then occurs to remove the intron sequence and fuse the two exons to generate a full-length functional transcript. The concept is outlined in Figure 4. Two halves of green fluorescent protein (GFP-L and GFP-R), which do not fluoresce when expressed individually, were conjugated to the intron sequence. The ribozyme-mediated RNA fusion construct was then cloned into the px600 AAV backbone. The two-intron model was first utilized with a longer intron sequence (>250 bp each) from the dihydrofolate reductase (DHFR) gene, which has previously been shown to undergo efficient splicing when joined.

[0126] When each intron was cloned between one half of a GFP transcript and one construct of a ribozyme-mediated RNA fusion pair and transfected into HEK293T cells, fluorescence was observed only when both sequences were delivered in combination with each other (see Figure 5A). RNA was isolated from these cells. Using RT-PCR and Sanger sequencing, we demonstrated that the intron was efficiently spliced ​​out and a full-length GFP transcript was generated from the two halves (see Figure 5B-C). The ability to generate functional fluorescent protein through flow cytometry was further demonstrated in Figure 7.

[0127] Ribozyme-mediated exonuclease-resistant and circular RNA fusion constructs. Because this fusion barcode approach is mediated at the RNA level, various RNA processing mechanisms can be used to further engineer the construct to obtain desirable properties. The ribozyme-mediated fusion barcodes can be engineered to contain short "exonuclease-resistant" RNA (xrRNA) structures derived from viral genomes (Figure 4). Once transcribed, these RNA elements serve to prevent the progressive extracellular nucleotide degradation of RNA. Their integration into the fusion barcode can improve the robustness and persistence of the barcode structure in the cellular environment.

[0128] To further improve the persistence of RNA barcodes in the cellular environment, the circularized RNA barcode design is shown in Figure 9A. In this design, the left fragment design was modified to include a "designer exon" and a chimeric intron at the 3' end of the construct (circ-fragL). The designer exon can be optimized to the appropriate length using various exon splicing enhancer (ESE) elements to promote splicing from the intron (sequence shown in Figure 10). The right fragment was also modified with an intron added to its 5' end, and another designer exon, followed by the original fragR construct shown in Figure 1 (circ-fragR). Once transcribed, the ribozyme can self-cleave, allowing the complementary sequences to hybridize to each other. The ends generated by ribozyme cleavage are recognized by the endogenous RNA ligase RtcB to form one fused end.

[0129] When the exon and intron are close to each other, the spliceosome machinery recognizes various intronic elements and splices out the two introns, joining the two designer exons together via this back-splicing mechanism. This fuses the other ends to form a circular structure. The constructs are circularized in cells by transfecting HEK293T cells with the circ-fragL and circ-fragR. RNA was isolated 48 hours after transfection and reverse transcribed. PCR was performed to determine whether the circular RNA persisted. By utilizing two directional primer pairs, the RNA barcodes could be circularized and recovered from a pool of cellular RNA (Figure 9B). Using these constructs, the barcodes are robust in complex cellular environments, allowing them to be used in a wider variety of screening platforms.

[0130] Use of a chimeric intron approach for gene expression A gene expression system was tested utilizing a chimeric intron (pCI) approach in which GFP-L was fused to the 5' end of an intron from the β-hemoglobin gene, including a 5' splice site; GFP-R was fused to the 3' end of an intron from the human IgG gene, including a branch point, a polypyrimidine tract, and a 3' splice site (Figure 11). Using the pCI vector containing the ribozyme-mediated RNA fusion construct, robust expression of GFP was observed upon transfection of both plasmids. Fluorescence levels were comparable to full-length GFP transcripts expressed individually, demonstrating the robust ability of this approach to generate functional protein (see Figure 6A). When GFP total fluorescence was quantified by flow cytometry, the pCI system produced 37% cellular GFP+, whereas the full GFP transcript in the px600 AAV backbone produced 67% cellular GFP+ (see Figure 6B). This is impressive considering that in the pCI approach, each cell must be transfected with two plasmids.

[0131] Next, we investigated whether the high expression levels of GFP using the pCI system required the complementary regions of RNA from the fragL and fragR constructs to hybridize with each other in order to obtain efficient splicing of the intron. For this investigation, constructs were generated that contained only the intron and GFP half, lacking the complementary region and ribozyme (termed "LinkFree"). These constructs and their full-length counterparts were used to transfect HEK293T cells. After 48 hours, RNA was isolated and RT-qPCR was performed to analyze the relative expression of GFP transcripts normalized to GAPDH. As shown in Figure 6C, the GFP-DHFR intron sequence could be spliced ​​out without the need for hybridization, since the relative expression did not change significantly in the LinkFree system. Alternatively, the pCI system showed a much higher requirement for hybridization with an activation ratio (full-length expression / unlinked expression) of >6 (see Figure 6D-E).

[0132] To deliver various therapeutic modalities, it may be advantageous to administer one viral vector and deliver the gene of interest. Therefore, the construct was further engineered using an inducible gene expression system contained in one plasmid so that the entire mechanism could be packaged into a single AAV vector. To achieve this, a dual promoter-based system was used in which GFP-pCI-L was driven by the U6 promoter and GFP-pCI-R was driven by the CMV promoter (Figure 11A). Transfection of the dual promoter plasmid construct in HEK293T cells resulted in expression of GFP with an activation ratio of >80-fold when compared to the design without the dual promoter linkage (Figure 11B).

[0133] Thus, using the concept of RNA fusion using ribozymes, separately delivered elements can be assembled to generate full-length functional proteins. Using the pCI intron sequence, high expression levels were achieved with greatly increased expression due to hybridization of the ribozyme-generated complementary regions. The constructs were further engineered and packaged into a single plasmid so that they could be packaged into individual AAV capsids and delivered as a single vector. It is expected that controlled inducible expression of effectors with the addition of synthetic riboswitches or other inducible elements to one or both constructs can also be utilized.

[0134] This disclosure demonstrates the feasibility of using a ribozyme-based approach that includes a tetracycline-responsive ribozyme embedded in the 3' untranslated region (UTR) of a gene of interest (GOI). In the system, at baseline levels, the hammerhead ribozyme self-cleaves during transcription, cleaving the 3'-poly(A) tail, destabilizing the RNA transcript and resulting in relatively low gene expression. Upon addition of the tetracycline ligand, the ligand binds with high affinity to the tetracycline-responsive aptamer. This induces a conformational change in the secondary structure of the RNA molecule, disrupting the tertiary loop-loop interaction of the hammerhead ribozyme, preventing cleavage of the poly(A) tail, stabilizing the mRNA transcript, and allowing increased expression of the GOI (Figure 12A).

[0135] The present disclosure illustrates tetracycline-induced insulin expression by cloning the tetracycline hammerhead aptazyme into the 3'UTR of modified insulin constructs. The proinsulin sequence was modified with H10D, K29R, R31K, and L62R mutations to allow for processing of proinsulin and maturation of insulin by furin protease in the outer pancreatic organ. As shown in FIG. 12B, upon transfection into HEK293T cells in vitro, the addition of tetracycline (125 μM) achieved induction of insulin secretion into the cell culture supernatant. This effect was shown to be dependent on the presence of the tetracycline hammerhead aptazyme, as the positive control (AAV-insulin) showed no tetracycline-induced fold change in the expression of secreted insulin. As it is advantageous to control insulin expression on a patient-by-patient basis, the present disclosure further demonstrates that tetracycline-induced expression of mature insulin occurs in a tetracycline dose-dependent manner (FIG. 12C). To demonstrate the tunability of insulin expression kinetics with this ribozyme-based approach, several versions of the insulin riboswitch design were generated and cloned into a pZac AAV backbone. The number and position of the tetracycline hammerhead aptazymes (Figure 13A) were then quantified for the amount of insulin secreted into cell culture supernatants of HEK293T cells 48 hours after transfection upon addition of DMEM or tetracycline (125 μM) 4-6 hours after transfection. As shown in Figure 13B, the addition of multiple aptazymes reduced background expression of insulin (no tetracycline). Because these background levels were low, the addition of tetracycline induced insulin secreted by one of the constructs by more than 20-fold.

[0136] Through the 3'UTR aptazyme-based approach, the present disclosure demonstrates the feasibility of utilizing the approach to address insulin needs in diabetic patients. The present disclosure shows that in vitro, mature insulin expression levels are dose-dependent and adjustable by engineering the construct.

[0137] The feasibility of this disclosure is also shown by first demonstrating its efficacy with a 3'UTR tetracycline hammerhead aptazyme, as shown in Figure 12A. Using the px601 plasmid backbone, the aptazyme riboswitch was cloned into the 3'UTR between the SaCas9 protein and the bGH poly(A) sequence (Figure 14A). HEK293T cells were transfected with the plasmid and tetracycline-induced expression of SaCas9 was demonstrated in vitro by replacing the medium with DMEM alone or DMEM containing 125 μM tetracycline 4-6 hours after transfection. RNA was isolated 48 hours after transfection, reverse transcribed, and the relative expression of the SaCas9 transcript to GAPDH was quantified by qPCR assay. As shown in Figure 14B, a reduction in background was observed and no ligand condition was associated with an increase in expression (approximately 6-fold) with the addition of medium containing tetracycline.

[0138] This disclosure demonstrates tetracycline-induced genome editing by SaCas9 in vivo using AAV8 capsids carrying the construct in FIG. 14B. For these in vivo experiments, SaCas9-specific single guide RNAs targeting the mouse Pcsk9 gene were cloned into the vector. These capsids were then administered to 6-8 week old C57Bl / 6 mice via retro-orbital injection at a dose of 5E12 viral genomes per mouse. Serum was collected weekly and tetracycline treatments (30 mg / Kg) were administered at the 2 and 5 week time points. Livers were also harvested from a subset of mice at the 5 and 7 week time points, DNA was isolated, and Cas9-induced Pcsk9 genome editing was quantified using Synthego's CRISPR Editing Inference (ICE) tool (FIG. 14C). As shown in Figure 14D, a decrease in serum PCSK9 protein levels was observed after the first 3 days of tetracycline administration, as measured by a mouse PCSK9 ELISA kit. This suggested that gene editing induced by Cas9-mediated gene knockdown was ligand-dependent. Livers were harvested and the percentage of DNA edited at the Pcsk9 locus was quantified, and the effect of DNA editing based on the tetracycline therapy that the mice received was observed. At the 5-week time point after one round of 3-day tetracycline therapy, less than 5% editing at the Pcsk9 locus was quantified, but with an additional 3 days of tetracycline therapy, the percentage of edited DNA increased to approximately 7% (Figure 14E). Through this experiment, it was shown that when utilizing an AAV-Cas9-riboswitch-based approach, the level of SaCas9-mediated DNA editing can be adjusted based on the tetracycline regimen.

[0139] Persistence of RNA constructs An RNA construct was generated by transcribing a DNA nucleic acid having a T7 promoter operably linked to a DNA construct encoding a 5' P3 twister ribozyme, a 5' linking sequence, an IRES operably linked to a coding sequence for GFP, a linker, a 3' linking sequence, a 3' twister ribozyme, and a poly-T tail, resulting in a linear RNA construct (see FIG. 16A).

[0140] 293T cells were seeded at 25% confluency in 12-well plates and transfected with lipid-RNA complexes consisting of 1 μg of mutated circular or circular GFP RNA and 3.5 μL of Lipofectamine Messenger Max. RNA was isolated from cells over 3 days using the Qiagen RNeasy kit and qPCR was performed to measure the amount of circularized RNA. **p<0.01 t-test comparison within each day (see Figure 16B-D).

[0141] 293T cells were seeded at 25% confluency in 12-well plates and transfected with lipid-RNA complexes consisting of 1 μg of mutated circular or circular GFP RNA and 3.5 μL of Lipofectamine Messenger Max. RNA was isolated from cells over 3 days using the Qiagen RNeasy kit and qPCR was performed to measure the amount of GFP RNA. *p<0.05 t-test comparison within each day.

[0142] 293T were seeded in 12 wells at 25% confluency and transfected with lipid-RNA complexes consisting of 1 μg of mutated circular or circular zinc finger RNA and 3.5 μL of Lipofectamine Messenger Max. DNA was isolated using the Qiagen DNeasy Blood & Tissue Kit, the edited region was amplified, Sanger sequenced, and editing efficiency was quantified using the Synthego ICE CRISPR analysis tool. 293T were seeded in 12 wells at 25% confluency and transfected with lipid-RNA complexes consisting of 1 μg of Cas wild-type or mutant RNA, 1 μg of T2 guide RNA, and 3.5 μL of Lipofectamine Messenger Max. After 3 days, genomic DNA was isolated using the Qiagen DNeasy Blood & Tissue Kit, the edited region was amplified, Sanger sequenced, and editing efficiency was quantified using the Synthego ICE CRISPR analysis tool (see Figure 17A-C).

[0143] To generate a circular format for a vaccine, a self-amplifying RNA system, such as that associated with alphaviruses, was used downstream of the IRES (see Figures 18A-B).

[0144] It will be understood that various modifications can be made without departing from the spirit and scope of the disclosure and, accordingly, other embodiments are within the scope of the following claims.

Claims

1. A ribozyme-activated RNA construct comprising: one or more ribozymes, and comprising one or more RNA coding sequences for at least one polypeptide of interest, Transcription of said one or more RNA coding sequences into said at least one polypeptide of interest is activated by or dependent on the activity of said one or more ribozymes. Ribozyme-activated RNA constructs.

2. a first artificial RNA element comprising an optional primer region, an optional barcode region, an RNA coding sequence for a polypeptide of interest, and a complementary sequence to the sequence of a second artificial RNA element, and a first self-cleaving ribozyme; a second artificial RNA element comprising an optional primer region, an optional barcode region, an RNA coding sequence for a polypeptide of interest, and a complementary sequence to the sequence of the first artificial RNA element, and a second self-cleaving ribozyme. Further comprising: Cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes, respectively, provides a hybridization construct comprising a region of dsRNA from the commentary sequence that hybridizes together; The hybridization constructs can be further ligated by an RNA ligase to form an RNA fusion construct; Expression from said RNA fusion construct produces said at least one polypeptide of interest. The ribozyme-activated RNA construct of claim 1.

3. 3. The ribozyme-activated RNA construct of claim 2, wherein the first artificial RNA element comprises a barcode sequence or a specific molecular identity (UMI) sequence and / or the second artificial RNA element comprises a barcode sequence or a UMI sequence.

4. 4. The ribozyme-activated RNA construct of claim 3, wherein the barcode sequence or the UMI sequence of the first artificial RNA element has a different sequence than the barcode region or the UMI sequence from the second artificial RNA element.

5. 3. The ribozyme-activated RNA construct of claim 2, wherein the first artificial RNA element comprises a primer sequence and / or the second artificial RNA element comprises a primer sequence.

6. 6. The ribozyme-activated RNA construct of claim 5, wherein the primer sequence of the first artificial RNA element is different from the primer sequence from the second artificial RNA element.

7. The ribozyme-activated RNA construct of claim 2, wherein the first and second complementary sequences are 30-60 bp in length.

8. The ribozyme-activated RNA construct of claim 2 , wherein the first and second ribozymes are twister ribozymes.

9. The ribozyme-activated RNA construct of claim 8, wherein the first ribozyme is a P3 twister ribozyme.

10. The ribozyme-activated RNA construct of claim 9, wherein the second ribozyme is a P1 twister ribozyme.

11. The ribozyme-activated RNA construct of claim 2 , wherein the RNA ligase is RtcB.

12. 3. The ribozyme-activated RNA construct of claim 2, wherein the vector or plasmid comprises the first artificial element, the first artificial element being located downstream of a first RNA promoter and a first perturbation element, and / or the vector or plasmid comprises the second artificial element, the second artificial element being located downstream of a second RNA promoter and a second perturbation element.

13. The ribozyme-activated RNA construct of claim 12, wherein the first RNA promoter and / or the second RNA promoter is a polymerase III promoter.

14. The ribozyme-activated RNA construct of claim 12, wherein the first perturbation element and / or the second perturbation element is an sgRNA utilized in a CRISPR knockout screen.

15. a first artificial RNA element comprising an RNA coding sequence for a polypeptide of interest, an intron sequence, a sequence of a second artificial RNA element and a complementary sequence to a 3' aptamer, and a first self-cleaving ribozyme, said 3' aptamer interacting with the first self-cleaving ribozyme to stabilize said first self-cleaving ribozyme; and a second artificial RNA element comprising an RNA coding sequence for a polypeptide of interest, an intron sequence, a complementary sequence to the sequence of the first artificial RNA element, and a 3' aptamer, said second artificial RNA template being tethered to a second self-cleaving ribozyme, said 3' aptamer interacting with the second self-cleaving ribozyme to stabilize said second self-cleaving ribozyme; Including, Cleavage of the first and second artificial RNA elements by the first and second self-cleaving ribozymes, respectively, provides a hybridization construct comprising a region of dsRNA from the commentary sequence that hybridizes together; The hybridization construct can be further joined by an RNA ligase, and the intron sequence is removed by the spliceosome to form an RNA fusion construct; Expression from said RNA fusion construct produces said at least one polypeptide of interest. The ribozyme-activated RNA construct of claim 1.

16. 16. The ribozyme-activated RNA construct of claim 15, wherein the intron sequence is derived from dihydrofolate reductase.

17. 16. The ribozyme-activated RNA construct of claim 15, wherein an RNA coding sequence for a polypeptide of interest is flanked by each of said intron sequences.

18. The ribozyme-activated RNA construct of claim 15, wherein the RNA coding sequence for a polypeptide of interest encodes a polypeptide / protein selected from insulin, coagulation factor IX, cystic fibrosis transmembrane conductance regulator protein, and dystrophin protein.

19. A pharmaceutical composition comprising the ribozyme-activated RNA construct of claim 1, wherein the ribozyme-activated RNA construct is linearized and 5' ribozyme, 5' linking sequence, internal ribosome entry site (IRES) sequence, an RNA coding sequence for at least one polypeptide of interest; 3' linking sequence, a 3' ribozyme sequence, and including a pharma- ceutically acceptable carrier; Pharmaceutical compositions.

20. 20. The pharmaceutical composition of claim 19, wherein the linear ribozyme-activated RNA construct lacks a polymerase binding region.

21. 20. The pharmaceutical composition of claim 19, wherein the 5' and 3' ribozymes are selected from the group consisting of twister ribozyme, hammerhead ribozyme, hatchet ribozyme, hepatitis delta virus ribozyme, ligase ribozyme, pistol ribozyme, twister sister ribozyme, Vg1 ribozyme, VS ribozyme, and derivatives of any of the foregoing.

22. 20. The pharmaceutical composition of claim 19, wherein the 5' and 3' linking sequences are substrates for a natural ligase in situ.

23. The pharmaceutical composition of claim 22, wherein the native ligase is RtcB.

24. The pharmaceutical composition of claim 19, wherein the IRES comprises any of the sequences of SEQ ID NOs: 1 to 1328.

25. 20. The pharmaceutical composition of claim 19, wherein the at least one polypeptide of interest comprises two or more polypeptides of interest separated by a self-cleaving peptide.

26. 26. The pharmaceutical composition of claim 25, wherein the self-cleaving peptide comprises a 2A or a 2A-like peptide.

27. 20. The pharmaceutical composition of claim 19, wherein the at least one polypeptide of interest is selected from the group consisting of a prodrug activating enzyme, a biological response modifier, a receptor ligand, an immunoglobulin-derived binding polypeptide, a non-immunoglobulin binding polypeptide, an antigenic polypeptide, a genome editing enzyme, and any combination thereof, and wherein the multiple polypeptides are separated by a 2A or a 2A-like peptide.

28. 28. The pharmaceutical composition of claim 27, wherein the biological response modifier is an immune enhancing cytokine.

29. 29. The pharmaceutical composition of claim 28, wherein the immune enhancing cytokine is selected from the group consisting of interleukins 1-38, interferons, tumor necrosis factors (TNF), and granulocyte-macrophage colony stimulating factors (GM-CSF).

30. The pharmaceutical composition of claim 26, wherein the 2A or 2A-like peptide further comprises a GSG linker moiety.

31. The pharmaceutical composition of claim 27, wherein the genome editing enzyme is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases (TALENs), artificial meganucleases, and RNA-guided DNA endonuclease (Cas) polypeptides.

32. 20. The pharmaceutical composition of claim 19, wherein the 5' and 3' ribozyme sequences are independently selected from a sequence that is at least 85-100% identical to either 5'-GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT-3' (SEQ ID NO: 1354) or 5'-AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC-3' (SEQ ID NO: 1355).

33. 20. The pharmaceutical composition of claim 19, wherein the 5' and 3' linked sequences are independently selected from a sequence that is at least 85-100% identical to either 5'-AACCATGCCGACTGATGGCAG-3' (SEQ ID NO: 1356) or 5'-CTGCCATCAGTCGGCGTGGACTGTAG-3' (SEQ ID NO: 1357).

34. The pharmaceutical composition of claim 19, wherein the IRES sequence is at least 85-100% identical to 5'-3' (SEQ ID NO: 1358).

35. A vaccine composition comprising the ribozyme-activated RNA construct of claim 1, wherein the ribozyme-activated RNA construct is linearized and 5' ribozyme, 5' linking sequence, internal ribosome entry site (IRES) sequence, an RNA coding sequence for at least one antigenic polypeptide; 3' linking sequence, a 3' ribozyme sequence, and including a pharma- ceutically acceptable carrier; Vaccine compositions.

36. 36. The vaccine composition of claim 35, wherein the linearized ribozyme-activated RNA construct lacks a polymerase binding region.

37. 36. The vaccine composition of claim 35, wherein the 5' and 3' ribozymes are selected from the group consisting of twister ribozyme, hammerhead ribozyme, hatchet ribozyme, hepatitis delta virus ribozyme, ligase ribozyme, pistol ribozyme, twister sister ribozyme, Vg1 ribozyme, VS ribozyme, and derivatives of any of the foregoing.

38. 36. The vaccine composition of claim 35, wherein the 5' and 3' linking sequences are substrates for a natural ligase in situ.

39. 39. The vaccine composition of claim 38, wherein the native ligase is RtcB.

40. The vaccine composition of claim 35, wherein the IRES comprises any of the sequences of SEQ ID NOs: 1 to 1328.

41. 36. The vaccine composition of claim 35, wherein the at least one antigenic polypeptide comprises two or more antigenic polypeptides separated by a self-cleaving peptide.

42. 42. The vaccine composition of claim 41, wherein the self-cleaving peptide comprises a 2A or a 2A-like peptide.

43. The vaccine composition of claim 42, wherein the 2A or 2A-like peptide further comprises a GSG linker moiety.

44. 36. The vaccine composition of claim 35, wherein the 5' and 3' ribozyme sequences are independently selected from a sequence that is at least 85-100% identical to 5'-GCCATCAGTCGCCGGTCCCAAGCCCGGATAAAATGGGAGGGGGCGGGAAACCGCCT-3' (SEQ ID NO: 1354) or 5'-AACACTGCCAATGCCGGTCCCAAGCCCGGATAAAAGTGGAGGGTACAGTCCACGC-3' (SEQ ID NO: 1355).

45. 36. The vaccine composition of claim 35, wherein the 5' and 3' linked sequences are independently selected from sequences that are at least 85-100% identical to 5'-AACCATGCCGACTGATGGCAG-3' (SEQ ID NO: 1356) or 5'-CTGCCATCAGTCGGCGTGGACTGTAG-3' (SEQ ID NO: 1357).

46. The vaccine composition of claim 35, wherein the IRES sequence is at least 85-100% identical to 5'--3' (SEQ ID NO: 1358).

47. 36. The vaccine composition of claim 35, wherein the antigenic polypeptide comprises a SARS-CoV-2 spike protein.

48. The vaccine composition of claim 35, wherein the at least one polypeptide of interest or antigenic polypeptide is contained within a self-amplifying RNA construct.

49. 49. The vaccine composition of claim 48, wherein the self-amplifying RNA construct comprises an alphavirus or a paramyxovirus.

50. 2. The ribozyme-activated RNA construct of claim 1, comprising: one or more promoter sequences, one or more RNA coding sequences for at least one polypeptide of interest, one or more aptazyme-based riboswitches; a 3'UTR sequence comprising the aptazyme-based riboswitch; and Poly(A) sequence Including, the aptazyme-based riboswitch, when not bound to a target ligand, destabilizes the ribozyme-activated RNA construct, resulting in reduced expression of the at least one polypeptide of interest; The aptazyme-based riboswitch, upon binding to a target ligand, stabilizes the ribozyme-activated RNA construct, resulting in increased expression of the at least one polypeptide of interest. Ribozyme-activated RNA constructs.

51. 51. The ribozyme-activated RNA construct of claim 50, wherein the aptazyme-based riboswitch is a hammerhead aptazyme.

52. 51. The ribozyme-activated RNA construct of claim 50, wherein the targeting ligand is selected from tetracycline, theophylline, and guanine.

53. 51. The ribozyme-activated RNA construct of claim 50, wherein the at least one polypeptide of interest is selected from the group consisting of a prodrug activating enzyme, a biological response modifier, a receptor ligand, an immunoglobulin-derived binding polypeptide, a non-immunoglobulin binding polypeptide, an antigenic polypeptide, a genome editing enzyme, and any combination thereof, and wherein the multiple polypeptides are separated by a 2A or 2A-like peptide.

54. 54. The ribozyme-activated RNA construct of claim 53, wherein the biological response modifier is an immune enhancing cytokine.

55. 55. The ribozyme-activated RNA construct of claim 54, wherein the immune enhancing cytokine is selected from the group consisting of interleukins 1-38, interferons, tumor necrosis factors (TNF), and granulocyte-macrophage colony-stimulating factors (GM-CSF).

56. 54. The ribozyme-activated RNA construct of claim 53, wherein the 2A or 2A-like peptide further comprises a GSG linker moiety.

57. The ribozyme-activated RNA construct of claim 53, wherein the genome editing enzyme is selected from the group consisting of zinc finger nucleases, transcription activator-like effector nucleases (TALENs), artificial meganucleases, and RNA-guided DNA endonuclease (Cas) polypeptides.

58. 51. The ribozyme-activated RNA construct of claim 50, wherein the one or more promoter sequences is a polymerase II (pol-II) promoter sequence.

59. 51. The ribozyme-activated RNA construct of claim 50, wherein the one or more promoter sequences have sequences for EF1α, hU6, SV40, CMV, RSV, NEUROD2, and / or TBX20.

60. 51. The ribozyme-activated RNA construct of claim 50, wherein the poly(A) sequence is a bGH poly(A) sequence.

61. A plasmid or capsid comprising the ribozyme-activated RNA construct of claim 50.

62. 62. The plasmid or capsid of claim 61 , wherein the plasmid or capsid is an AAV-based plasmid or capsid.

63. 62. The plasmid or capsid of Claim 61, wherein the plasmid expresses a Cas9 protein and a gRNA.

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